α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes

α-Poly-l-lysine (PLL) has been used for various purposes such as cell attachment, immunization, and molecular delivery, and is known to be cytotoxic to several cell lines. Here, we studied the effect of PLL on the adipogenesis of 3T3-L1 cells and investigated the underlying mechanism. Differentiation media containing PLL with a molecular weight (MW) greater than 4 kDa enhanced lipid droplet formation and increased adipogenic marker levels, indicating an increase in adipocyte differentiation. PLL with a molecular weight between 30 and 70 kDa was more effective than PLL of other sizes in 3T3-L1 cell differentiation. Moreover, PLL induced 3T3-L1 adipogenesis in insulin-free adipocyte differentiation medium. Incubation with insulin and PLL exhibited greater adipogenesis than insulin treatment only even at a high concentration. PLL stimulated insulin signaling and augmented the signaling pathway when it was added with insulin. While PLL did not activate the glucocorticoid receptor, which is phosphorylated by dexamethasone (DEX), it showed a positive effect on the cAMP signal pathway when preadipocytes were treated with PLL and 3-isobutyl-1-methylxanthine (IBMX). Consistent with these results, incubation with PLL and DEX without IBMX induced adipocyte differentiation. We also observed that the mitotic clonal expansion phase was the critical stage in adipogenesis for inducing the effects of PLL. These results suggest that PLL functions as an adipogenic inducer in 3T3-L1 preadipocytes and PLL has a direct effect on insulin signaling, one of the main regulatory pathways.

     

Environmental Endocrine Disruptors Promote Adipogenesis in the 3T3‐L1 Cell Line through Glucocorticoid Receptor Activation

The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3‐L1 cell line. Various EDCs were screened for glucocorticoid‐like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator‐activated receptor‐γ. 3T3‐L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte‐specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes.     

促酰化蛋白(ASP)诱导3T3-L1前脂肪细胞分化

促酰化蛋白 (ASP)代替经典激素“鸡尾酒”诱导法中胰岛素 ,通过形态学观察、油红染色分化百分比测定、脂肪细胞甘油三酯合成率和甘油三酯总量测定 ,并与经典激素“鸡尾酒”法诱导前脂肪细胞分化情况比较 ,探讨ASP是否具有诱导 3T3 L1前脂肪细胞分化作用 .ASP组诱导分化第 6d ,3T3 L1前脂肪细胞变大、变圆 ,出现大量脂肪滴 ,形态由前脂肪细胞向成熟脂肪细胞转变 ;随着诱导分化时间延长 ,胞浆中脂滴进一步积累 .分化 9d时 ,3T3 L1前脂肪细胞分化完全 .油红染色结果显示 ,ASP组分化率很高 (85 % ) ,与胰岛素组分化率 (90 % )相似 ,明显高于IBMX +DEX组 (4 0 % ) .ASP不仅促进 3T3 L1前脂肪细胞形态向成熟脂肪细胞转化 ,同时促进细胞中甘油三酯的合成和积累 .ASP组诱导分化第 3d时 ,脂肪细胞甘油三酯合成率明显高于对照组和IBMX +DEX组 ,但仍低于胰岛素组 ;在分化第 6d和第 9d时 ,ASP组甘油三酯合成率进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,与胰岛素组相比无显著性差异 .ASP组诱导分化 3d时 ,脂肪细胞中甘油三酯总量明显高于对照组和IBMX +DEX组 ;分化 6d和 9d时 ,甘油三酯总量进一步升高 ,与对照组和IBMX +DEX组相比差异有极显著性 ,而与胰岛素组相比无显著性差异 .结果表明 ,新型脂源性激     

Induction of Adipocyte Differentiation by Polybrominated Diphenyl Ethers (PBDEs) in 3T3-L1 Cells

Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants that were extensively used in commercial products. PBDEs are ubiquitous environmental contaminants that are both lipophilic and bioaccumulative. Effects of PBDEs on adipogenesis were studied in the 3T3-L1 preadipocyte cell model in the presence and absence of a known adipogenic agent, dexamethasone (DEX). A PBDE mixture designed to mimic body burden of North Americans was tested, in addition to the technical mixture DE-71 and the individual congener BDE-47. The mixture, DE-71, and BDE-47 all induced adipocyte differentiation as assessed by markers for terminal differentiation [fatty acid binding protein 4 (aP2) and perilipin] and lipid accumulation. Characterization of the differentiation process in response to PBDEs indicated that adipogenesis induced by a minimally effective dose of DEX was enhanced by these PBDEs. Moreover, C/EBPα, PPARγ, and LXRα were induced late in the differentiation process. Taken together, these data indicate that adipocyte differentiation is induced by PBDEs; they act in the absence of glucocorticoid and enhance glucocorticoid-mediated adipogenesis.     

Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways

Because leptin has recently been shown to induce proliferation and/or differentiation of different cell types through different pathways, the aim of the present study was to investigate, in vitro, the influence of leptin on adipogenesis in rat preadipocytes. A prerequisite to this study was to identify leptin receptors (Ob-Ra and Ob-Rb) in preadipocytes from femoral subcutaneous fat. We observed that expressions of Ob-Ra and Ob-Rb increase during adipogenesis. Furthermore, leptin induces an increase of p42/p44 mitogen-activated protein kinase phosphorylated isoforms in both confluent and differentiated preadipocytes and of STAT3 phosphorylation only in confluent preadipocytes. Moreover, exposure to leptin promoted activator protein-1 complex DNA binding activity in confluent preadipocytes. Finally, exposure of primary cultured preadipocytes from the subcutaneous area to leptin (10 nM) resulted in an increased proliferation ([(3)H]thymidine incorporation and cell counting) and differentiation (glycerol-3-phosphate dehydrogenase activity and mRNA levels of lipoprotein lipase, peroxisome proliferator-activated receptor-gamma2, and c-fos). Altogether, these results indicate that, in vitro at least, leptin through its functional receptors exerts a proadipogenic action in subcutaneous preadipocytes.     

Activation of MEK/ERK signaling promotes adipogenesis by enhancing peroxisome proliferator-activated receptor gamma (PPARgamma ) and C/EBPalpha gene expression during the differentiation of 3T3-L1 preadipocytes

We demonstrate that exposure of post-confluent 3T3-L1 preadipocytes to insulin, isobutylmethylxanthine (MIX), dexamethasone (DEX), and fetal bovine serum induces a rapid but transient activation of MEK1 as indicated by extensive phosphorylation of ERK1 and ERK2 during the initial 2 h of adipogenesis. Inhibition of this activity by treating the cells with a MEK1-specific inhibitor (U0126 or PD98059) prior to the induction of differentiation significantly attenuated the expression of peroxisome proliferator-activated receptor (PPAR) gamma, CCAAT/enhancer-binding protein (C/EBP) alpha, perilipin, and adipocyte-specific fatty acid-binding protein (aP2). Treating the preadipocytes with troglitazone, a potent PPARgamma ligand, could circumvent the inhibition of adipogenic gene expression by U0126. Fibroblast growth factor-2 (FGF-2), in the presence of dexamethasone, isobutylmethylxanthine, and insulin, induces a prolonged activation of the MEK/ERK signaling pathway, which lasts for at least 12 h post-induction, and this activity is less sensitive to the MEK inhibitors. Consequently, preadipocytes treated with U0126 in the presence of fibroblast growth factor-2 (FGF-2) express normal post-induction levels of MEK activity, and, in so doing, are capable of undergoing adipogenesis. We further show that activation of MEK1 significantly enhances the transactivation of the C/EBPalpha minimal promoter during the early phase of the differentiation process. Our results suggest that activation of the MEK/ERK signaling pathway during the initial 12 h of adipogenesis enhances the activity of factors that regulate both C/EBPalpha and PPARgamma expression.     

Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.