RESUMEN
The mechanisms whereby prolonged plasma free fatty acids elevation, as found in obesity, causes hepatic insulin resistance are not fully clarified. We herein investigated whether inhibition of p38 mitogen-activated protein kinase (MAPK) prevented hepatic insulin resistance following prolonged lipid infusion. Chronically cannulated rats were subdivided into one of four intravenous (i.v.) treatments that lasted 48 h: Saline (5.5 µl min(-1)), Intralipid plus heparin (IH, 20% Intralipid+20 U ml(-1) heparin; 5.5 µl min(-1)), IH+p38 MAPK inhibitor (SB239063) and SB239063 alone. During the last 2 h of treatment, a hyperinsulinemic (5 mU kg(-1) min(-1)) euglycemic clamp together with [3-(3)H] glucose methodology was carried out to distinguish hepatic from peripheral insulin sensitivity. We found that SB239063 prevented IH-induced hepatic insulin resistance, but not peripheral insulin resistance. SB239063 also prevented IH-induced phosphorylation of activating transcription factor 2 (ATF2), a marker of p38 MAPK activity, in the liver. Moreover, in another lipid infusion model in mice, SB239063 prevented hepatic but not peripheral insulin resistance caused by 48 h combined ethyloleate plus ethylpalmitate infusion. Our results suggest that inhibition of p38 MAPK may be a useful strategy in alleviating hepatic insulin resistance in obesity-associated disorders.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Ácidos Grasos no Esterificados/sangre , Imidazoles/farmacología , Resistencia a la Insulina , Hígado/efectos de los fármacos , Pirimidinas/farmacología , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Factor de Transcripción Activador 2/genética , Factor de Transcripción Activador 2/metabolismo , Animales , Glucemia/metabolismo , Emulsiones/administración & dosificación , Emulsiones/efectos adversos , Técnica de Clampeo de la Glucosa , Heparina/administración & dosificación , Heparina/efectos adversos , Insulina/sangre , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Fosfolípidos/administración & dosificación , Fosfolípidos/efectos adversos , Fosforilación , Ratas , Ratas Wistar , Aceite de Soja/administración & dosificación , Aceite de Soja/efectos adversosRESUMEN
BACKGROUND: We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR). METHODS: Human NSCLC cells, mouse embryonic fibroblasts from wild-type and AMP-activated kinase (AMPK) α1/2-subunit(-/-) embryos (AMPKα1/2(-/-)-MEFs) and NSCLC tumours grafted into Balb/c-nude mice were treated with IR and MET and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays and immunohistochemistry (IHC). RESULTS: Metformin (2.5 µM-5 mM) inhibited proliferation and radio-sensitised NSCLC cells. Metformin (i) activated the ataxia telengiectasia-mutated (ATM)-AMPK-p53/p21(cip1) and inhibited the Akt-mammalian target of rapamycin (mTOR)-eIF4E-binding protein 1 (4EBP1) pathways, (ii) induced G1 cycle arrest and (iii) enhanced apoptosis. ATM inhibition blocked MET and IR activation of AMPK. Non-small cell lung cancer cells with inhibited AMPK and AMPKα1/2(-/-)-MEFs were resistant to the antiproliferative effects of MET and IR. Metformin or IR inhibited xenograft growth and combined treatment enhanced it further than each treatment alone. Ionising radiation and MET induced (i) sustained activation of ATM-AMPK-p53/p21(cip1) and inhibition of Akt-mTOR-4EBP1 pathways in tumours, (ii) reduced expression of angiogenesis and (iii) enhanced expression of apoptosis markers. CONCLUSION: Clinically achievable MET doses inhibit NSCLC cell and tumour growth and sensitise them to IR. Metformin and IR mediate their action through an ATM-AMPK-dependent pathway. Our results suggest that MET can be a clinically useful adjunct to radiotherapy in NSCLC.
Asunto(s)
Adenilato Quinasa/fisiología , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Proteínas de Ciclo Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Neoplasias Pulmonares/radioterapia , Metformina/uso terapéutico , Proteínas Serina-Treonina Quinasas/fisiología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Proteínas Supresoras de Tumor/fisiología , Adenilato Quinasa/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/patología , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Embrión de Mamíferos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metformina/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Proteínas Serina-Treonina Quinasas/metabolismo , Fármacos Sensibilizantes a Radiaciones/farmacología , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Insulina/farmacología , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Músculo Esquelético/enzimología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Proteínas Serina-Treonina Quinasas , Actinas/metabolismo , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Citoesqueleto/metabolismo , Inhibidores Enzimáticos/farmacología , Glucógeno Sintasa Quinasa 3 , Glucógeno Sintasa Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , Músculo Esquelético/efectos de los fármacos , Organofosfonatos/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Fosforilación , Proteína Quinasa C/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/metabolismoRESUMEN
Insulin has diverse effects on cells, including stimulation of glucose transport, gene expression, and alterations of cell morphology. The hormone mediates these effects by activation of signaling pathways which utilize, 1) adaptor molecules such as the insulin receptor substrates (IRS), the Src and collagen homologs (Shc), and the growth factor receptor binding protein 2 (Grb2); 2) lipid kinases such as phosphatidylinositol 3-kinase (PI 3-Kinase); 3) small G proteins; and 4) serine, threonine, and tyrosine kinases. The activation of such signaling molecules by insulin is now well established, but we do not yet fully understand the mechanisms integrating these seemingly diverse pathways. Here, we discuss the involvement of the actin cytoskeleton in the propagation and regulation of insulin signals. In muscle cells in culture, insulin induces a rapid actin filament reorganization that coincides with plasma membrane ruffling and intense accumulation of pinocytotic vesicles. Initiation of these effects of insulin requires an intact actin cytoskeleton and activation of PI 3-kinase. We observed recruitment PI 3-kinase subunits and glucose transporter proteins to regions of reorganized actin. In both muscle and adipose cells, actin disassembly inhibited early insulin-induced events such as recruitment of glucose transporters to the cell surface and enhanced glucose transport. Additionally, actin disassembly inhibited more prolonged effects of insulin, including DNA synthesis and expression of immediate early genes such as c-fos. Intact actin filaments appear to be essential for mediation of early events such as association of Shc with Grb2 in response to insulin, which leads to stimulation of gene expression. Preliminary observations support a role for focal adhesion signaling complexes in insulin action. These observations suggest that the actin cytoskeleton facilitates propagation of the morphological, metabolic, and nuclear effects of insulin by regulating proper subcellular distribution of signaling molecules that participate in the insulin signaling pathway.
Asunto(s)
Actinas/fisiología , Insulina/fisiología , Proteínas Musculares , Transducción de Señal/fisiología , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/efectos de los fármacos , Células Cultivadas , Citocalasina D/farmacología , Proteínas del Citoesqueleto/metabolismo , ADN/biosíntesis , Endocitosis , Proteína-Tirosina Quinasas de Adhesión Focal , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Immunoblotting , Insulina/farmacología , Proteínas de Microfilamentos/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Paxillin , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor de Insulina/fisiologíaRESUMEN
BACKGROUND: High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44). METHODS: Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation. RESULTS: In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1. CONCLUSION: In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.
Asunto(s)
Endotelina-1/farmacología , Mesangio Glomerular/enzimología , Glucosa/farmacología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Activación Enzimática/efectos de los fármacos , Mesangio Glomerular/citología , Masculino , Concentración Osmolar , Factor de Crecimiento Derivado de Plaquetas/farmacología , Ratas , Ratas Sprague-Dawley , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Vanadate and pervanadate (pV) are protein tyrosine phosphatase (PTP) inhibitors that mimic insulin to stimulate glucose transport. To determine whether phosphatidylinositol (PI) 3-kinase is required for vanadate and pV, as it is for insulin, cultured L6 myotubes were treated with vanadate and pV. The two compounds stimulated glucose transport to levels similar to those stimulated by insulin; however, while PI 3-kinase activity and the increase in the lipid products PI 3,4-bisphosphate and PI 3,4,5-trisphosphate were inhibited by wortmannin after stimulation by all three agents--insulin, vanadate, and pV--wortmannin blocked glucose transport stimulated by insulin but not vanadate or pV. Vanadate and pV stimulated the translocation of GLUTs from an intracellular compartment to the plasma membrane; this stimulation was not blocked by wortmannin, but insulin-induced GLUT translocation was inhibited. Similar results were obtained in cultured H9c2 cardiac muscle cells in which wortmannin did not inhibit glucose transport or the vanadate-induced translocation of GLUT4 in c-myc-GLUT4 transfected cells. The ser/thr kinase PKB (Akt/PKB/RAC-PK) is activated by insulin, lies downstream of PI 3-kinase, and has been implicated in signaling of glucose transport. Insulin and pV stimulated PKB activity, and both were inhibited by wortmannin. In contrast, vanadate, at concentrations that maximally stimulated glucose transport, did not significantly increase PKB activity. To determine the potential role of protein kinase C (PKC), L6 cells were incubated chronically with phorbol myristate acetate (PMA) or acutely with the PKC inhibitors calphostin C and bisindolylmaleimide. There was no inhibition of glucose transport stimulation by insulin, vanadate, or pV, and a combination of wortmannin and PKC inhibitors also failed to block the effect of vanadate and pV. In contrast, disassembly of the actin network with cytochalasin D blocked the stimulation of glucose transport by all three agents. In conclusion, vanadate and pV are able to stimulate glucose transport and GLUT translocation by a mechanism independent of PI 3-kinase and PKC. Similar to that by insulin, glucose transport stimulation by vanadate and pV requires the presence of an intact actin network.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Vanadatos/farmacología , Androstadienos/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Citocalasina D/farmacología , Genes myc , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/genética , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Transfección , WortmaninaRESUMEN
The pathophysiologic importance of insulin resistance in diseases such as obesity and diabetes mellitus has led to great interest in defining the mechanism of insulin action as well as the means to overcome the biochemical defects responsible for the resistance. Vanadium compounds have been discovered to mimic many of the metabolic actions of insulin both in vitro and in vivo and improve glycemic control in human subjects with diabetes mellitus. Apart from its direct insulinmimetic actions, we found that vanadate modulates insulin metabolic effects by enhancing insulin sensitivity and prolonging insulin action. All of these actions appear to be related to protein tyrosine phosphatase (PTP) inhibition. However, in contrast to its stimulatory effects, vanadate inhibits basal and insulin-stimulated system A amino acid uptake and cell proliferation. The mechanism of these actions also appears to be related to PTP inhibition, consistent with the multiple roles of PTPs in regulating signal transduction. While the precise biochemical pathway of vanadate action is not yet known, it is clearly different from that of insulin in that the insulin receptor and phosphatidylinositol 3'-kinase do not seem to be essential for vanadate stimulation of glucose uptake and metabolism. The ability of vanadium compounds to 'bypass' defects in insulin action in diseases characterized by insulin resistance and their apparent preferential metabolic versus mitogenic signaling profile make them attractive as potential pharmacological agents.
Asunto(s)
Insulina/metabolismo , Insulina/fisiología , Mitógenos/farmacología , Mitosis/fisiología , Transducción de Señal/efectos de los fármacos , Compuestos de Vanadio/farmacología , Animales , Humanos , Mitosis/efectos de los fármacosRESUMEN
Vanadium is an element found in low concentrations in mammals, for which a function remains to be discovered. Over the past century, vanadium compounds have been suggested anecdotally as therapeutic agents for a variety of diseases. The discovery that vanadate inhibits various enzymes, in particular protein tyrosine phosphatases, and mimics many of the biological actions of insulin suggested a potential role in the therapy of diabetes mellitus. Successful use and an enhancement of insulin sensitivity in rodents and human diabetic subjects, as well as the finding that these agents are capable of stimulating metabolic effects while bypassing the insulin receptor and the early steps in insulin action, target these agents preferentially toward type II diabetes mellitus. Long-term safety remains a major concern, as tissue accumulation and relative nonspecificity of enzyme inhibition may result in adverse effects. Continued research into mechanism of action, consequences of chronic administration, and improvement of specificity is warranted. Regardless of their ultimate success or failure as therapeutic agents, vanadium compounds continue to be useful probes of enzyme structure and function in various biological processes. (Trends Endocrinol Metab 1997;8:51-58). (c) 1997, Elsevier Science Inc.
RESUMEN
The protein tyrosine phosphatase (PTP) inhibitors vanadate and pervanadate (pV) exert insulin-like biologic effects. In cultured differentiated rat L6 skeletal muscle cells, vanadate and pV stimulated 2-deoxy-D-[3H]glucose uptake in a dose- and time-dependent manner. There was no increase in maximum stimulation by additional insulin. In contrast, whereas insulin stimulated [14C]methylaminoisobutyric acid (MeAIB) uptake, basal uptake was inhibited by vanadate and pV. Insulin-stimulated MeAIB uptake was also inhibited in a dose-dependent manner and completely abolished by 5 mM vanadate or 0.1 mM pV. The inhibitory effect on basal MeAIB uptake was associated with a decrease in transporter affinity and a small decrease in maximum transport capacity, whereas the insulin-stimulated increase in maximum transport capacity was completely inhibited. Inhibition of MeAIB uptake by vanadate and pV was not blocked by cycloheximide, and oubain did not inhibit uptake. Vanadate also inhibited amino acid deprivation-stimulated MeAIB uptake. Insulin-stimulated MeAIB uptake was also inhibited in rat hepatoma cells. Thus vanadate and pV mimic insulin to stimulate glucose uptake but inhibit system A amino acid uptake. The relative inhibitory concentrations of vanadate and pV suggest that the mechanism may involve PTP inhibition.
Asunto(s)
Aminoácidos/antagonistas & inhibidores , Glucosa/farmacocinética , Hipoglucemiantes/farmacología , Insulina/farmacología , Vanadatos/farmacología , Aminoácidos/farmacocinética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular , Cicloheximida/farmacología , Desoxiglucosa/farmacocinética , Combinación de Medicamentos , Inhibidores Enzimáticos/farmacología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Ouabaína/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Ratas , beta-Alanina/análogos & derivados , beta-Alanina/farmacocinéticaRESUMEN
Many studies suggest that sulfonylureas (SUs) have direct extrapancreatic actions. The action of gliclazide, a new SU, was examined and compared to that of glyburide in L6 myotubes, a model of skeletal muscle. Gliclazide and glyburide increased 2-deoxy-D-glucose (2DG) uptake in a time- and dose-dependent fashion after 24 h to a maximum of 179% and 202% of the basal value, respectively (P < 0.001). Acute (30-min) insulin (10(-7) M) stimulated 2DG uptake to similar levels (203% of basal), but this effect was absent after maximum stimulation by SU. SU action did not require insulin and was not blocked by the protein synthesis inhibitor cycloheximide. To investigate the mechanism of stimulation of 2DG uptake, cells were fractionated, and total plasma membrane and internal membrane levels of glucose transporter (GLUT) isoforms were determined by immunoblotting. Both drugs significantly increased the total content (1.7-fold) and plasma membrane level (1.8-fold) of GLUT1, with no change in internal membrane. Total content and plasma membrane levels of GLUT4 and GLUT3 did not change or showed a small decrease. We conclude that the stimulation of glucose uptake in L6 cells by gliclazide and glyburide is associated not with a redistribution but, rather, with an increase in the total membrane content and plasma membrane level of GLUT1, which is independent of protein synthesis. These data suggest a novel action of SU to stabilize GLUT1 protein at the plasma membrane.