RESUMEN
Correlative studies have indicated that hyperinsulinemia is present in many individuals with atherosclerosis. Insulin resistance has also been linked to cardiovascular disease. It has proved to be difficult to decipher whether hyperinsulinemia or insulin resistance plays the most important role in the pathogenesis of atherosclerosis and coronary artery disease. In this study, we demonstrate that insulin increases the amount of farnesylated p21Ras in vascular smooth muscle cells (VSMC), thereby augmenting the pool of cellular Ras available for activation by platelet-derived growth factor (PDGF). In VSMC incubated with insulin for 24 h, PDGF's influence on GTP-loading of Ras was significantly increased. Furthermore, in cells preincubated with insulin, PDGF increased thymidine incorporation by 96% as compared with a 44% increase in control cells (a 2-fold increment). Similarly, preincubation of VSMC with insulin increased the ability of PDGF to stimulate gene expression of vascular endothelial growth factor 5- to 8-fold. The potentiating influence of insulin on PDGF action was abrogated in the presence of a farnesyltransferase inhibitor. Thus, the detrimental influence of hyperinsulinemia on the arterial wall may be related to the ability of insulin to augment farnesyltransferase activity and provide greater amounts of farnesylated p21Ras for stimulation by various growth promoting agents.
Asunto(s)
Insulina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Células Cultivadas , Sinergismo Farmacológico , Factores de Crecimiento Endotelial/genética , Factor I del Crecimiento Similar a la Insulina/biosíntesis , Linfocinas/genética , Músculo Liso Vascular/citología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/análisis , Ratas , Ratas Wistar , Porcinos , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Farnesylation of p21Ras by farnesyltransferase (FTase) is obligatory for anchoring p21Ras to the plasma membrane, where it can be activated by growth factors. Insulin significantly stimulates the phosphorylation of the alpha-subunit of FTase (4-fold) and the enzymatic activity of FTase in 3T3-L1 fibroblasts and adipocytes. FTase activity was assessed by the amount of [3H] mevalonate (a precursor of farnesyl) incorporated into p21Ras in vivo and by quantitating the amount of farnesylated p21Ras before and after insulin administration. Insulin-stimulated phosphorylation of the alpha-subunit of FTase in 3T3-L1 fibroblasts and adipocytes was blocked by the mitogen-activated protein/extracellular-signal regulated kinase-kinase inhibitor, PD98059, but not by wortmannin or bisindolylmaleimide. Additionally, PD98059 blocked insulin-stimulated [3H]mevalonic incorporation and farnesylation of unprocessed p21Ras in both cell lines. Furthermore, expression of the dominant negative mutant of p21Ras precluded insulin-stimulated phosphorylation of the FTase alpha-subunit and activation of its enzymatic activity. In contrast, 3T3-L1 fibroblasts, expressing the constitutively active Raf-1, exhibited enhanced phosphorylation of the FTase alpha-subunit. It seems that insulin's effect on the phosphorylation and activation of FTase in both fibroblasts and adipocytes is mediated via the Ras pathway, resulting in a positive feedback augmentation of the cellular pool of farnesylated p21Ras.
Asunto(s)
Transferasas Alquil y Aril/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Insulina/farmacología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células 3T3 , Adipocitos/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Farnesiltransferasa , Fibroblastos/metabolismo , Flavonoides/farmacología , Ácido Mevalónico/antagonistas & inhibidores , Ácido Mevalónico/metabolismo , Ratones , Fosforilación/efectos de los fármacosRESUMEN
Incubation of 3T3-L1 fibroblasts with insulin (10 nM or 100 nM) for 24 or 48 hours resulted in a significant increase in the amount of farnesylated p21Ras with a concomitant increase in the amount of GTP-loaded p21Ras. Cells preincubated with 100 nM insulin for 24 or 48 hours exhibited further 5-8 fold increases in p21Ras.GTP loading in response to an acute (10 minute) challenge with either insulin, EGF, or IGF-1. Effects of hyperinsulinemia were completely abolished by the presence of 1 microM alpha-hydroxyfarnesylphosphonic acid, a potent inhibitor of farnesyltransferase. These novel observations indicate that hyperinsulinemia increases the cellular pool of farnesylated p21Ras and thereby potentiates activation of p21Ras by growth factors.
Asunto(s)
Transferasas Alquil y Aril , Sustancias de Crecimiento/farmacología , Insulina/farmacología , Organofosfonatos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células 3T3 , Animales , Sinergismo Farmacológico , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/farmacología , Farnesol/análogos & derivados , Farnesol/farmacología , Farnesiltransferasa , Guanosina Trifosfato/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Ratones , Compuestos Organofosforados/farmacología , Prenilación de Proteína , Transferasas/antagonistas & inhibidoresRESUMEN
To characterize tissue-specific differences in insulin signaling, we compared the mechanisms of mitogen-activated protein (MAP) kinase activation by insulin in the mitogenically active 3T3-L1 fibroblasts with the metabolically active 3T3-L1 adipocytes. In both cell lines, insulin significantly increased p21(ras).GTP loading (1.5-2-fold) and MAP kinase activity (5-8-fold). Inhibition of Ras farnesylation with lovastatin blocked activation of p21(ras) and Raf-1 kinase in both 3T3-L1 fibroblasts and 3T3-L1 adipocytes. In 3T3-L1 fibroblasts, this was accompanied by an inhibition of the stimulatory effect of insulin on MAP kinase. In contrast, in 3T3-L1 adipocytes, despite an inhibition of activation of p21(ras) and Raf-1 by lovastatin, insulin continued to stimulate MAP kinase activity. Fractionation of the cell lysates on the FPLC Mono-Q column revealed that lovastatin inhibited insulin stimulation of ERK2 (and, to a lesser extent, ERK1) in 3T3-L1 fibroblasts and had no effect on the insulin-stimulated ERK2 in 3T3-L1 adipocytes. These results demonstrate an important distinction between the mechanism of insulin signaling in the metabolically and mitogenically active cells. Insulin activates MAP kinase by the Ras-dependent pathway in the 3T3-L1 fibroblasts and by the Ras-independent pathway in the 3T3-L1 adipocytes.
Asunto(s)
Adipocitos/enzimología , Transferasas Alquil y Aril , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Insulina/farmacología , Organofosfonatos , Animales , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Farnesol/análogos & derivados , Farnesol/farmacología , Farnesiltransferasa , Guanosina Trifosfato/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Lovastatina/farmacología , Ratones , Compuestos Organofosforados/farmacología , Proteína Quinasa C/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Ratas , Transducción de Señal , Transferasas/antagonistas & inhibidoresRESUMEN
In this study, we investigated the influence of the protein kinase C (PKC)-dependent system upon the ability of insulin to stimulate p21(ras).GTP loading in 3T3-L1 adipocytes. Activation of PKC by 12-0-tetradecanoylphorbol-13-acetate (TPA) did not affect the basal amount of p21(ras).GTP but significantly reduced insulin-induced increases in p21(ras).GTP. This reduction was due to inhibition of the insulin's ability to stimulate guanine nucleotide exchange activity of Sos in cells incubated with 100 nM TPA for either 30 min or 3 h. TPA had no effect on basal activity of Sos. Depletion of PKC by an 18-h incubation with TPA or inhibition by bisindolylmaleimide resulted in profound inhibition of the insulin-induced p21(ras).GTP loading. In contrast to PKC activation, removal of PKC did not influence Sos activity but resulted in a 2-fold stimulation of GTPase activating protein (GAP). This effect of PKC depletion is unique to 3T3-L1 adipocytes and was not observed in either 3T3-L1 fibroblasts or Rat-1 fibroblasts. Thus, it appears that in 3T3-L1 adipocytes, PKC has a constitutive inhibitory effect on GAP that permits insulin to activate Sos and p21(ras). Removal of this inhibitory influence activates GAP and reduces insulin-stimulated p21(ras).GTP loading.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteína Quinasa C/fisiología , Receptor de Insulina/fisiología , Células 3T3 , Animales , Células Cultivadas , Proteínas Activadoras de GTPasa , Guanosina Trifosfato/metabolismo , Ratones , Proteína Quinasa C/antagonistas & inhibidores , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/fisiología , Ratas , Proteínas Recombinantes , Sistemas de Mensajero Secundario , Transducción de Señal , Proteínas Son Of Sevenless , Proteínas Activadoras de ras GTPasaRESUMEN
Insulin-stimulated activity of Raf-1 kinase was examined in Rat-1 fibroblasts transfected with wild-type and mutant human insulin receptors. Insulin stimulated Raf-1 binding to p21Ras in HIRc (wild-type), delta CT (insulin receptor lacking a 43-amino acid C-terminal domain), and Y/F2 (tyrosine 1316 and 1322 replaced by phenylalanine) cells. Despite equal binding to p21Ras, the activity of Raf-1 kinase (measured by phosphorylation of its downstream substrate, mitogen-activated protein/extracellular receptor kinase (MEK) was significantly reduced in the delta CT cells. As an association of Raf-1 with p21Ras does not activate Raf-1 kinase, but merely targets Raf-1 to the plasma membrane, we examined the binding of Raf-1 to 14-3-3 proteins and to the insulin receptor itself. Raf-1 was detected in both 14-3-3 and insulin receptor immunoprecipitates. Association of Raf-1 with either 14-3-3 protein or insulin receptor was not influenced by insulin and was similar in all control and insulin-treated cell lines. These results indicate that the delta CT cells are deficient in stimulating Raf-1 activity despite normal binding of Raf-1 to p21Ras. Thus, an unidentified mechanism of Raf-1 activation at the plasma membrane must be impaired in these cells.
Asunto(s)
Insulina/farmacología , Proteínas Quinasas/metabolismo , Receptor de Insulina/metabolismo , Células 3T3 , Animales , Activación Enzimática/efectos de los fármacos , Guanosina Trifosfato/metabolismo , Humanos , Técnicas de Inmunoadsorción , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-raf , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.
Asunto(s)
Adipocitos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Células 3T3 , Animales , Western Blotting , Células CHO , Cromonas/farmacología , Cricetinae , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fibroblastos , Proteínas Activadoras de GTPasa , Glucosa/metabolismo , Humanos , Immunoblotting , Insulina/farmacología , Ratones , Mitógenos/farmacología , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Ratas , Proteínas Activadoras de ras GTPasaRESUMEN
Insulin's interaction with its receptor initiates a multitude of cellular effects on metabolism, growth, and differentiation. We recently described an insulin-mediated inhibition of nuclear protein phosphatase 2A (PP-2A), which is associated with an increase in phosphorylation of the transcription factor cAMP response element-binding protein. To clarify the role of nuclear PP-2A inhibition in the insulin signaling cascade, we examined the regulation of this phosphatase activity by insulin in Rat-1 fibroblasts overexpressing normal (HIRc) or mutant human insulin receptors (delta CT cells, deletion of a 43-amino acid C-terminal domain). The delta CT cells represent an excellent model of impaired metabolic and intact mitogenic action of insulin. Insulin inhibited nuclear PP-2A activity and enhanced cAMP response element-binding protein phosphorylation in HIRc cells, but not in delta CT cells. The delta CT cells exhibited normal ras activation and blunted mitogen-activating protein kinase phosphorylation and activation in response to insulin (16-fold in HIRc cells vs. 3-fold in delta CT cells), indicating that the mitogen-activating protein kinase pathway is important for the regulation of nuclear PP-2A activity by insulin. We conclude that insulin inhibits nuclear PP-2A activity, and that the carboxy-terminal domain of the insulin receptor is important for this effect.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas de Unión al ADN , Insulina/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Receptor de Insulina/metabolismo , Factor de Transcripción Activador 1 , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , Proteína Fosfatasa 2 , Ratas , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/genética , Eliminación de Secuencia , Factores de Transcripción/metabolismo , TransfecciónRESUMEN
To evaluate the role of the "Ras pathway" in mediating metabolic signaling by insulin, we employed lovastatin to exhibit isoprenilation of Ras proteins in Rat-1 fibroblasts transfected with human insulin receptors (HIRc cells) and in differentiated 3T3-L1 adipocytes. Lovastatin blocked an ability of insulin to activate p21ras and mitogen-activated protein kinase. Lovastatin also significantly (p < 0.01) reduced insulin effects on thymidine incorporation and glucose incorporation into glycogen. Nevertheless, an effect of insulin on glucose uptake remained unaffected. It appears that in contrast to its mitogenic action and to its effect on glycogenesis, an effect of insulin on glucose uptake does not require p21ras activation.