Ankrd26 gene disruption enhances adipogenesis of mouse embryonic fibroblasts

We previously reported that partial disruption of the Ankrd26 gene in mice leads to hyperphagia and leptin-resistant obesity. To determine whether the Ankrd26 mutation can affect the development of adipocytes, we studied mouse embryo fibroblasts (MEFs) from the mutant mice. We found that Ankrd26(-/-) MEFs have a higher rate of spontaneous adipogenesis than normal MEFs and that adipocyte formation is greatly increased when the cells are induced with troglitazone alone or with a mixture of troglitazone, insulin, dexamethasone, and methylisobutylxanthine. Increased adipogenesis was detected as an increase in lipid droplet formation and in the expression of several markers of adipogenesis. There was an increase in expression of early stage adipogenesis genes such as Krox20, KLF5, C/EBPβ, C/EBPδ, and late stage adipogenesis regulators KLF15, C/EBPα, PPARγ, and aP2. There was also an increase in adipocyte stem cell markers CD34 and Sca-1 and preadipocyte markers Gata2 and Pref-1, indicating an increase in both stem cells and progenitor cells in the mutant MEFs. Furthermore, ERK was found constitutively activated in Anrd26(-/-) MEFs, and the addition of MEK inhibitors to mutant cells blocked ERK activation, decreased adipogenesis induction, and significantly reduced expression of C/EBPδ, KLF15, PPARγ2, CD34, and Pref-1 genes. We conclude that Ankrd26 gene disruption promotes adipocyte differentiation at both the progenitor commitment and differentiation steps and that ERK activation plays a role in this process.     

A Role for Bone Morphogenetic Protein-4 in Adipocyte Development

Obesity is characterized by increases in the number of mature adipocytes. Nascent adipocytes arise from mesenchymal stem cells (MSCs) by a multi-step process — MSCs are recruited to the adipocyte lineage forming determined preadipocytes, these committed progenitors proliferate, undergo growth arrest, and finally differentiate into mature adipocytes. Although the genetic mechanisms that control the differentiation of preadipocytes into mature adipocytes are understood to a large extent, the earliest events in adipogenesis — especially the commitment of MSCs into preadipocytes — are largely unknown. Recently, bone morphogenetic protein-4 (BMP-4) has been implicated in the commitment of pluripotent MSCs to the adipocyte lineage by two independent lines of investigation. First, growth-arrested 10T1/2 cells do not normally respond to a hormonal cocktail that causes various growth-arrested preadipocyte cell lines to differentiate into adipocytes, but if 10T1/2 cells are first treated with BMP-4 they will respond to these hormonal inducers by undergoing terminal adipocyte differentiation. Second, a preadipocyte cell line, A33 cells, derived from 10T1/2 cells after exposing the cells to the DNA methyltransferase inhibitor 5-azacytidine was shown to express BMP-4, and this endogenous BMP-4 expression is required for acquisition of the preadipocyte phenotype of these cells. A role for the BMP-4 signaling pathway in adipogenesis is discussed.     

Genome-wide survey identifies TNNI2 as a target of KLF7 that inhibits chicken adipogenesis via downregulating FABP4

Krüppel-like factor 7 (KLF7) negatively regulates adipocyte differentiation; however, the mechanism underlying its activity in mammals and birds remains poorly understood. To identify genome-wide KLF7-binding motifs in preadipocytes, we conducted a chromatin immunoprecipitation-sequencing analysis of immortalized chicken preadipocytes (ICP2), which revealed 11,063 specific binding sites. Intergenic binding site analysis showed that KLF7 regulates several novel factors whose functions in chicken and mammal adipogenesis are underexplored. We identified a novel regulator, troponin I2 (TNNI2), which is positively regulated by KLF7. TNNI2 is downregulated during preadipocyte differentiation and acts as an adipogenic repressor at least in part by repressing FABP4 promoter activity. In conclusion, we demonstrated that KLF7 functions through cis-regulation of TNNI2, which inhibits adipogenesis. Our findings not only provide the first genome-wide picture of KLF7 associations in preadipocytes but also identify a novel function of TNNI2.     

An Evi1-C/EBPβ complex controls peroxisome proliferator-activated receptor γ2 gene expression to initiate white fat cell differentiation

Fibroblastic preadipocyte cells are recruited to differentiate into new adipocytes during the formation and hyperplastic growth of white adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ), the master regulator of adipogenesis, is expressed at low levels in preadipocytes, and its levels increase dramatically and rapidly during the differentiation process. However, the mechanisms controlling the dynamic and selective expression of PPARγ in the adipocyte lineage remain largely unknown. We show here that the zinc finger protein Evi1 increases in preadipocytes at the onset of differentiation prior to increases in PPARγ levels. Evi1 expression converts nonadipogenic cells into adipocytes via an increase in the predifferentiation levels of PPARγ2, the adipose-selective isoform of PPARγ. Conversely, loss of Evi1 in preadipocytes blocks the induction of PPARγ2 and suppresses adipocyte differentiation. Evi1 binds with C/EBPβ to regulatory sites in the Pparγ locus at early stages of adipocyte differentiation, coincident with the induction of Pparγ2 expression. These results indicate that Evi1 is a key regulator of adipogenic competency.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.     

Beta-Mecaptoethanol Suppresses Inflammation and Induces Adipogenic Differentiation in 3T3-F442A Murine Preadipocytes

Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is "metabolically healthy". Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.     

Knockdown of KLF9 promotes the differentiation of both intramuscular and subcutaneous preadipocytes in goat

ABSTRACT

KLF9 is reported to promote adipocyte differentiation in 3T3-L1 cells and pigs. However, the roles of KLF9 in adipocytes differentiation of goat remain unknown. In this study, the expression profiles of KLF9 were different between subcutaneous and intramuscular preadipocytes of goat during differentiation process. After silencing KLF9 gene, the lipid droplets were increased in both two types of adipocytes. In subcutaneous preadipocyte with silencing KLF9, the expressions of C/EBPβ, PPARγ, LPL, KLF1-2, KLF5, and KLF17 genes were up-regulated, while KLF12, KLF4, and KLF13 genes were down-regulated in expression level. In intramuscular preadipocyte, aP2, C/EBPα, KLF2-3, KLF5, and KLF7 gene were up-regulated, and Pref-1 gene was down-regulated. In addition, the binding sites of KLF9 existed in the promoters of aP2, C/EBPα, C/EBPβ, LPL and Pref-1. Taken together, KLF9 play a negative role in the differentiation of both intramuscular and subcutaneous preadipocytes in goats, but the functional mechanism may be different.     

Insulin-like growth factor-I is an essential regulator of the differentiation of 3T3-L1 adipocytes

Murine 3T3-L1 preadipocytes proliferate normally in medium containing fetal calf serum depleted of insulin, growth hormone, and insulin-like growth factor-I (IGF-I). However, the cells do not differentiate into adipocytes in the presence of the hormone-depleted serum. Supplementation of the growth medium with 10-20 nM IGF-I or 2 microM insulin restores the ability of 3T3-L1 cells to develop into adipocytes. The cells acquire an adipocyte morphology, accumulate triglycerides, and express a 450-fold increase in the activity of the lipogenic enzyme glycerol-3-phosphate dehydrogenase. The increase in glycerol-3-phosphate dehydrogenase activity is paralleled by the accumulation of glycerol-3-phosphate dehydrogenase mRNA and mRNA for the myelin P2-like protein aP2, another marker for fat cell development. IGF-I or insulin-stimulated adipogenesis in 3T3-L1 cells is not dependent on growth hormone. Occupancy of preadipocyte IGF-I receptors by IGF-I (or insulin) is implicated as a central step in the differentiation process. The IGF-I receptor binds insulin with a 70-fold lower affinity than IGF-I, and 30-70-fold higher levels of insulin are required to duplicate the effects of an optimal amount of IGF-I. The effects of 10-20 nM IGF-I are likely to be mediated by high affinity (KD = 5 nM) IGF-I receptors that are expressed at a density of 13,000 sites/preadipocyte. In undifferentiated cells the IGF-I receptor concentration is twice that of the insulin receptor. After adipocyte differentiation is triggered, the number and affinity of IGF-I receptors remain constant while insulin receptor number increases approximately 25-fold as developing adipocytes become responsive to insulin at the level of metabolic regulation. Thus, preadipocytes have the potential for a maximal response to IGF-I, whereas the accumulation of more than 95% of adipocyte insulin receptors and the appearance of responsiveness to insulin are consequences of differentiation. IGF-I or insulin is essential for the induction of a variety of abundant and nonabundant mRNAs characteristic of 3T3-L1 adipocytes.     

Carbonic anhydrase III regulates peroxisome proliferator-activated receptor-γ2

Carbonic anhydrase III (CAIII) is an isoenzyme of the CA family. Because of its low specific anhydrase activity, physiological functions in addition to hydrating CO(2) have been proposed. CAIII expression is highly induced in adipogenesis and CAIII is the most abundant protein in adipose tissues. The function of CAIII in both preadipocytes and adipocytes is however unknown. In the present study we demonstrate that adipogenesis is greatly increased in mouse embryonic fibroblasts (MEFs) from CAIII knockout (KO) mice, as demonstrated by a greater than 10-fold increase in the induction of fatty acid-binding protein-4 (FABP4) and increased triglyceride formation in CAIII(-/-) MEFs compared with CAIII(+/+) cells. To address the underlying mechanism, we investigated the expression of the two adipogenic key regulators, peroxisome proliferator-activated receptor-γ2 (PPARγ2) and CCAAT/enhancer binding protein-α. We found a considerable (approximately 1000-fold) increase in the PPARγ2 expression in the CAIII(-/-) MEFs. Furthermore, RNAi-mediated knockdown of endogenous CAIII in NIH 3T3-L1 preadipocytes resulted in a significant increase in the induction of PPARγ2 and FABP4. When both CAIII and PPARγ2 were knocked down, FABP4 was not induced. We conclude that down-regulation of CAIII in preadipocytes enhances adipogenesis and that CAIII is a regulator of adipogenic differentiation which acts at the level of PPARγ2 gene expression.     

The adipokine Chemerin induces lipolysis and adipogenesis in bovine intramuscular adipocytes

The adipokine Chemerin is reported to regulate adipogenesis and glucose homeostasis in vivo and in 3T3-L1 cells. Our team is focused on the role of Chemerin in metabolism and intramuscular adipocyte differentiation because intramuscular fat is the basic material for the formation of marbling in livestock and poultry meat. In this study, bovine intramuscular mature adipocytes were cultured in medium with Chemerin, and the process of lipolysis of mature adipocytes and the adipogenesis of de-differentiated preadipocytes were investigated. The results showed that Chemerin induced significant lipolytic metabolism in intramuscular mature adipocytes, indicated by increased levels of glycerol, FFA, and up-regulated expression of the lipolysis critical factors HSL, LPL, and leptin. Meanwhile, the expressions of adipogenic key factors PPARγ, C/EBPα, and A-FABP were decreased by Chemerin during lipolysis or dedifferentiation in mature adipocytes. The de-differentiated preadipocytes could re-differentiate into mature adipocytes. Intriguingly, the formation of cells’ lipid droplets was promoted by Chemerin during preadipocyte differentiation. In addition, mRNA and protein expressions of PPARγ, C/EBPα, and A-FABP were up-regulated by Chemerin during preadipocytes differentiation. These results suggest that Chemerin promotes lipolysis in mature adipocytes and induces adipogenesis during preadipocyte re-differentiation, further indicating a dual role for Chemerin in the deposition of intramuscular fat in ruminant animals.

     

Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein beta gene during adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.     

2,2′,4,4′,5‐Pentabromodiphenyl ether induces lipid accumulation throughout differentiation in 3T3‐L1 and human preadipocytes in vitro

Flame retardants, specifically polybrominated diphenyl ethers (PBDEs), are chemical compounds widely used for industrial purposes and household materials. NHANES data indicate that nearly all Americans have trace amounts of PBDEs in serum, with even higher levels associated with occupational exposure. PBDEs are known to bioaccumulate in the environment due to their lipophilicity and stability, and more importantly, they have been detected in human adipose tissue. The present study examined whether the PBDE congener, BDE‐99 (2,2′,4,4′,5‐pentabromodiphenyl ether; 0.2‐20 μM), enhances the adipogenesis of mouse and human preadipocyte cell models in vitro via induced lipid accumulation. 3T3‐L1 mouse preadipocytes and human visceral preadipocytes demonstrated enhanced hormone‐induced lipid accumulation upon BDE‐99 treatment. In addition, BDE‐99 (20 μM) induced preadipocyte differentiation and lipid development in nondifferentiated human preadipocytes. BDE‐99, the second most abundant congener in human adipose tissue, increased total lipids in differentiating adipocytes and therefore showed a potential role in the regulation of adipogenesis. This warrants more research to further understand the impact of lipophilic persistent pollutants on adipose tissue homeostasis.     

An Adenosine Receptor-Krüppel-like Factor 4 Protein Axis Inhibits Adipogenesis

Adipogenesis represents a key process in adipose tissue development and remodeling, including during obesity. Exploring the regulation of adipogenesis by extracellular ligands is fundamental to our understanding of this process. Adenosine, an extracellular nucleoside signaling molecule found in adipose tissue depots, acts on adenosine receptors. Here we report that, among these receptors, the A2b adenosine receptor (A2bAR) is highly expressed in adipocyte progenitors. Activation of the A2bAR potently inhibits differentiation of mouse stromal vascular cells into adipocytes, whereas A2bAR knockdown stimulates adipogenesis. The A2bAR inhibits differentiation through a novel signaling cascade involving sustained expression of Krüppel-like factor 4 (KLF4), a regulator of stem cell maintenance. Knockdown of KLF4 ablates the ability of the A2bAR to inhibit differentiation. A2bAR activation also inhibits adipogenesis in a human primary preadipocyte culture system. We analyzed the A2bAR-KLF4 axis in adipose tissue of obese subjects and, intriguingly, found a strong correlation between A2bAR and KLF4 expression in both subcutaneous and visceral human fat. Hence, our study implicates the A2bAR as a regulator of adipocyte differentiation and the A2bAR-KLF4 axis as a potentially significant modulator of adipose biology.     

用成熟脂肪建立一种新的猪前体脂肪细胞培养模型

用去分化的成熟脂肪细胞建立一种新的具有再增殖和再分化能力的猪前体脂肪细胞模型. 用“天花板” 培养法分离、培养1～3日龄仔猪皮下成熟脂肪细胞, 显微镜下观察细胞形态变化并计数, 流式细胞术检测细胞周期;油红O染色法检测脂肪细胞分化率, RT-PCR分析前体脂肪细胞标志基因Pref-1及成熟脂肪细胞关键转录因子PPARγ和C/EBPα等mRNA表达情况. 发现刚贴壁的细胞为单室脂滴成熟脂肪细胞, 油红O染色完全阳性; 14d后这种成熟脂肪细胞完全去分化为无脂滴的纤维状细胞, 并表达前体脂肪细胞标志基因Pref-1, 油红O染色阴性. 这种去分化的前体脂肪细胞在成脂诱导剂作用下,可重新分化为成熟的脂肪细胞. 结果证实,成熟脂肪细胞去分化后的前体脂肪细胞可重新增殖、分化为成熟脂肪细胞, 是一种新的有效的前体脂肪细胞模型.     

miR-128-3p regulates 3T3-L1 adipogenesis and lipolysis by targeting <Emphasis Type="Italic">Pparg</Emphasis> and <Emphasis Type="Italic">Sertad2</Emphasis>

Differentiation of adipocytes and their aggregation to adipose tissue are critical for mammalian growth and development. MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs that play important roles in adipogenesis and lipid metabolism. miR-128-3p may contribute to adipose tissue development according to the previous studies. However, the role of miR-128-3p in the process of preadipocyte differentiation and lipid metabolism is not yet understood. The purpose of this research was to investigate the biological function and molecular mechanism of miR-128-3p in 3T3-L1 cells. In the present study, we found that miR-128-3p was downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-128-3p obstructed the expressions of adipogenic marker genes as well as the lipid droplets accumulation and triglyceride content, suggesting the importance of miR-128-3p for adipogenesis. Moreover, miR-128-3p could lead to the retardation of cell proliferation in 3T3-L1 preadipocytes. Further evidences showed that, as a negative regulator of adipogenesis, miR-128-3p could directly target peroxisome proliferator-activated receptor γ (Pparg) which resulted in the suppression of 3T3-L1 preadipocyte differentiation, and miR-128-3p could also bind with SERTA domain containing 2 (Sertad2) which drove triglyceride hydrolysis and lipolysis. In addition, inhibition of Sertad2 with siRNA displayed the same effects as overexpression of miR-128-3p. Our research demonstrated that miR-128-3p impeded 3T3-L1 adipogenesis by targeting Pparg and Sertad2, resulting in the obstruction of preadipocyte differentiation and promotion of lipolysis. Taken together, this study offers profound insight into the mechanism of miRNA-mediated adipogenesis and lipid metabolism.