Preadipocyte Determination Research Articles

Suppression of preadipocyte determination by SOX4 limits white adipocyte hyperplasia in obesity

Preadipocyte determination expanding the pool of preadipocytes is a vital process in adipocyte hyperplasia, but the molecular mechanisms underlying this process are yet to be elucidated. Herein, SRY-related HMG box transcription factor 4 (SOX4) was identified as a critical target in response to BMP4- and TGFβ-regulated preadipocyte determination. SOX4 deficiency is sufficient to promote preadipocyte determination in mesenchymal stem cells (MSCs) and acquisition of preadipocyte properties in nonadipogenic lineages, while its overexpression impairs the adipogenic capacity of preadipocytes and converts them into nonadipogenic lineages. Mechanism studies indicated that SOX4 activates and cooperates with LEF1 to retain the nuclear localization of β-catenin, thus mediating the crosstalk between TGFβ/BMP4 signaling pathway and Wnt signaling pathway to regulate the preadipocyte determination. Invivo studies demonstrated that SOX4 promotes the adipogenic-nonadipogenic conversion and suppresses the adipocyte hyperplasia. Together, our findings highlight the importance of SOX4 in regulating the adipocyte hyperplasia in obesity.

Low lysine stimulates adipogenesis through ZFP423 upregulation in bovine stromal vascular cells

Adipogenesis is a complex process comprising commitment and a differentiation stages. Through research, many different transcriptional factors were found to mediate preadipocyte commitment and differentiation. Lysine has a potential of regulating the commitment and differentiation of preadipocytes. In the present study, intramuscular stromal vascular cells (SVC) isolated from Hanwoo beef cattle were used to elucidate the effects of low lysine level on adipogenesis. SVC were isolated and incubated with various concentrations of lysine (0, 37.5, 75, 150 and 300 µg/mL). No significant difference were observed in the proliferation of SVC after 24 and 48 h of incubation with different concentration of lysine. On preadipocyte determination, reducing the level of lysine significantly increased the expression of preadipocyte commitment gene Zinc finger protein 423 and Preadipocyte factor-1. Upon differentiation, Oil Red O staining revealed that lipid accumulation and triglyceride content significantly increased with the decreasing lysine levels in the media. Expression levels of peroxisome proliferator-activated receptor-γ, CCAAT enhancer binding protein-α, sterol regulatory element binding protein-1c, Fatty Acid Binding Protein 4 and stearoyl CoA desaturase were upregulated by the decreased level of lysine. These data suggest the potential mechanism of action for the improved preadipocyte commitment and adipocyte differentiation in bovine intramuscular SVC upon treatment with low levels of lysine. These findings may be valuable in developing feed rations that promote deposition of intramuscular fat in beef cattle through lysine level modification.

Pre-Adipocyte Determination and Adipocyte Differentiation of Stromal Vascular Cells Isolated From Intramuscular Tissue of Hanwoo Beef Cattle Treated by Acetate and Propionate

The effects of acetate and propionate in enhancing intramuscular fat (marbling) deposition in beef cattle are poorly understood. This study aimed to evaluate the effects of acetate and propionate on pre-adipocyte determination and adipocyte differentiation in stromal vascular cells (SVC) isolated from the intramuscular tissue of Hanwoo beef cattle. SVC were isolated and treated with different concentrations of acetate and propionate (0.125 to 1 mM). Pre-adipocyte determination was observed through the Zfp423 (Zinc finger protein 423) and Pref-1 (Pre-adipocyte factor-1) genes expressions, which were important transcription factors used to identify committed pre-adipocytes. Adipocyte differentiation was determined based on the lipid accumulation, triglyceride contents, and their molecular activities related to adipogenic markers. The results suggest that acetate supports pre-adipocyte determination in SVC which is observed to increase the expression of Zfp423 and Pref-1 along with the increasing acetate concentration. Propionate treatment was seen to significantly affect lipid accumulation as well as triglyceride content in SVC, compared to acetate treatment. Furthermore, propionate treatment very significantly increased the expressions of PPARγ, C/EBPα, SREBP-1c, and FABP4. In conclusion, acetate likely enhances pre-adipocyte determination, while propionate effectively promotes adipogenesis in SVC isolated from the intramuscular tissue of Hanwoo beef cattle.

Siah2 Expression in Adipocyte Progenitor Cells

Obesity-related insulin resistance is associated with adipose tissue inflammation and visceral adipose tissue expansion via adipocyte hypertrophy and impaired recruitment of new progenitor cells to undergo adipogenesis (hyperplasia). Deletion of the ubiquitin ligase Seven-in-absentia homolog 2 (Siah2) in obese mice significantly reduced adipose tissue inflammation and insulin resistance compared to obese wild type mice. However, larger adipocyte size accompanied this phenotype and subsequent experiments with adipose tissue stromal vascular fraction (SVF) cells showed adipogenesis required Siah2. In an earlier study, we found Siah2 affects adipogenesis by enhancing gene expression of the PPARγ coactivator, Zfp423 via proteasome-dependent degradation of ZFP521, a transcriptional repressor of Zfp423. ZFP423 is an early marker of preadipocyte determination that is expressed in adipocyte progenitor cells. Although Siah2 mediates adipogenesis and is highly expressed in the adipose tissue stromal vascular cell fraction, cell-specific expression of Siah2 in adipose tissue is not well characterized. We found that Siah2 is highly expressed in macrophage. RNA-seq and microarray data from the Immunological Genome Project (Immgen.org) also reported Siah2 expression in lymphocytes. To identify Siah2 expression profile in adipose tissue, unsorted cells and magnetically sorted macrophage, T-cell, B-cell, and SCA-1 positive progenitor cells from mouse adipose tissues SVF were analyzed for Siah2 expression. In parallel, in situ hybridization RNAscope of fat pads was employed to inspect Siah2 mRNA localization in adipose tissue. Since our evidence indicates Siah2 acts upstream of ZFP423, we predict Siah2 is expressed in adipocyte progenitor cells as well as immune cells in adipose tissue. This work will provide the groundwork to better understand how Siah2 controls commitment of progenitor cells to the adipocyte lineage and links obesity to adipose tissue inflammation and insulin resistance. Disclosure T. Dang: None. G.E. Kilroy: None. J.L. Taylor: None. Y. Yu: None. E. Floyd: None.

Amplification of Adipogenic Commitment by VSTM2A

SUMMARYDespite progress in our comprehension of the mechanisms regulating adipose tissue development, the nature of the factors that functionally characterize adipose precursors is still elusive. Defining the early steps regulating adipocyte development is needed for the generation of tools to control adipose tissue size and function. Here, we report the discovery of V-set and transmembrane domain containing 2A (VSTM2A) as a protein expressed and secreted by committed preadipocytes. VSTM2A expression is elevated in the early phases of adipogenesis in vitro and adipose tissue development in vivo. We show that VSTM2A-producing cells associate with the vasculature and express the common surface markers of adipocyte progenitors. Overexpression of VSTM2A induces adipogenesis, whereas its depletion impairs this process. VSTM2A controls preadipocyte determination at least in part by modulating BMP signaling and PPARγ2 activation. We propose a model in which VSTM2A is produced to preserve and amplify the adipogenic capability of adipose precursors.

Origin of fat cells

Adipose tissue expansion involves the enlargement of existing adipocytes, the formation of new cells from committed preadipocytes, and the coordinated development of the tissue vascular network. Here we find that murine endothelial cells (ECs) of classic white and brown fat depots share ultrastructural characteristics with pericytes, which are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-cadherin promoter reveal localization of reporter genes in ECs and also in preadipocytes and adipocytes of white and brown fat depots. Furthermore, capillary sprouts from human adipose tissues, which have predominantly EC characteristics, are found to express Zfp423, a recently identified marker of preadipocyte determination. In response to PPARg activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Together these data support an endothelial origin of murine and human adipocytes, suggesting a model for how adipogenesis and angiogenesis are coordinated during adipose tissue expansion.

Editorial: Control of Adipocyte Differentiation and Metabolism.

EDITORIAL article Front. Endocrinol., 28 August 2015Sec. Experimental Endocrinology Volume 6 - 2015 | https://doi.org/10.3389/fendo.2015.00132

Double SET point: G9a makes its mark in adipogenesis

EMBO J (2013) 32 1, 45–59 doi:10.1038/emboj.2012.306; published online November232012 Sequence-specific DNA-binding transcription factors (TFs) bind throughout the genome to orchestrate the process of adipocyte differentiation in an exquisitely timed and precise manner. TF binding recruits histone-modifying enzymes, resulting in dynamic epigenomic modifications that correlate with extensive gene expression changes. In this issue of The EMBO Journal, Wang et al (2012) demonstrate a critical role for the SET-containing histone methyltransferase G9a in generating repressive chromatin marks that are lost in the process of adipogenesis.

The Vascular Endothelium of the Adipose Tissue Gives Rise to Both White and Brown Fat Cells

Adipose tissue expansion involves the enlargement of existing adipocytes, the formation of new cells from committed preadipocytes, and the coordinated development of the tissue vascular network. Here we find that murine endothelial cells (ECs) of classic white and brown fat depots share ultrastructural characteristics with pericytes, which are pluripotent and can potentially give rise to preadipocytes. Lineage tracing experiments using the VE-cadherin promoter reveal localization of reporter genes in ECs and also in preadipocytes and adipocytes of white and brown fat depots. Furthermore, capillary sprouts from human adipose tissue, which have predominantly EC characteristics, are found to express Zfp423, a recently identified marker of preadipocyte determination. In response to PPARγ activation, endothelial characteristics of sprouting cells are progressively lost, and cells form structurally and biochemically defined adipocytes. Together these data support an endothelial origin of murine and human adipocytes, suggesting a model for how adipogenesis and angiogenesis are coordinated during adipose tissue expansion.

Zfp423 Expression Identifies Committed Preadipocytes and Localizes to Adipose Endothelial and Perivascular Cells

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression invivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.

CREBL2, interacting with CREB, induces adipogenesis in 3T3-L1 adipocytes

The factors that influence preadipocyte determination remain poorly understood. In the present paper, we report that CREBL2 [CREB (cAMP-response-element-binding protein)-like 2], a novel bZIP_1 protein, is up-regulated during MDI-induced preadipocyte differentiation. During both overexpression and under physiological conditions, CREBL2 interacted and was entirely co-localized with CREB. Overexpression of CREBL2 was sufficient to promote adipogenesis via up-regulating the expression of PPARγ (peroxisome-proliferator-activated receptor γ) and C/EBPα (CCAAT/enhancer-binding protein α) and accelerate lipogenesis accompanied with increased GLUT (glucose transporter) 1 and GLUT4. CREBL2 knockdown restrained adipogenic conversion and lipogenesis. Additionally, depletion of CREB could completely block the effects of overexpressed CREBL2, whereas an increase in CREB could not drive adipogenesis in the absence of CREBL2, indicating that the roles for CREBL2 on adipogenesis were CREB-dependent. Furthermore, siCREBL2 [siRNA (short interfering RNA) against CREBL2] could down-regulate CREB transcriptional activity and suppress CREB phosphorylation. CREB knockdown decreased the CREBL2 protein levels and vice versa. Collectively, the results of the present study indicate that CREBL2 plays a critical role in adipogenesis and lipogenesis via interaction with CREB.

Transcriptional control of preadipocyte determination by Zfp423

The worldwide epidemic of obesity has increased the urgency of developing a deeper understanding of physiological systems related to energy balance and energy storage, including the mechanisms controlling the development of fat cells (adipocytes). The differentiation of committed preadipocytes to adipocytes is controlled by PPARγ and several other transcription factors 1, but the molecular basis for preadipocyte determination is not understood. Using a novel method for the quantitative analysis of transcriptional components, we identified the zinc-finger protein Zfp423 as a factor enriched in preadipose versus non-preadipose fibroblasts. Ectopic expression of Zfp423 in non-adipogenic NIH 3T3 fibroblasts robustly activates expression of PPARγ in undifferentiated cells and permits cells to undergo adipocyte differentiation under permissive conditions. ShRNA-mediated reduction of Zfp423 expression in 3T3-L1 cells blunts preadipocyte PPARγ expression and diminishes the ability of these cells to differentiate. Furthermore, both brown and white adipocyte differentiation is strikingly impaired in Zfp423-deficient mouse embryos. Zfp423 regulates PPARγ expression, in part, through amplification of the BMP signaling pathway, an effect dependent on the SMAD binding capacity of Zfp423. This study identifies Zfp423 as a transcriptional regulator of preadipocyte determination.

Ectopic Overexpression of C/EBP-alpha in Primary Human Mesenchymal Stem Cells Is Sufficient To Induce Preadipocyte Determination but Not Terminal Differentiation without Hormonal Enhancement.

Background: Bone marrow Mesenchymal Stem Cells (MSC) have been identified as multipotent non-hematopoietic progenitor cells that can differentiate into osteoblasts, myocytes, chondrocytes and adipocytes. MSC also play an important role for the normal hematopoiesis and bone marrow insufficiency goes along with an increase of fat involution, which might origin from MSC. One important factor that regulates later stages of adipogenesis is the CCAAT/enhancer binding protein alpha (C/EBP-alpha). Whereas most insights into the regulation of adipogenesis come from the analysis of murine cell lines or human preadipoyctes under the influence of adipogenic hormonal growth medium, data on primary human MSC under the influence of a single adipogenic transcription factor are lacking - especially without hormonal enhancement. In this study, we examined, if ectopic expression of C/EBP-alpha is able to induce adipogenic differentiation of primary human MSC.Methods / Results: Transient overexpression of C/EBP-alpha was performed in primary human MSC from bone marrow aspirates after two culture passages. MSC were cultured in DMEM medium (+10% FCS) without hormonal substitution. Total RNA was prepared from different time points (12h, 24h, 48h, 72h) and the expression of C/EBP-alpha as well as adipose genes (PPAR-gamma, FABP-4) was analyzed by semiquantitative RT-PCR. MSC transfected with the empty control vector had no detectable amounts of C/EBP-alpha, PPAR-gamma or FABP-4. In contrast, MSC under ectopic expression of C/EBP-alpha expressed FABP-4 after 48 hours with further increase over the time, whereas PPAR-gamma expression could be detected first after 72 hours. These MSC showed only slight morphologic changes without distinct intracellular lipid drops. Red Oil-O staining for fat cells was negative in all groups. Furthermore, permanent lentiviral overexpression of C/EBP-alpha for 7 to 14 days showed no significant morphological changes from MSC to adipocytes, when grown in normal culture medium. Adipogenic growth medium (Insulin 10ug/ml, Dexamethason 10uM, Indomethacin 200uM, Isobutylxanthine 500uM) was added after 3 days of C/EBP-alpha overexpression and led to terminal differentiated adipocytes after 3 further days, whereas in the control group the amount of differentiated adipocytes increased only after 7 days. The expression of C/EBP-alpha, PPAR-gamma and FABP-4 was determined again by semiquantitative RT-PCR and showed an earlier and higher upregulation of all factors in the C/EBP-alpha group compared to the control. Red Oil-O staining showed a significant higher amount of terminal differentiated adipocytes in the C/EBP-alpha group.Conclusion: This work shows that ectopic expression of C/EBP-alpha in primary human Mesenchymal Stem Cells is able to determine MSC only to preadipocytes, which do not undergo morphologic terminal differentiation until other factors like PPAR-gamma are upregulated. Moreover, ectopic expression of C/EBP-alpha in primary human MSC accelerates the process of terminal adipogenic differentiation dramatically as soon as other factors are induced by hormonal enhancement.

Prediction of preadipocyte differentiation by gene expression reveals role of insulin receptor substrates and necdin

The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differentiation via complex signalling networks. Here, using microarray analysis of brown preadipocytes that are derived from wild-type and insulin receptor substrate (Irs) knockout animals that exhibit progressively impaired differentiation, we define 374 genes/expressed-sequence tags whose expression in preadipocytes correlates with the ultimate ability of the cells to differentiate. Many of these genes, including preadipocyte factor-1 (Pref-1) and multiple members of the Wnt signalling pathway, are related to early adipogenic events. Necdin is also markedly increased in Irs knockout cells that cannot differentiate, and knockdown of necdin restores brown adipogenesis with downregulation of Pref-1 and Wnt10a expression. Insulin receptor substrate proteins regulate a necdin-E2F4 interaction that represses peroxisome-proliferator-activated receptor gamma (PPARgamma) transcription via a cyclic AMP response element binding protein (CREB)-dependent pathway. Together these define a key signalling network that is involved in brown preadipocyte determination.

Pre-adipocyte determination either by insulin or by 5-azacytidine.

CHEF/18 is a diploid Chinese hamster cell line of embryonic origin, which is fibroblastic in structure, but behaves like a mesenchymal stem cell line in its ability to differentiate into adipocytes, myoblasts, and chondrocytes. With these cells, adipocyte formation has been divided experimentally into two stages: (i) determination of pre-adipocytes, which have lost the ability to form other cell types while retaining their fibroblast structure; and (ii) commitment or terminal differentiation, in which lipids accumulate, adipocyte structure develops, and cells lose the ability to divide. This paper reports that the first stage can be induced by exposure to 5-azacytidine or 2'-deoxy-5-azacytidine, drugs that also induce CHEF cells to form other mesenchymal cell types, or by growth with added insulin. Pre-adipocytes are distinguished from CHEF stem cells by (i) their inability to form other mesenchymal cell types; and (ii) their rapid accumulation of lipid in response to added insulin. The possibility is discussed that both insulin and the cytidine analogs promote differentiation by the same mechanism, namely changes in the pattern of DNA methylation.