Inhibition of gap-junctional intercellular communication and enhanced binding of fibronectin-coated latex beads by stimulation of DNA synthesis in quiescent 3T3-L1 cells

To clarify the modulation of intercellular communication via gap junctions, associated with the growth induction of quiescent 3T3-L1 cells, we investigated the gap-junctional intercellular communication in growth-stimulated cells that were able to bind fibronectin-coated beads. When quiescent 3T3-L1 cells were incubated with fibronectin-coated beads for the first 2 h after the addition of calf serum, 24.0% of the cells bound and phagocytosed beads. Among the cells with bound beads, the percentage of the cells labeled concurrently with bromodeoxyuridine was 63.7% when examined 13 h after the addition of calf serum. Transient reduction of dye-coupling, measured with Lucifer Yellow CH, was observed only in the cells with bound beads 2 h after addition of calf serum, but it was not observed in the cells without bound beads. When the quiescent cells were incubated with fibronectin-coated beads for 2 h from 4-6 h after the addition of calf serum, the percentage of cells with bound beads increased to 53.1%, but the decrease in dye-coupling among the cells with bound beads was slight. These results suggest that the induction of cell growth causes a transient reduction in gap-junctional intercellular communication in 3T3-L1 cells with bound fibronectin-coated beads.     

12-O-tetradecanoylphorbol-13-acetate disrupts actin filaments and focal contacts and enhances binding of fibronectin-coated latex beads to 3T3-L1 cells

The effect of a tumor-promoting phorbol ester on the binding of fibronectin-coated beads to 3T3-L1 cells was studied to clarify the relationship between the binding of fibronectin to the cells, cell adhesion, and the organization of actin filaments. Interference-reflection microscopy revealed focal contacts of 3T3-L1 cells with the substratum. Stress fibers observed after rhodamine-phalloidin staining were well-developed in the cells. Treatment of the cells for 20 min with 12-O-tetradecanoylphorbol-13-acetate (TPA), but not with phorbol, disrupted focal contacts and caused a reorganization of stress fibers to generate actin ribbons. Treatment of the cells with TPA enhanced the binding of beads coated with human plasma fibronectin to the cells, as observed after incubation for 6 h with the beads. The TPA-induced increase in the percentage of cells with bound beads was dependent on the duration of treatment with TPA and on the concentration of TPA. Treatment of the cells with TPA also enhanced proliferation of cells in a dose-dependent manner. The enhancement of binding of the beads by TPA was suppressed by addition of an adhesion-inhibitory peptide (Gly-Arg-Gly-Asp-Ser-Pro). Treatment with TPA did not enhance nonspecific binding of beads coated with heat-denatured bovine serum albumin. Furthermore, treatment of the cells with phorbol did not enhance the binding of beads coated with fibronectin. These results suggest that TPA specifically enhances the binding of fibronectin-coated beads to 3T3-L1 cells, and that TPA-induced binding of the beads may be related to disruption of focal contacts and reorganization of actin filaments.     

dlk1 specifically interacts with insulin-like growth factor binding protein 1 to modulate adipogenesis of 3T3-L1 cells

dlk1 is an epidermal growth factor (EGF)-like homeotic protein containing an intracellular region, a single transmembrane domain, and an extracellular region possessing six EGF-like repeats and a protease-target sequence. dlk1 functions as a modulator of adipogenesis, and other differentiation processes. The molecular mechanisms by which dlk1 regulates these processes are unclear. It has been reported that different Dlk1 mRNA spliced variants, encoding for isoforms possessing the protease-target sequence or not, determine the production of membrane-associated or soluble, secreted extracellular dlk1 proteins that appear to affect adipogenesis of 3T3-L1 cells differently. In particular, only soluble variants inhibit this process. Some recent evidence suggest that dlk1 may modulate extracellular stimuli inducing differentiation. Thus, an enforced decrease of Dlk1 expression in BALB/c 3T3 cells, which results in an increase of their adipogenic potential in response to insulin-like growth factor 1 (IGF-1), modifies the kinetics and levels of activation of ERK1/2 triggered by it. In this work, we identified a strong and specific interaction between the protease-target dlk1 region and the non-IGF binding region of IGF binding protein 1 (IGFBP1), a protein that binds to IGFs and modulates their action. We also observed that the increased adipogenic potential of 3T3-L1 cells caused by diminishing Dlk1 expression through transfection with an antisense Dlk1 expression construct was inhibited by the presence of IGFBP1 in the differentiation medium. On the other hand, the presence of IGFBP1 in the culture medium slightly increased the adipogenic potential of control 3T3-L1 cells, expressing regular levels of Dlk1. These data suggest that membrane dlk1 variants bind to extracellular IGFBP1/IGF-1 complexes, which may favor the release of IGF-1 and increase the local concentration of free IGF-1 that can enhance IGF receptor signaling, leading to adipogenesis.     

Polyamine metabolism is involved in adipogenesis of 3T3-L1 cells

Polyamines spermidine and spermine are known to be required for mammalian cell proliferation and for embryonic development. Alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC) a limiting enzyme of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation in 3T3-L1 cells. In this study, to explore the function of polyamines in adipogenesis, we examined the effect of polyamine biosynthesis inhibitors on adipocyte differentiation and lipid accumulation of 3T3-L1 cells. The spermidine synthase inhibitor trans-4-methylcyclohexylamine (MCHA) increased spermine/spermidine ratios, whereas the spermine synthase inhibitor N-(3-aminopropyl)-cyclohexylamine (APCHA) decreased the ratios in the cells. MCHA was found to decrease lipid accumulation and GPDH activity during differentiation, while APCHA increased lipid accumulation and GPDH activity indicating the enhancement of differentiation. The polyamine-acetylating enzyme, spermidine/spermine N ¹-acetyltransferase (SSAT) activity was increased within a few hours after stimulus for differentiation, and was found to be elevated by APCHA. In mature adipocytes APCHA decreased lipid accumulation while MCHA had the opposite effect. An acetylpolyamine oxidase and spermine oxidase inhibitor MDL72527 or an antioxidant N-acetylcysteine prevented the promoting effect of APCHA on adipogenesis. These results suggest that not only spermine/spermidine ratios but also polyamine catabolic enzyme activity may contribute to adipogenesis.     

Adenovirus transduction of 3T3-L1 cells

3T3-L1 cells offer an excellent model system for studies of differentiation and biochemistry of fat cells. However, these cells are limited in their utility by the low efficiency with which DNA can be introduced by transfection. Gene delivery by viral vectors, particularly adenovirus, has proven a powerful means for introduction of genes into certain cell types. Furthermore, adenovirus transduction has been used to study mechanisms involved in the differentiation of 3T3-L1 cells into mature fat cells. We show in this study that 3T3-L1 cells are inefficiently transduced by adenovirus. The potential advantages offered by adenovirus transduction led us to examine methods designed to enhance transduction of 3T3-L1 cells by adenovirus. Of these methods, polylysine-mediated enhancement demonstrates considerable promise because it permits up to 100% of cells to be transduced and because it does not inhibit differentiation of 3T3-L1 cells. -- Orlicky D. J., and J. Schaack. Adenovirus transduction of 3T3-L1 cells. J. Lipid Res. 2001. 42: 460--466.     

小鼠3T3-L1前脂肪细胞系的增强绿色荧光蛋白标记

细胞模型是研究细胞分化原理以及进行高通量筛选的有效工具。为了建立特异性标记的脂肪细胞分化模型,构建了包括脂肪细胞分化特异性表达基因PPARγ2的启动子在内的载体（pPPARγ2-promoter-EGFP）,用电穿孔方法转染小鼠3T3L1 前脂肪细胞,用显微荧光观察和RT-PCR确认PPARγ2基因的内源表达。结果显示,EGFP基因成功转入3T3-L1前脂肪细胞,观察到细胞分化过程中EGFP表达和脂肪积累,RTPCR分析表明EGFP代表了稳定而真实的PPARγ2基因的内源性表达。建立了由脂肪组织特异表达基因PPARγ2的表达控制的EGFP标记的小鼠3T3-L1前脂肪细胞系,目前国内外尚未见用同样方法对前脂肪细胞进行特异性标记。该细胞系将为脂肪细胞分化机理研究以及为抗肥胖症和抗糖尿病药物筛选提供有力工具。     

Protocol for effective differentiation of 3T3-L1 cells to adipocytes

In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 μM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.     

Visfatin is released from 3T3-L1 adipocytes via a non-classical pathway

Visfatin is a secretory protein which exerts insulin mimetic and proinflammatory effects, also functioning as an intracellular enzyme to produce NAD. Plasma visfatin levels and visfatin mRNA expression in adipose tissues are increased in obese subjects. Visfatin does not have a decent cleavable signal sequence, and the mechanism, that mediates release of visfatin from adipocytes, remains poorly understood. In this study, we demonstrate that visfatin is released abundantly into culture medium from 3T3-L1 adipocytes. Subcellular fractionation analysis showed that visfatin was localized in the cytosol, but not in nucleus, membrane, vesicles, or mitochondria fractions. Visfatin release was not reduced by Brefeldin A and Monensin, inhibitors of endoplasmic reticulum (ER)-Golgi-dependent secretion. In addition, visfatin was not released on microvesicles. These results suggest that visfatin should be released from 3T3-L1 adipocytes via an ER-Golgi or microvesicles independent pathway.     

Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3–4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.     

Interferon-alpha induction of STATs1, -3 DNA binding and growth arrest is independent of Lck and active mitogen-activated kinase in T cells

Type I interferons (IFNs) are a family of cytokines that have antiviral and antiproliferative effects. Data regarding the processes by which these cytokines transduce signals from the cell membrane to the nucleus are becoming increasingly complex. The most characterized pathway is via JAK-STAT signaling. Previous studies established a potential role for the Src-family kinase Lck in JAK-STAT signaling. Therefore, this study was designed to analyze the role of Lck in IFN-alpha signaling by using the Jurkat, JCam (an Lck-defective cell line derived from Jurkat), and JCam/Lck (JCam cells with Lck restored). The results show that IFN-alpha can induce MAPK activity, but only in cells containing Lck. Furthermore, STATs1 and -3 are effectively phosphorylated and activated to bind DNA in the absence of Lck expression in IFN-alpha-treated cells. Finally, the results demonstrate that IFN-alpha exerts an antiproliferative effect in all three cell lines. These data indicate that Lck and active MAPK do not affect IFN-alpha-induced growth arrest or induction of STAT1s1 and -3 DNA binding ability.     

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.     

Anti-adipogenic chromone glycosides from Cnidium monnieri fruits in 3T3-L1 cells

Seven new chromone glycosides, monnierisides A (3), B (10), C (11), D (12), E (13), F (15) and G (16) were isolated from Cnidium. monnieri, together with ten known chromone derivatives, undulatoside A (1), cnidimol C (2), saikochromoside A (4), cnidimoside A (5), cnidimoside B (6), 2-methyl-5-hydroxy-6-(2-butenyl-3-hydroxymethyl)-7-(β-d-glucopyranosyloxy)-4H-1-benzopyran-4-one (7), cnidimol D (8), hydroxycnidimoside A (9), umtatin (14) and 6'-hydroxylangelicain (17). The structures of isolated compounds were determined on the basis of spectroscopic analysis including 1D, 2D NMR and HR-MS. Among the compounds isolated, compounds 5, 6, 9 and 10 significantly inhibited adipocyte differentiation as measured by fat accumulation in 3T3-L1 cells using Oil Red O staining.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

3T3-L1 adipocytes promote the growth of mammary epithelium

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material ('extracellular matrix') derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.     

Effects of amide constituents from pepper on adipogenesis in 3T3-L1 cells

Several amide constituents (piperlonguminine and retrofractamides A, B, and C) from the fruit of Piper chaba promoted adipogenesis of 3T3-L1 cells. Among them, retrofractamide A was the most active and significantly increased the amount of adiponectin released into the medium and the uptake of 2-deoxyglucose into the cells. Retrofractamide A also increased mRNA levels of adiponectin, peroxisome proliferator-activated receptor gamma2 (PPARgamma2), glucose transporter 4 (GLUT4), and insulin receptor substrate 1 (IRS-1), but did not act as a PPARgamma agonist different from troglitazone.