灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

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.     

Impact of stress hormone on adipogenesis in the 3T3-L1 adipocytes

Stress hormone is known to play a vital role in lipolysis and adipogenesis in fat cells. The present study was carried out to evaluate the effect of epinephrine on adipogenesis in the 3T3-L1 cells. The investigation on adipogenesis was done in both mono and co-cultured 3T3-L1 cells. 3T3-L1 preadipocytes and C2C12 cells were grown independently on transwell plates and transferred to differentiation medium. Following differentiation, C2C12 cells transferred to 3T3-L1 plate and treated with medium containing 10 μg/ml of epinephrine. Adipogenic markers such as fatty acid binding protein 4, peroxisome proliferator activating receptor, CCAAT/enhancer-binding protein, adiponectin, lipoprotein lipase and fatty acid synthase mRNA expressions were evaluated in the 3T3-L1 cells. Epinephrine treatment reduced adipogenesis, evidenced by reducing adipogenic marker mRNA expression in the 3T3-L1 cells. In addition, glycerol accumulation and oil red-O staining supported the reduced rate of adipogenesis. Taking all together, it is concluded that the stress hormone, epinephrine reduces the rate of adipogenesis in the mono and co-cultured 3T3-L1 cells. In addition, the rate of adipogenesis is much reduced in the co-cultured 3T3-L1 cells compared monocultured 3T3-L1 cells.     

Construction of stable coxsackievirus and adenovirus receptor-expressing 3T3-L1 cells

3T3-L1 cells have been used as a model to study the differentiation and physiology of adipocytes. Exogenous expression of proteins in these cells offers the prospect of understanding the protein's function(s) in adipose tissue. Viral vectors, in particular, adenovirus, have proven to be a powerful means for introduction of genes into many cell types. However, we have previously shown that 3T3-L1 cells are inefficiently transduced by adenovirus (Orlicky, D. J., and J. Schaack. 2001. J. Lipid Res. 42: 460-466). To overcome the inefficient transduction, we have stably introduced the gene-encoding coxsackie and adenovirus receptor (CAR), which was modified by deletion of the region encoding the cytoplasmic tail, into 3T3-L1 cells. 3T3-L1 CARDelta1 cells are transduced approximately 100-fold more efficiently than parental 3T3-L1 cells. 3T3-L1 CARDelta1 cells should prove to be a useful tool for examination of exogenous protein expression in fat cells.     

脂联素siRNA对3T3-L1细胞基础葡萄糖转运的影响

目的：构建携带小鼠脂联素（Acrp30）siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法：设计并化学合成小鼠脂肪细胞Acrp30 siRNA片断,将其亚克隆入AdEaxy XL腺病毒载体系统,在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2Deoxy-[3H]D—glucose掺入法测定脂肪细胞葡萄糖转运。结果：设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制Acrp30 mRNA和蛋白表达,影响3T3-L1脂肪细胞基础葡萄糖的转运,与对照组相比,差异有显著性意5C（P〈0．05）。结论：构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素在3T3-L1脂肪细胞中的表达,从而影响3T3-L1脂肪细胞基础葡萄糖转运。     

脂联素siRNA对3T3-L1细胞基础葡萄糖转运的影响(英文)

目的:构建携带小鼠脂联素(Acrp30)siRNA腺病毒载体,并检测其对小鼠脂肪细胞Acrp30表达以及对3T3-L1脂肪细胞基础葡萄糖转运的影响。方法:设计并化学合成小鼠脂肪细胞Acrp30 siRNA片断,将其亚克隆入AdEaxy XL腺病毒载体系统,在293细胞内包装扩增为重组腺病毒。用此重组腺病毒感染3T3-L1脂肪细胞,用RT-PCR和ELISA检测其Acrp30 mRNA和蛋白表达。采用2 Deoxy-[3H]D-glucose掺入法测定脂肪细胞葡萄糖转运。结果:设计并构建了小鼠Acrp30基因特异性siRNA腺病毒载体,该载体感染脂肪细胞后,能显著抑制Acrp30 mRNA和蛋白表达,影响3T3-L1脂肪细胞基础葡萄糖的转运,与对照组相比,差异有显著性意义(P<0.05)。结论:构建的Acrp30基因特异性siRNA腺病毒载体能有效的抑制脂联素在3T3-L1脂肪细胞中的表达,从而影响3T3-L1脂肪细胞基础葡萄糖转运。     

Changes in the guanine nucleotide-binding proteins, Gi and Go, during differentiation of 3T3-L1 cells

Differentiation of 3T3-L1 cells from fibroblasts to adipocytes is accompanied by increased adenylate cyclase response to lipolytic agents. We used pertussis toxin and specific antibodies to measure the inhibitory guanine nucleotide-binding protein, Gi, and the novel G-protein, Go, in membranes from 3T3-L1 cells. Pertussis toxin-dependent labeling of a 39-40 kDa protein showed an initial 30% rise, followed by an 80% fall during differentiation. Immunoblots showed that 3T3-L1 cells contain Go, as well as Gi, and that changes in the former parallel the changes in pertussis toxin labeling. Changes in Gi and GO may contribute to altered adenylate cyclase response during 3T3-L1 cell differentiation.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

Co-culture of C2C12 and 3T3-L1 preadipocyte cells alters the gene expression of calpains, caspases and heat shock proteins

The present study was carried out to understand the co-culture effect of C2C12 and 3T3-L1 preadipocyte cells on calpain, caspase, and heat shock protein (Hsp) systems. Calpains, caspases, and heat shock proteins play critical roles in the growth and development of mammalian cells. Cells were co-cultured using transwell inserts with a 0.4-??m porous membrane to separate C2C12 and 3T3-L1 preadipocyte cells. Each cell type was grown independently on the transwell plates. Following cell differentiation, inserts containing 3T3-L1 cells were transferred to C2C12 plates and inserts containing C2C12 transferred to 3T3-L1 plates. Following co-culture for 24 and 48?h, the cells in the lower well were harvested for analysis. Calpains include ??-calpain, m-calpain, and their specific inhibitor calpastatin. The expression pattern of ??-calpain did not change in the co-cultured C2C12 and 3T3-L1 cells, whereas m-capain mRNA expression significantly reduced in the 48-h co-cultured 3T3-L1 cells. Calpastatin mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Caspase-7 mRNA expression did not change in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells. Caspase-3 mRNA expression significantly reduced in the 24- and 48-h co-cultured 3T3-L1 cells; caspase-9 mRNA had a significant reduction only at 48?h, whereas caspase-9 mRNA expression significantly increased in the 48-h co-cultured C2C12 cells. Hsp27 and Hsp90 mRNA expressions are significantly reduced in the 24- and 48-h co-cultured C2C12 and 3T3-L1 cells, whereas Hsp70 mRNA expression significantly increased in the 48-h co-cultured 3T3-L1 cells. The co-culture reflects three-dimensional views of C2C12 and 3T3-L1 cell types as in vivo, which is quite distinct from the one-dimensional monocultured C2C12 and 3T3-L1 cells.     

辣椒碱对3T3-L1 前脂肪细胞葡萄糖摄取的影响

目的:探讨辣椒碱对3T3-L1前脂肪细胞葡萄糖摄取的影响。方法:不同浓度的辣椒碱作用于3T3-L1前脂肪细胞,采用MTT测定细胞活性,GLU Test试剂盒法测定葡萄糖摄取,Western Blot法检测葡萄糖转运蛋白1(GLUT-1)表达的变化。结果:25μM辣椒碱作用72 h和50μM、100μM辣椒碱作用48 h、72 h,可显著抑制3T3-L1细胞增殖,6.25、12.5、25μM辣椒碱作用可显著促进3T3-L1细胞的葡萄糖摄入,Western Blot结果显示辣椒碱能够显著增加GLUT1蛋白表达量,差异均具有统计学意义(P0.05)。结论:低剂量辣椒碱具有降糖作用,其作用机制可能与增加GLUT-1蛋白表达有关。     

Prostaglandin F(2)alpha enhances glucose consumption through neither adipocyte differentiation nor GLUT1 expression in 3T3-L1 cells

Arachidonic acid (AA) at 0.2 mM enhances glucose uptake through increased levels of glucose transporter (GLUT) 1 protein in 3T3-L1 adipocytes. Since AA is a precursor of prostaglandins (PGs), we investigated the effect of PGs on glucose consumption in 3T3-L1 cells. Among several PGs, only prostaglandin F(2)alpha (PGF(2)alpha) enhanced glucose consumption in 3T3-L1 cells treated with dexamethasone (DEX), 3-isobutyl-1-methyl-xanthine (IBMX), and insulin. To study the mechanism of PGF(2)alpha-enhanced glucose consumption, we investigated the effect of PGF(2)alpha on glycerol-3-phosphate dehydrogenase (GPDH) activity, triglycerides (TGs) content, and the expression of GLUT1 protein. PGF(2)alpha suppressed GPDH activity and did not increase the expression of GLUT1 protein in 3T3-L1 cells treated with DEX, IBMX, and insulin. These results suggest that AA-stimulated glucose uptake is not through the effect of PGF(2)alpha. Our results indicate that PGF(2)alpha is a unique regulator of adipocyte differentiation (suppression) and glucose consumption (enhancement) in 3T3-L1 cells.     

Aortic carboxypeptidase-like protein is regulated by transforming growth factor beta in 3T3-L1 preadipocytes

Adipogenesis is characterized by early remodeling of the extracellular matrix, allowing preadipocytes to adopt a more spherical shape and optimize lipid accumulation as they mature. Aortic carboxypeptidase-like protein (ACLP), found in collagen-rich tissues including adipose tissue, is expressed in 3T3-L1 and 3T3-F442A preadipocytes, and is downregulated during adipogenesis. We now report that ACLP is found in medium conditioned by 3T3-L1 preadipocytes. Transforming growth factor (TGF) beta, a known modulator of fibrillar matrix protein production, increased ACLP expression by 2.4+/-0.4-fold (mean+/-SE; n=3) in 3T3-L1 preadipocytes, through a mechanism that requires p42/44 MAPK activity. Addition of TGFbeta to differentiation medium, which inhibits adipogenesis, raised ACLP levels in 3T3-L1 cells. However, sustained expression of ACLP in stable clones of 3T3-L1 or 3T3-F442A preadipocytes did not interfere with adipogenesis.     

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

Differential modulation of 3T3-L1 adipogenesis mediated by 11beta-hydroxysteroid dehydrogenase-1 levels

The localized activation of circulating glucocorticoids in vivo by the enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) plays a critical role in the development of the metabolic syndrome. However, the precise contribution of 11beta-HSD1 in the initiation of adipogenesis by inactive glucocorticoids is not fully understood. 3T3-L1 fibroblasts can be terminally differentiated to mature adipocytes in a glucocorticoid-dependent manner. Both inactive rodent dehydrocorticosterone and human cortisone were able to substitute for the synthetic glucocorticoid dexamethasone in 3T3-L1 adipogenesis, suggesting a potential role for 11beta-HSD1 in these effects. Differentiation of 3T3-L1 cells caused a strong increase in 11beta-HSD1 protein levels, which occurred late in the differentiation protocol. Reduction of 11beta-HSD1 activity in 3T3-L1 fibroblasts, achieved by pharmacological inhibition or adenovirally mediated delivery of short hairpin RNA constructs, specifically blocked the ability of inactive glucocorticoids to drive 3T3-L1 differentiation. However, even modest increases in exogenous 11beta-HSD1 expression in 3T3-L1 fibroblasts, to levels comparable with endogenous 11beta-HSD1 in differentiated 3T3-L1 adipocytes, were sufficient to block adipogenesis. Luciferase reporter assays indicated that overexpressed 11beta-HSD1 was catalyzing the inactivating dehydrogenase reaction, because the ability of both active and inactive glucocorticoids to activate the glucocorticoid receptor were largely suppressed. These results suggest that the temporal regulation of 11beta-HSD1 expression is tightly controlled in 3T3-L1 cells, so as to mediate the initiation of differentiation by inactive glucocorticoids and also to prevent the inhibitory activity of prematurely expressed 11beta-HSD1 during adipogenesis.     

TSH signaling and cell survival in 3T3-L1 preadipocytes

Thyroid-stimulating hormone (TSH) action in adipose tissue remains largely unknown. Our previous work indicates that human preadipocytes express functional TSH receptor (TSHR) protein, demonstrated by TSH activation of p70 S6 kinase (p70 S6K). We have now studied murine 3T3-L1 preadipocytes to further characterize TSH signaling and cellular action. Western blot analysis of 3T3-L1 preadipocyte lysate revealed the 100-kDa mature processed form of TSHR. TSH activated p70 S6K and protein kinase B (PKB/Akt), as measured by immunoblot analysis. Preincubation with wortmannin or LY-294002 completely blocked TSH activation of p70 S6K and PKB/Akt, implicating phosphoinositide 3-kinase (PI3K) in their regulation. TSH increased phosphotyrosine protein(s) in the 125-kDa region and augmented the associated PI3K activity fourfold. TSH had no effect on cAMP levels in 3T3-L1 preadipocytes, suggesting that adenylyl cyclase is not involved in TSH activation of the PI3K-PKB/Akt-p70 S6K pathway. 3T3-L1 preadipocyte cell death was reduced by 29-76% in serum-deprived (6 h) preadipocytes treated with 1-20 microM TSH. In the presence of 20 microM TSH, an 88% reduction in terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)-positive cells was observed in serum-starved (3 h) 3T3-L1 preadipocytes as well as a 93% reduction in the level of cleaved activated caspase 3. In summary, TSH acts as a survival factor in 3T3-L1 preadipocytes. TSH does not stimulate cAMP accumulation in these cells but instead activates a PI3K-PKB/Akt-p70 S6K pathway.     

Identification and characterization of a transcript for a novel Rac GTPase-activating protein in terminally differentiating 3T3-L1 adipocytes

Using differential display, we sought to identify novel genes expressed in the early stages of 3T3-L1 adipocyte differentiation. A gene which we have named "band25" was identified, and a full-length cDNA sequence was assembled. Sequence analysis revealed that the 2842-bp cDNA encodes a putative 628-amino acid protein product, which is a member of the GTPase-activating protein (GAP) family. This gene may be the murine homolog of the human MgcRacGAP protein, which was identified in male germ cells. Other closely related proteins include the Drosophila protein Rotund, several chimerins, and the human breakpoint cluster region (Bcr) protein. These GAP proteins all specifically inactivate Rac, a member of the Ras-like family of proteins. A consensus sequence for a diacyl glycerol/phorbol ester-binding domain was also found in the Band25 sequence. The expression of band25 mRNA is regulated during the differentiation of both adipocytes and myoblasts. Its mRNA was shown to be expressed at a low level in confluent 3T3-L1 preadipocytes and in differentiated 3T3-L1 adipocytes. Expression of band25 was increased 15.5 fold by 24 h after the induction of differentiation, when 3T3-L1 cells undergo several rounds of postconfluent cell division. Expression was also high in growing 3T3-L1 and C2C12 cells but decreased progressively as C2C12 cells underwent differentiation. These observations suggest that the expression of band25 is growth regulated and that the protein could play a role in the regulation of growth-related processes.     

A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

The protein product of the c-Cbl proto-oncogene is prominently tyrosine phosphorylated in response to insulin in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. After insulin-dependent tyrosine phosphorylation, c-Cbl specifically associates with endogenous c-Crk and Fyn. These results suggest a role for tyrosine-phosphorylated c-Cbl in 3T3-L1 adipocyte activation by insulin. A yeast two-hybrid cDNA library prepared from fully differentiated 3T3-L1 adipocytes was screened with full-length c-Cbl as the target protein in an attempt to identify adipose-specific signaling proteins that interact with c-Cbl and potentially are involved in its tyrosine phosphorylation in 3T3-L1 adipocytes. Here we describe the isolation and the characterization of a novel protein that we termed CAP for c-Cbl-associated protein. CAP contains a unique structure with three adjacent Src homology 3 (SH3) domains in the C terminus and a region showing significant sequence similarity with the peptide hormone sorbin. Both CAP mRNA and proteins are expressed predominately in 3T3-L1 adipocytes and not in 3T3-L1 fibroblasts. CAP associates with c-Cbl in 3T3-L1 adipocytes independently of insulin stimulation in vivo and in vitro in an SH3-domain-mediated manner. Furthermore, we detected the association of CAP with the insulin receptor. Insulin stimulation resulted in the dissociation of CAP from the insulin receptor. Taken together, these data suggest that CAP represents a novel c-Cbl binding protein in 3T3-L1 adipocytes likely to participate in insulin signaling.