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.     

Constitutively active mitogen-activated protein kinase kinase 6 (MKK6) or salicylate induces spontaneous 3T3-L1 adipogenesis

Although much has been learned regarding the importance of p38 mitogen-activated protein kinase in inflammatory and stress responses, relatively little is known concerning its role in differentiation processes. Recently, we demonstrated that p38 mitogen-activated protein kinase activity is necessary for the differentiation of 3T3-L1 fibroblasts into adipocytes (Engelman, J. A., Lisanti, M. P., and Scherer, P. E. (1998) J. Biol. Chem. 273, 32111-32120). p38 activity is high during the initial stages of differentiation but decreases drastically as the fibroblasts undergo terminal differentiation into adipocytes. However, it remains unknown whether activation of p38 is sufficient to stimulate adipogenesis and whether the down-regulation of p38 activity in mature adipocytes is critical for maintaining adipocyte homeostasis. In this report, we have directly addressed these questions by analyzing 3T3-L1 cell lines harboring a specific upstream activator of p38 (a constitutively active mitogen-activated protein kinase kinase 6 (MKK6) mutant, MKK6(Glu)) under the control of an inducible promoter. Induction of MKK6(Glu) in 3T3-L1 fibroblasts spurs adipocyte conversion in the absence of the hormonal mixture normally required for efficient differentiation of wild-type cells. However, activation of p38 in adipocytes leads to cell death. Furthermore, treatment of 3T3-L1 fibroblasts with salicylate, a potent stimulator of p38, produces adipocyte-specific changes consistent with those observed with induction of MKK6(Glu). Expression of MKK6(Glu) in NIH-3T3 fibroblasts (cells that do not differentiate into adipocytes under normal conditions) is capable of converting these fibroblasts into lipid-laden fat cells following hormonal stimulation. Thus, p38 activation has pro-adipogenic effects in multiple fibroblast cell lines.     

Rheb过表达在前体脂肪细胞株3T3-L1分化中的作用

目的:利用前体脂肪细胞株3T3-L1细胞观察mTOR(mammalian target of rapamycin)信号通路中上游调控因子Rheb(Ras homolog enriched in brain)对其分化的影响。方法:利用高表达Rheb的基因重组质粒转染前体脂肪细胞株,3T3-L1。通过蛋白质免疫印迹实验鉴定质粒成功转染细胞后,诱导该细胞脂肪分化。予以分化第8天的3T3-L1细胞油红染色,并检测细胞内甘油三酯的含量。另外,我们用Western blot方法检测脂肪细胞特异性转录因子PPAR-γ(Peroxisome proliferator-activated receptor-γ)和C/EBP-α(CCAAT-enhancer-binding protein-α)的表达情况来研究Rheb在脂肪细胞分化过程中的作用。结果:我们成功构建了高表达Rheb的3T3-L1细胞株,发现高表达Rheb后可以促进脂滴的生成,油红O染色有显著区别,与对照组相比Rheb高表达组的三酰甘油含量明显升高(P0.05);C/EBP-α和PPAR-γ等脂肪细胞特异性的转录因子蛋白表达量与对照组相比也均有升高(P0.05)。结论:Rheb基因作为mTOR通路上游调控因子,可以促进脂肪细胞的分化。     

Growth hormone has dual stage-specific effects on the differentiation of 3T3-L1 preadipocytes

Reports vary on the role of growth hormone (GH) in adipocyte differentiation. In this study, we showed that GH exerted dual effects depending on the stage of differentiation, using a serum-free culture of 3T3-L1 preadipocytes. GH promoted the differentiation when added to the medium during differentiation-inducing treatment with a hormone cocktail, but apparently suppressed it when added after the treatment. Only the suppressive effect was observed in the presence of 10% fetal bovine serum (FBS). Immunodepletion study showed that GH contributes to the differentiation-promoting activity of FBS. Insulin-like growth factor-1 could not replicate either the stimulative or the suppressive effect of GH. Stimulation of differentiation by GH involved the enhanced expression of mRNA of middle to late adipocyte markers. Among the key regulators of adipogenesis, peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha, but not C/EBPbeta, were stimulated for mRNA expression by GH added during the treatment with hormone cocktail. The stimulation of adipogenesis by GH was indeed due to the increase in the ratio of differentiated cells, though GH also promoted cell growth.     

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.

     

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.     

Neudesin, an extracellular heme-binding protein, suppresses adipogenesis in 3T3-L1 cells via the MAPK cascade

Adult mice abundantly express neudesin, an extracellular heme-binding protein with neurotrophic activity, in white adipose tissues. At the early stage of adipocyte differentiation during adipogenesis, however, the expression of neudesin decreased transiently. Neudesin-hemin significantly suppressed adipogenesis in 3T3-L1 cells. The knockdown of neudesin by RNA interference markedly promoted adipogenesis in 3T3-L1 cells and decreased MAPK activation during adipocyte differentiation. The addition or knockdown of neudesin affected the expression of C/EBPα and PPARγ but not of C/EBPβ. These findings suggest that neudesin plays a critical role in the early stage of adipocyte differentiation in which C/EBPβ induces PPARγ and C/EBPα expressions, by controlling the MAPK pathway.     

An association between glutamine synthetase activity and adipocyte differentiation in cultured 3T3-L1 cells

Confluent 3T3-L1 Swiss mouse fibroblasts acquired morphological and biochemical characteristics of adipocytes when maintained in medium containing 10% calf serum and added insulin. Identical cultures maintained in the absence of added insulin did not differentiate into adipocytes. Incubation of confluent cultures for 48 h with 0.25 μm dexamethasone and 0.5 mm 1-methyl-3-isobutylxanthine yielded subsequent adipocyte differentiation when the culture medium contained 10% fetal calf serum. In contrast, differentiation did not occur when similarly treated cultures were maintained in medium containing 10% calf serum. The increase in glutamine synthetase which occurred during adipocyte differentiation was closely associated with an increased rate of triglyceride synthesis from acetate, with increased protein, and with increases in the activities of glycerol-3-P dehydrogenase and glucose-6-P dehydrogenase. Glutamine synthetase activity remained undetectable in insulin-treated confluent 3T3-C2 cells maintained under conditions which yielded high glutamine synthetase activity in 3T3-L1 cells. (3T3-C2 cells did not differentiate into adipocytes.) Glutamine accumulated in the culture medium of 3T3-L1 adipocytes, but it did not accumulate in the medium from identically treated 3T3-C2 cells. A half-maximal increase in glutamine synthetase specific activity occurred at a culture medium insulin concentration of 10 ng/ml. Neither adipocyte differentiation nor the rise in glutamine synthetase activity were substantially altered by maintaining confluent cultures in medium lacking added glutamine. Incubation of confluent 3T3-L1 cultures with 3 mml-methionine sulfone, a reversible inhibitor of glutamine synthetase, increased by two-fold both the activity and the cellular content of glutamine synthetase. Incubation of confluent 3T3-L1 cultures with 4 mml-glutamine and l-methionine-dl-sulfoximine, an irreversible inhibitor of glutamine synthetase activity, decreased glutamine synthetase activity to less than 5% of the activity in control cultures; however, neither cellular content of the enzyme nor synthesis rate of the enzyme were substantially altered. In the presence of added glutamine, neither methionine sulfone nor methionine sulfoximine had a significant effect on phenotypic adipocyte conversion. By contrast, when confluent cultures were incubated with methionine sulfoximine and no added glutamine, glutamine synthetase remained absent and there was no evidence of adipocyte conversion. Our data indicate (1) that added insulin is required for adipocyte differentiation of 3T3-L1 cells maintained in medium containing calf serum, (2) that glutamine synthetase activity increases during adipocyte conversion regardless of the culture conditions employed to achieve differentiation, and (3) that glutamine synthetase activity may be required for adipocyte differentiation when cultures are maintained in medium lacking added glutamine.     

Effect of differentiation on the adenylate cyclase system of 3T3-C2 and 3T3-L1 cells. Determination of choleragen substrates in differentiating 3T3-L1 and nondifferentiating 3T3-C2 cells

3T3-L1 preadipocytes, when treated with 3-isobutyl-1-methylxanthine, dexamethasone, and insulin, differentiate into cells with the morphological and biochemical properties of adipocytes; the closely related 3T3-C2 cells, under identical conditions, exhibit a low frequency of adipocyte conversion. During differentiation, 3T3-L1 preadipocytes acquire an increased responsiveness to certain agonists (e.g. isoproterenol and adrenocorticotropic hormone) that influence lipolysis and lipogenesis through activation of adenylate cyclase, whereas 3T3-C2 cells do not. It has been suggested that changes in hormone responsiveness of 3T3-L1 cells during differentiation result from increased amounts of the guanyl nucleotide-binding protein of adenylate cyclase, as demonstrated by choleragen-catalyzed [32P]ADP ribosylation of 42 and 49-50-kilodalton particulate peptides. Particulate fractions from nondifferentiating 3T3-C2 cells, like those from 3T3-L1 cells, contained choleragen substrates of 42 and 46-47 (doublet) kilodaltons. Incubation of intact 3T3-L1 or 3T3-C2 cells with choleragen prior to preparation of particulate fractions prevented the subsequent in vitro choleragen-dependent [32P]ADP ribosylation of only these peptides. Increased incorporation of radioactivity into both the 42 and 46-47-kilodalton peptides was observed during differentiation of 3T3-L1 cells. However, a similar increase was also observed in nondifferentiating 3T3-C2 cells subjected to the differentiation protocol. Therefore, increased hormone responsiveness of 3T3-L1 adipocytes cannot be explained solely on the basis of increased labeling, and perhaps increased amounts, of the guanyl nucleotide-binding protein.     

peg10, an imprinted gene, plays a crucial role in adipocyte differentiation

An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPbeta and C/EBPdelta, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation.     

H-89 potentiates adipogenesis in 3T3-L1 cells by activating insulin signaling independently of protein kinase A

Among four kinds of protein kinase A (PKA) inhibitors tested, H-89 exhibited a unique action to remarkably enhance adipocyte differentiation of 3T3-L1 cells, whereas the other three PKA inhibitors, PKA inhibitor Fragment 14-22 (PKI), Rp-cAMP, and KT 5720, did not enhance adipocyte differentiation. H-85, which is an inactive form of H-89, exhibited a similar enhancing effect on adipocyte differentiation. H-89 also potentiated the phosphorylation of Akt and extracellular signal-regulated kinase (ERK) 1/2 in 3T3-L1 cells, which function as downstream signaling of insulin. Phosphoinositide 3-kinase (PI3K) inhibitor wortmannin and mitogen-activated protein kinase kinase (MEK) inhibitor PD 98059 suppressed both the H-89-induced promotion of adipocyte differentiation and the H-89-induced potentiation of phosphorylation of Akt and ERK1/2. Rho kinase inhibitor Y-27632 also promoted the phosphorylation of both Akt and ERK1/2 and enhanced adipocyte differentiation, although its effect was somewhat less than that of H-89. Even when cells were treated with a mixture of Y-27632 and H-89, the additive enhancing effects on both the insulin signaling and adipocyte differentiation were not detected. Therefore, it is suggested that the major possible mechanism whereby H-89 potentiates adipocyte differentiation of 3T3-L1 cells is activation of insulin signaling that is elicited mostly by inhibiting Rho/Rho kinase pathway.