Altered subcellular distribution of estrogen receptor alpha is implicated in estradiol-induced dual regulation of insulin signaling in 3T3-L1 adipocytes

We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17beta-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10(-8) m, but inhibited these effects at 10(-5) m. A concentration of 10(-5) m E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser(307), which was abolished by treatment with a c-Jun NH(2)-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10(-8) m, but inhibited it at 10(-5) m. Furthermore, E2 decreased the amount of estrogen receptor alpha at the plasma membrane at 10(-8) m, but increased it at 10(-5) m. In contrast, the subcellular distribution of estrogen receptor beta was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser(307) via a c-Jun NH(2)-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor alpha in response to E2 may explain the dual effect of E2.     

Latent insulin receptors and possible receptor precursors in 3T3-L1 adipocytes.

Cell surface and cryptic insulin receptors were solubilized from the particulate fraction of murine 3T3-L1 adipocytes with buffer containing 1% Triton X-100. Solubilized receptors were affinity crosslinked with 125I-labeled insulin and disuccinimidyl suberate and characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and autoradiography after specific immunoprecipitation. Two insulin-binding polypeptides were identified: the more abundant protein had a Mr of 130,000, corresponding to the size of the hormone-binding subunit of insulin receptors on the surface of target cells; the second polypeptide exhibited a Mr of 200,000 and appears to be a component of the latent pool because it was unaffected when 3T3-L1 adipocytes were exposed to trypsin under conditions that result in a 95% reduction in cell surface insulin-binding activity and the loss of the Mr 130,000 polypeptide in crosslinking experiments. Unexpectedly, the population of Mr 200,000 molecules in intact cells was accessible for limited cleavage by chymotrypsin, yielding a Mr 195,000 insulin-binding polypeptide. When 3T3-L1 adipocytes received a 15-min pulse of [35S]methionine, the predominant immunoprecipitated polypeptide had a Mr of 180,000. During a 1.5-hr chase, radioactivity in the Mr 180,000 species rapidly declined while the latent Mr 200,000 polypeptide became intensely labeled. After a 5-hr chase period, broad protein bands with Mrs of 130,000 and 90,000 were visualized as the major immunoprecipitated radioactive polypeptides. Thus, the Mr 180,000 species may be a very early biosynthetic precursor that may be subsequently processed to a Mr 200,000 form and one or both of the smaller receptor subunits at the cell surface.     

Alterations in insulin signalling pathway induced by prolonged insulin treatment of 3T3-L1 adipocytes

Summary Insulin-induced glucose transport stimulation, which results from the translocation of glucose transporter 4 (GLUT 4)-containing vesicles, is completely blocked after prolonged insulin treatment of 3T3-L1 adipocytes. Since GLUT 4 expression was reduced by only 30%, we looked at the insulin signalling pathway in this insulin-resistant model. Insulin-induced tyrosine phosphorylation of the major insulin receptor substrate IRS 1 was reduced by 50±7%, while its expression was decreased by 70±4%. When cells were treated with wortmannin (a PI3-kinase inhibitor) together with insulin, the expression of IRS 1 diminished to a much lower extent. Associated with the decrease in IRS 1 expression and phosphorylation, the activation by insulin of antiphosphotyrosine immunoprecipitable PI3-kinase activity and of p44^mapk and p42^mapk activities was altered. However, the expression of these proteins was normal and p44^mapk activity remained responsive to the tumour promoter TPA. Those results indicate that prolonged insulin treatment of 3T3-L1 adipocytes induces an insulin-resistant state with a reduced ability of insulin to stimulate the PI3-kinase and the MAP-kinases and a blockade of glucose transporter translocation.Abbreviations GLUT Glucose transporter - TPA tumour promoter - MAPK mitogen-activated protein kinase - IRS insulin receptor substrate - SH2 src homology 2 - GRB GRB: Growth factor Receptor bound protein - PVDF polyvinyliden difluoride - HDM/LDM high density/low density microsomes - MBP myelin basic protein - DMEM Dulbecco's modified Eagle's medium - PMSF phenylmethanesulphonyl fluoride - PI3-kinase phosphatidylinositol 3-kinase     

Inhibition of endogenous SHIP2 ameliorates insulin resistance caused by chronic insulin treatment in 3T3-L1 adipocytes

Aims/hypothesis SHIP2 is a physiologically important negative regulator of insulin signalling hydrolysing the PI3-kinase product, PI(3,4,5)P3, which also has an impact on insulin resistance. In the present study, we examined the effect of inhibiting the endogenous SHIP2 function on the insulin resistance caused by chronic insulin treatment.Methods The endogenous function of SHIP2 was inhibited by expressing a catalytically inactive SHIP2 (IP-SHIP), and compared with the effect of treatments designed to restore the levels of IRS-1 in insulin signalling systems of 3T3-L1 adipocytes.Results Chronic insulin treatment induced the large (86%) down-regulation of IRS-1 and the modest (36%) up-regulation of SHIP2. Subsequent stimulation by insulin of Akt phosphorylation, PKC activity, and 2-deoxyglucose (2-DOG) uptake was markedly decreased by the chronic insulin treatment. Coincubation with the mTOR inhibitor, rapamycin, effectively inhibited the proteosomal degradation of IRS-1 caused by the chronic insulin treatment. Although the coincubation with rapamycin and advanced overexpression of IRS-1 effectively ameliorated subsequent insulin-induced phosphorylation of Akt, insulin stimulation of PKC activity and 2-DOG uptake was partly restored by these treatments. Similarly, expression of IP-SHIP2 effectively ameliorated the insulin-induced phosphorylation of Akt without affecting the amount of IRS-1. Furthermore, the decreased insulin-induced PKC activity and 2-DOG uptake following chronic insulin treatment were ameliorated by the expression of IP-SHIP2 more effectively than by the treatment with rapamycin.Conclusions/interpretation Our results indicate that the inhibition of endogenous SHIP2 is effective in improving the state of insulin resistance caused by chronic insulin treatment.     

Estrogen receptor alpha regulates insulin sensitivity through IRS-1 tyrosine phosphorylation in mature 3T3-L1 adipocytes

There are many clinical and experimental reports demonstrating that estrogens and insulin interact when affecting their target organs. Estrogen receptors consist of two isoforms, estrogen receptors-alpha (ER-alpha) and -beta (ER-beta), but their roles in insulin-induced glucose uptake in mature adipose tissue have yet to be clarified. To evaluate the roles of ER-alpha, expressed predominantly in adipocytes, we have investigated the effects of estradiol (E2), an ER-alpha selective agonist (PPT), and its selective antagonist (MPP) on glucose uptake and insulin action in 3T3-L1 adipocytes. 3T3-L1 adipocytes were exposed to E2 or PPT and/or MPP at different concentrations. The cells were then subjected to 2-deoxy-D-glucose transport assay, western blot analysis, or RT-PCR analysis. Treatment of these cells with E2 or PPT resulted in biphasic effects on glucose transport, that is high (10(-5) M or 3 x 10(-6) M each) and low (10(-8) M) doses produced inhibition and stimulation, respectively. The favorable effect observed at 10(-8) M of E2 was diminished by treatment with MPP. Western bolt analysis revealed that these effects of E2, PPT and MPP paralleled the level of IRS-1 tyrosine phosphorylation. However, IRS-1 serine phosphorylation, suppressor of cytokine signaling (SOCS)-1,-2,-3 and protein tyrosine phosphatase 1B (PTP1B) expression did not change compared to control subjects. Our data clearly show that ER-alpha contributes to insulin stimulated glucose uptake through regulation of the tyrosine phosphorylation of IRS-1 protein.     

beta-Adrenergic desensitization by chronic insulin exposure in 3T3-L1 cultured adipocytes

Cultured 3T3-L1 cells provide a model system for studies of the long-term regulation of lipolysis. Insulin acutely inhibits isoproterenol-stimulated lipolysis primarily by decreasing the apparent affinity apparent Km for isoproterenol. In contrast, chronic insulin exposure inhibits lipolysis by a reduction in the maximal effect of isoproterenol Vmax. The decrease in Vmax can be observed with insulin concentrations that are as low as 10(-9) mol/L at the time of addition. The effect is stable to washing, and the cells' responsiveness to isoproterenol returns partially with continued culture. Chronic insulin exposure also markedly reduced dibutyryl-cAMP-stimulated lipolysis indicating an insulin-induced change distal to cAMP concentration in the cascade of reactions controlling lipolysis in these cells. Time course and insulin dose-response experiments indicate an additional proximal alteration. These results indicate that: (1) 3T3-L1 cells are a useful model for studying the long-term regulation of lipolysis. (2) Chronic insulin exposure inhibits lipolysis by a mechanism that differs from the acute effect of insulin. (3) The chronic effects of insulin may be mediated through changes at multiple levels in the lipolytic cascade.     

吡格列酮减轻肿瘤坏死因子α所致3T3-L1脂肪细胞胰岛素抵抗

目的观察吡格列酮对TNF-α介导的胰岛素抵抗和胰岛素信号通路的影响方法经或未经吡格列酮预处理的3T3-L1细胞与TNF-α作用24h后,分别与对照组比较细胞对胰岛素刺激的葡萄糖摄取,IRS-1及其酪氨酸磷酸化以及PKB,PKCλ及其磷酸化的变化。结果TNF-α抑制胰岛素刺激的葡萄糖摄取以及IRS-1酪氨酸及PKB磷酸化,并使IRS-1蛋白水平下降,对PKC-λ磷酸化无影响。吡格列酮预处理可以逆转TNF-α导致的胰岛素抵抗,部分恢复IRS-1蛋白水平,增强胰岛素刺激的IRS-1酪氨酸、PKB磷酸化及PKC-λ磷酸化。结论TNF-α导致胰岛素抵抗与IRS-1酪氨酸磷酸化水平下降密切相关,吡格列酮可以逆转TNF-α的上述作用。     

GLP-1 amplifies insulin signaling by up-regulation of IRbeta, IRS-1 and Glut4 in 3T3-L1 adipocytes

Glucagon-like peptide-1 (7–36) amide (GLP-1) is an insulin secretagogue. Recently, many studies have shown GLP-1 can improve insulin resistance in peripheral tissues. In the present study, we investigated glucose uptake in 3T3-L1 adipocytes in either basal or insulin resistant state and dissected insulin signaling pathway in order to elucidate the molecular mechanisms of GLP-1 mediated improvement of insulin resistance. We found GLP-1 and its long lasting analogue, exendin 4 up-regulated basal IR, IRS-1 and Glut 4 expressions although they did not increase basal glucose uptake alone. However, GLP-1 and exendin-4 increased insulin mediated glucose uptake in intact and TNF-α treated 3T3-L1 adipocytes by up-regulation of phophorylated IRβ, IRS-1, Akt and GSK-3β. These results indicate that GLP-1 and its analogue exendin-4 can amplify insulin signaling in 3T3-L1 adipocytes by up-regulation of some crucial insulin signaling molecules. Hong Gao and Xinjun Wang equally contributed to this work.     

Andrographolide attenuates tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes

Andrographolide (AG), the primary bioactive component of Andrographils paniculate Nees, has showed an anti-diabetic effect. However, the molecular mechanism has not been clarified. In this study, we demonstrated that AG increased glucose uptake in 3T3-L1 cells in a time- and dosedependent manner. The activation of insulin signaling by AG was initiated from phosphotyrosine of IRS-1 and further passed on through phosphatidylinositol 3-kinase (PI3K) and the downstream signaling cascades. Moreover importantly, pretreatment cells with AG suppressed the TNF-α induced activation of NF-κB signaling pathway and its downstream inflammatory factors expression, therefore ameliorating insulin resistance. In conclusion, AG can improve insulin sensitivity through inhibition of NF-κB pathway. These findings are helpful in understanding the anti-diabetic properties of AG and can be of interest for the therapeutic application of AG in glucose controlling.     

Role of glycosylation and protein synthesis in insulin receptor metabolism by 3T3-L1 mouse adipocytes.

The roles of glycosylation and protein synthesis in the maintenance of insulin receptor levels and turnover rates in 3T3-L1 adipocytes were investigated. The heavy isotope density-shift technique was employed to determine the effects of inhibitors of these processes on the rates of synthesis and degradation of cellular insulin receptors. Inhibitors of protein synthesis--i.e., cycloheximide and puromycin--markedly decreased the rate of degradation of the insulin receptor, the half-life for receptor decay increasing from 7.5 hr without to 25 hr with inhibitor. The continued synthesis of a short-lived protein appears to be necessary for normal insulin receptor turnover. Tunicamycin, a potent inhibitor of core oligosaccharide addition in the formation of N-glycosidically linked glycoproteins, caused the depletion of cell-surface and total cellular detergent-extractable insulin receptors. This inhibitor totally prevented the formation of functional newly synthesized insulin receptor, yet receptor degradation was affected minimally. Thus, glycosylation of the receptor appears to be required for its activation after translation.     

Persistent activation of phosphatidylinositol 3-kinase causes insulin resistance due to accelerated insulin-induced insulin receptor substrate-1 degradation in 3T3-L1 adipocytes

Recently, we have reported that the overexpression of a membrane-targeted phosphatidylinositol (PI) 3-kinase (p110CAAX) stimulated p70S6 kinase, Akt, glucose transport, and Ras activation in the absence of insulin but inhibited insulin-stimulated glycogen synthase activation and MAP kinase phosphorylation in 3T3-L1 adipocytes. To investigate the mechanism of p110CAAX-induced cellular insulin resistance, we have now studied the effect of p110CAAX on insulin receptor substrate (IRS)-1 protein. Overexpression of p110CAAX alone decreased IRS-1 protein levels to 63+/-10% of control values. Insulin treatment led to an IRS-1 gel mobility shift (most likely caused by serine/threonine phosphorylation), with subsequent IRS-1 degradation. Moreover, insulin-induced IRS-1 degradation was enhanced by expression of p110CAAX (61+/-16% vs. 13+/-15% at 20 min, and 80+/-8% vs. 41+/-12% at 60 min, after insulin stimulation with or without p110CAAX expression, respectively). In accordance with the decreased IRS-1 protein, the insulin-stimulated association between IRS-1 and the p85 subunit of PI 3-kinase was also decreased in the p110CAAX-expressing cells, and IRS-1-associated PI 3-kinase activity was decreased despite the fact that total PI 3-kinase activity was increased. Five hours of wortmannin pretreatment inhibited both serine/threonine phosphorylation and degradation of IRS-1 protein. These results indicate that insulin treatment leads to serine/threonine phosphorylation of IRS-1, with subsequent IRS-1 degradation, through a PI 3-kinase-sensitive mechanism. Consistent with this, activated PI 3-kinase phosphorylates IRS-1 on serine/threonine residues, leading to IRS- 1 degradation. The similar finding was observed in IRS-2 as well as IRS-1. These results may also explain the cellular insulin-resistant state induced by chronic p110CAAX expression.     

Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDP beta-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3 beta at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and beta-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.     

小檗碱改善3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究小檗碱对游离脂肪酸诱导的3T3-L1脂肪细胞胰岛素抵抗的作用，探讨小檗碱改善胰岛素抵抗的分子机制。方法以0．5mmol／L软脂酸诱导3T3-L1脂肪细胞产生胰岛素抵抗，予以小檗碱进行干预，同时以阿司匹林作为阳性对照，用葡萄糖氧化酶法检测培液中的葡萄糖消耗量，以2-脱氧-[^3H]-D-葡萄糖摄入法观察葡萄糖的转运率，用Western印迹检测IκB激酶B（IKKβ）、胰岛素受体底物1（IRS-1）、磷酸肌醇3激酶p85（PI-3K p85），葡萄糖转运子4（Glut4）的蛋白表达和IKKβ 181位丝氨酸（IKKβ Ser181）、IRS-1 307位丝氨酸（IRS-1 Ser307）的磷酸化。结果0．5mmol／L软脂酸作用24h使3T3-L1脂肪细胞葡萄糖消耗降低41％，胰岛素刺激的葡萄糖转运抑制67％，IKKβSer181和IRS-1 Ser307的磷酸化增加，IRS-1和PI-3K p85蛋白的表达减少；同时加入小檗碱或阿司匹林则可逆转上述效应。但软脂酸、小檗碱、阿司匹林对3T3-L1脂肪细胞IKKβ蛋白、Glut4蛋白的表达无明显影响。结论小檗碱可以明显改善游离脂肪酸诱导的胰岛素抵抗，其分子机制可能是通过抑制IKKβ Ser181磷酸化实现的。     

Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.     

Regulation of adiponectin secretion by insulin and amino acids in 3T3-L1 adipocytes

Adiponectin is a fat cell-derived hormone with insulin-sensitizing properties. Low plasma adiponectin levels are associated with insulin resistance as found in obesity. One of the mechanisms for this finding is hampered insulin signaling via phosphatidylinositol 3-kinase (PI3K) with concomitant decreased adiponectin secretion. Because insulin can also stimulate signaling at the level of mammalian target of rapamycin (mTOR) by a mechanism that is dependent on the presence of amino acids, the role of mTOR signaling in adiponectin secretion was studied. In view of the vesicular nature of adiponectin secretion, the role of lysosomes was explored as well. In 3T3-L1 adipocytes, both insulin and amino acids stimulated adiponectin secretion. The stimulation by insulin was PI3K dependent but mTOR independent. The stimulation by amino acids was independent of both PI3K and mTOR. Whereas the effect of insulin via PI3K was mainly on adiponectin secretion from adipocytes, the effect of amino acids was predominantly due to their role as substrates for adiponectin synthesis. The acidotropic agents ammonia and methylamine, but not the lysosomal protease inhibitor leupeptin and the autophagy inhibitor 3-methyladenine, strongly inhibited adiponectin secretion and increased the intracellular adiponectin pool. In conclusion, adiponectin production is substrate driven. Phosphatidylinositol 3-kinase and an acidic lysosomal pH, but not amino acid-mediated mTOR signaling or lysosomal breakdown, are involved in adiponectin secretion.