胰岛素对3T3-L1脂肪细胞中极低密度脂蛋白受体基因表达的影响

体外培养3T3-L1细胞分化模型,研究不同浓度胰岛素及慢性胰岛素刺激对3T3-L1脂肪细胞中极低密度脂蛋白受体（VLDLR）基因表达的影响.在不同浓度胰岛素及胰岛素慢性刺激的干预下,用半定量RT-PCR检测细胞VLDLR mRNA水平的变化.微量化GOD-PAP法检测培养基中残存的葡萄糖.在细胞诱导分化过程中,胰岛素浓度的增高促进VLDLR的表达;胰岛素慢性刺激下,VLDLR表达因浓度差异呈现不同变化.研究结果表明,胰岛素的浓度及慢性刺激对3T3-L1脂肪细胞的成熟和VLDLR基因的表达有显著作用,而胰岛素抵抗明显减低成熟脂肪细胞VLDLR的表达.     

A role for soluble cAMP phosphodiesterases in differentiation of 3T3-L1 adipocytes

Differentiation of 3T3-L1 adipocytes, monitored by accumulation of neutral lipid and by increase in alpha-glycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3-L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its receptor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20-1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing alpha-glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8-phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6-(R phenyl-isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in alpha-glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3-L1 cells.     

Reciprocal regulation of tissue-type and urokinase-type plasminogen activators in the differentiation of murine preadipocyte line 3T3-L1 and the hormonal regulation of fibrinolytic factors in the mature adipocytes

Adipose tissue expresses a variety of genes including tumor necrosis factor alpha and type-1 plasminogen activator inhibitor (PAI-1); and these factors, produced by adipocytes, may be associated with the risk of coronary events in obesity. In this study, we characterized the production of fibrinolytic factors including tissue-type plasminogen activator (tPA), urokinase-type PA (uPA), and PAI-1 in the differentiation of preadipocytes, and examined the hormonal regulation of these fibrinolytic factors in mature adipocytes. Mouse 3T3-L1 preadipocytes were employed as a model of adipocytes. Adipocyte differentiation was induced by insulin, dexamethasone, and 3-isobutyl-1-methyl xanthine (IBMX). alpha-Glycerophosphate dehydrogenase (GPDH) activity and glucose transporter 4 (GLUT4) mRNA, indices for adipocyte maturation, were induced on Day 4, and gradually increased. GPDH activity reached its maximum level on Day 14. The level of tPA, a major PA in preadipocytes, dramatically decreased with differentiation. On the other hand, that of uPA reciprocally increased. PAI-1 production was also dramatically induced concomitant with differentiation. In mature adipocytes, uPA production was dominant (25 microg/ml/24 h vs. 0.8 microg/ml/24 h for tPA). Total PA activity in the mature adipocytes was reduced by insulin or dexamethasone, but not by glucagon. Insulin, IBMX, and dexamethasone significantly decreased both uPA and tPA production, and increased PAI-1 production. Glucagon had no effect on the production of these fibrinolytic factors. Our results reveal that uPA is one of the markers for the differentiation of 3T3-L1 cells and that insulin, IBMX, and dexamethasone are potent regulators of the fibrinolytic activity in differentiated 3T3-L1 cells, reciprocally affecting PA and PAI-1 levels in them.     

Identification of pyruvate carboxylase in 3T3-L1 cells by transblot and its correlation with differentiation into adipocytes.

The 3T3-L1 preadipocytes treated with insulin, dexamethasone and 3-methyl-1-isobutylxanthine (IBMX) two days before reaching monolayer undergo differentiation into adipocytes. Cell lysates were prepared from these cells under various conditions and analyzed by SDS-PAGE and transblot. Peroxidase-conjugated avidin used to detect endogenous proteins interacted strongly with a protein with an estimated molecular weight of 120 kDa, corresponding to pyruvate carboxylase, in the differentiated 3T3-L1 cells. On the other hand, this protein was not detected in undifferentiated 3T3-L1 cells.     

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.     

DNA synthesis and mitotic clonal expansion is not a required step for 3T3-L1 preadipocyte differentiation into adipocytes

Upon differentiation induction of 3T3-L1 preadipocytes by a hormone mixture containing 1-isobutyl-3-methylxanthine, dexamethasone, and insulin, the preadipocytes undergo approximately 2 rounds of mitotic clonal expansion, which just precedes the adipogenic gene expression program and has been thought to be an essential early step for differentiation initiation. By inducing 3T3-L1 preadipocytes with each individual hormone, it was determined that the mitotic clonal expansion was induced only by insulin and not by 1-isobutyl-3-methylxanthine or dexamethasone. Cell number counting and fluorescence-activated cell-sorting analysis indicated that a significant fraction of 3T3-L1 preadipocytes differentiated into adipocytes without mitotic clonal expansion when induced with the combination of 1-isobutyl-3-methylxanthine and dexamethasone. Furthermore, when normally induced 3T3-L1 preadipocytes were treated with PD98059 (an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1) to block the activation of extracellular signal-regulated kinase (Erk) 1 and Erk2, the mitotic clonal expansion was blocked, but adipocyte differentiation was not affected. These observations were confirmed by bromodeoxyuridine labeling. The differentiated adipocytes induced with 1-isobutyl-3-methylxanthine and dexamethasone or standard hormone mixture plus PD98059 were not labeled by bromodeoxyuridine. Thus, it is evident that 3T3-L1 preadipocytes could differentiate into adipocytes without DNA synthesis and mitotic clonal expansion. Our results also suggested that activation of Erk1 and Erk2 is essential to but not sufficient for induction of mitotic clonal expansion.     

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.     

Tyramine and benzylamine partially but selectively mimic insulin action on adipose differentiation in 3T3-L1 cells

Biogenic amines like tyramine, methylamine and the non-naturally occuring amine, benzylamine, have been described to promote adipose conversion of murine 3T3 preadipocytes. To further investigate these novel effects of amines, we studied whether they selectively mimic the long-term adipogenic action of insulin. To this aim, we decided to use the 3T3-L1 cell line since this model needs a complex combination of inducers to trigger the differentiation programme: insulin, isobutylmethylxanthine (IBMX, an activator of cAMP-signal transduction pathway) and the synthetic glucocorticoid, dexamethasone. A cell culture protocol was designed, by which each component of the differentiation cocktail was replaced with either benzylamine or tyramine, in order to determine whether these amine oxidase substrates could substitute any of the differentiation inducers in 3T3-L1 cells. The incomplete lipid accumulation found in cells grown under IBMX- or dexamethasone-free conditions was not improved by the daily addition of amines to the culture medium. Insulin was the only component of adipose differentiation cocktail of 3T3-L1 that could be replaced, although partially, by tyramine or benzylamine. When used at 0.5 mM, these amines resulted in a significant increase of triacylglycerol accumulated eight days after confluence, when compared to cells kept without insulin. This partial insulin replacement was totally abolished by SSAO-inhibitors, while MAO-blockade did not reduce lipid accumulation. As previously reported for other insulin-sensitive processes, such as stimulation of glucose transport or lipolysis inhibition in mature adipocytes, the stimulation of adipogenesis by tyramine and benzylamine was an SSAO-dependent mechanism that apparently shared common signaling pathways with insulin.     

Regulation of the expression of pp160, a putative insulin receptor signal protein, by insulin, dexamethasone, and 1-methyl-3-isobutylxanthine in 3T3-L1 adipocytes.

pp160, a cytosolic protein with Mr of approximately 160,000, is phosphorylated on tyrosine in response to insulin and is considered to be involved in signaling from the insulin receptor. The expression of pp160 during the differentiation of 3T3-L1 fibroblasts to adipocytes and in adipocytes has been investigated using quantitative immunoblotting with antibodies against a peptide from pp160. Between day 6 and day 8 of differentiation induced by insulin, dexamethasone (Dex), and 1-methyl-3-isobutylxanthine (Mix), pp160 expression increased 10-20-fold over the amount present in confluent fibroblasts. Omission of either insulin or Dex resulted in reduced expression of pp160 and in incomplete adipogenesis. Chronic treatment of fully differentiated adipocytes for 24 h with either insulin, Dex, or Mix alone in the presence of serum resulted in a decrease in the expression of pp160 by 70-85%. Chronic exposure to insulin caused a significant increase in the apparent size of pp160 to 172 kDa. Alkaline phosphatase treatment lowered the Mr of pp160 from both insulin-treated and basal cells to 150,000. These results demonstrate that pp160 is expressed in 3T3-L1 adipocytes during the time when insulin receptors are expressed in large numbers and that the maintenance of pp160 concentrations in adipocytes can be regulated by insulin, Mix, and Dex. The decreased expression of pp160 caused by these factors may be related to postreceptor insulin resistance.     

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.

     

Dexamethasone regulates the beta-adrenergic receptor subtype expressed by 3T3 L1 preadipocytes and adipocytes

The subtype of the beta-adrenergic receptor expressed in 3T3-L1 preadipocytes and adipocytes differentiated with dexamethasone and methylisobutylxanthine was determined by comparing the affinity of the receptors for epinephrine, norepinephrine, and beta-1 and beta-2 selective antagonist, 8-fold more avidly than adipocyte receptors. In contrast, adipocyte beta-receptors had a 10-fold higher affinity for epinephrine than for norepinephrine and complexed the beta-2 selective agonist zinterol with a 20-fold higher affinity than preadipocyte receptors. Hofstee plots and computer analyses of the binding data revealed that the populations of beta-1 receptors in preadipocytes and beta-2 receptors in adipocytes were nearly homogeneous. Preliminary characterizations of the beta-receptor phenotype in (nondifferentiating) 3T3-C2 cells treated with dexamethasone and methylisobutylxanthine and 3T3-422A adipocytes differentiated with insulin indicated that the expression of beta-2 receptors was not correlated with differentiation, but rather with exposure of the cells to dexamethasone and methylisobutylxanthine. The regulator of beta-receptor subtype was identified as the glucocorticoid analog, dexamethasone, by employing 3T3-L1 adipocytes which were stimulated to differentiate with methylisobutylxanthine and insulin. Detailed binding studies showed that under these conditions the adipocyte receptors retain beta-1 character. Subsequent treatment with 0.5 microM dexamethasone promoted the loss of beta-1 receptors, the appearance of beta-2 receptors, and a net 2- to 3-fold increase in the number of beta-receptors. Dexamethasone effected a complete switch from beta-1 to beta-2 subtype at concentrations as low as 2.5 nM while other steroids were ineffective below a concentration of 10 microM.     

Prolonged treatment with 3-isobutyl-1-methylxanthine improves the efficiency of differentiating 3T3-L1 cells into adipocytes

Until now, the low efficiency of current protocols or kits for the differentiation of 3T3-L1 preadipocytes makes it difficult to continue the studies of the cellular and molecular mechanisms in adipocytes. Here we present a productive and highly efficient protocol for the differentiation of 3T3-L1 cells that uses a prolonged treatment with 3-isobutyl-1-methylxanthine (IBMX) during the differentiated process. 3T3-L1 cells of unknown passage +3 and unknown passage +7 treated with a prolonged exposure to IBMX showed significantly increased differentiation efficiency by day 15, in contrast to low levels of differentiation seen with protocols that lacked additional IBMX.     

Induction of cell death by photodynamic therapy with a new synthetic photosensitizer DH-I-180-3 in undifferentiated and differentiated 3T3-L1 cells

To examine the photodynamic therapy (PDT) effect on adipocytes, we investigated whether PDT using DH-I-180-3, a new synthetic lipophilic photosensitizer, induced cell death of both undifferentiated and differentiated 3T3-L1. 3T3-L1 pre-adipocytes were differentiated into adipocytes in the culture medium containing pantothenate, insulin, dexamethasone, isobutylmethylxanthine, and troglitazone. PDT was applied to both undifferentiated and differentiated 3T3-L1. Photosensitizer uptake in fat cells was determined by measuring its mean fluorescence intensity. DH-I-180-3 mediated effectively PDT-induced cell death of both pre-adipocytes and adipocytes. And the photosensitizer was accumulated more rapidly in 3T3-L1 adipocytes, compared with other cancer cell lines. These results demonstrate that PDT is a potent cell death inducer in pre-adipocytes and adipocytes. Thus, PDT with DH-I-180-3 may be applied for a new therapeutic modality for obesity treatment.     

Glucose, dexamethasone, and the unfolded protein response regulate TRB3 mRNA expression in 3T3-L1 adipocytes and L6 myotubes

Tribbles 3 (TRB3) is a recently recognized atypical inactive kinase that negatively regulates Akt activity in hepatocytes, resulting in insulin resistance. Recent reports link TRB3 to nutrient sensing and regulation of cell survival under stressful conditions. We studied the regulation of TRB3 by glucose, insulin, dexamethasone (Dex), and the unfolded protein response (UPR) in 3T3-L1 adipocytes and in L6 myotubes. In 3T3-L1 adipocytes, incubation in high glucose with insulin did not increase TRB3 mRNA expression. Rather, TRB3 mRNA increased fourfold with glucose deprivation and two- to threefold after incubation with tunicamcyin (an inducer of the UPR). Incubation of cells in no glucose or in tunicamcyin stimulated the expression of CCAAT/enhancer-binding protein homologous protein. In L6 myotubes, absent or low glucose induced TRB3 mRNA expression by six- and twofold, respectively. The addition of Dex to 5 mM glucose increased TRB3 mRNA expression twofold in 3T3-L1 adipocytes but decreased it 16% in L6 cells. In conclusion, TRB3 is not the mediator of high glucose or glucocorticoid-induced insulin resistance in 3T3-L1 adipocytes or L6 myotubes. TRB3 is induced by glucose deprivation in both cell types as a part of the UPR, where it may be involved in regulation of cell survival in response to glucose depletion.     

Mitogen-activated protein kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced insulin resistance in 3T3-L1 adipocytes

Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-lambda remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulin-induced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per se.     

Inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes.

Lovastatin treatment caused down-regulation of the insulin-responsive glucose transporter 4 (Glut4) and up-regulation of Glut1 in 3T3-L1 adipocytes. These changes in protein expression were associated with a marked inhibition of insulin-stimulated glucose transport. Lovastatin had no effect on cell cholesterol levels, but its effects were reversed by mevalonate, demonstrating that inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes. These findings support the notion that whole body insulin resistance may arise as a result of perturbations in general biochemical pathways, rather than primary defects in insulin signalling.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.