RESUMEN
Beta3-adrenergic agonists are well-recognited to promote lipid mobilisation and adipose tissue remodeling in rodents, leading to multilocular fat cells enriched in mitochondria. However, effects of beta3-adrenergic agonists on glucose transport are still controversial. In this work, we studied in white adipose tissue (WAT) the influence of sustained beta3-adrenergic stimulation on the glucose transport and on the mitochondrial monoamine oxidase (MAO) activity. As one-week administration of CL 316243 (CL, 1 mg/kg/d) induces beta-adrenergic desensitization in rat but not in guinea pig adipocytes, attention was paid to compare these models. When expressing glucose uptake as nmoles of 2-deoxyglucose/100 mg cell lipids, maximally stimulated uptake was increased in adipocytes of WAT from treated rats but not from treated guinea pigs. However, basal hexose uptake was also increased in CL-treated rats and, as a consequence, the dose-dependent curves for insulin stimulation were similar in control and CL-treated rats when expressed as fold increase over basal. Insulin-induced lipogenesis was unchanged in rat or guinea pig adipocytes after CL-treatment. The glucose carriers GLUT4 and corresponding mRNA were increased in subcutaneous WAT or in brown adipose tissue (BAT) but not in visceral WAT or muscles of CL-treated rats. There was an increase of MAO activity in WAT and BAT, but not in liver, of CL-treated rats while no change was detected in guinea pigs. These findings show that only rat adipocytes, which are beta3-adrenergic-responsive, respond to chronic beta3-AR agonist by an increase of GLUT4 content and MAO activity, despite a desensitization of all beta-adrenoceptor subtypes.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacología , Dioxoles/farmacología , Transportador de Glucosa de Tipo 4/biosíntesis , Glucosa/metabolismo , Monoaminooxidasa/metabolismo , Adipocitos Blancos/efectos de los fármacos , Tejido Adiposo/patología , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Pardo/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Cobayas , Insulina/fisiología , Grasa Intraabdominal/metabolismo , Masculino , Ratas , Grasa Subcutánea/metabolismoRESUMEN
Plasma level of the protein VAP-1/SSAO (Vascular Adhesion Protein-1/Semicarbazide-Sensitive Amine Oxidase) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFalpha in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the VAP-1/SSAO membrane form. While TNFalpha produced a decrease on VAP-1/SSAO membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma VAP-1/SSAO levels observed in diabetes.
Asunto(s)
Adipocitos/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Moléculas de Adhesión Celular/metabolismo , Isoproterenol/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3-L1 , Adipocitos/enzimología , Adipocitos/metabolismo , Amina Oxidasa (conteniendo Cobre)/análisis , Animales , Western Blotting , Técnicas de Cultivo de Célula , Fraccionamiento Celular , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Ratones , SolubilidadRESUMEN
Plasma level of the protein SSAO/VAP-1 (samicarbazide-sensitive amine oxidase / vascular-adhesion protein-1) is increased in diabetes and/or obesity and may be related to vascular complications associated to these pathologies. The aim of this work was to complete a preceding study where we described the role played by some hormones or metabolites, implicated in diabetes and/or obesity, in the regulation of the release of VAP-1/SSAO by 3T3-L1 adipocytes. Here we focused on the previously observed effect produced by TNFa in the release of VAP-1/SSAO and studied the effect of a beta-adrenergic compound, isoproterenol. Both compounds stimulated the release of VAP-1/SSAO to the culture medium but had a different effect on the SSAO/VAP-1 membrane form. While TNFa produced a decrease on SSAO/VAP-1 membrane form content, isoproterenol did not modify it. We thus observed two different ways of regulation of the release of SSAO/VAP-1 by 3T3-L1 adipocytes by metabolites implicated in diabetes and adipose tissue physiopathology. Our work permits a better understanding of this increased plasma SSAO/VAP-1 levels observed in diabetes
Los niveles plasmáticos de la proteina SSAO/VAP-1 están aumentados en la diabetes y la obesidad, lo que podría estar relacionado con las complicaciones vasculares asociadas a estas patologías. En continuidad con trabajos anteriores acerca del papel de algunas hormonas o metabolitos, implicados en la diabetes y obesidad. Se estudia en este trabajo el efecto producido por el TNFa y del agonista beta-adrenérgico, isoproterenol en la regulación de la liberación de VAP-1/SSAO por adipocitos 3T3-L1. Ambos compuestos estimularon la liberación de VAP-1/SSAO al medio de cultivo, pero tuvieron un efecto diferente sobre la isoforma ligada a la membrana de SSAO/VAP-1. Así, mientras que el TNFa produjo una disminución significativa en la actividad SSAO/VAP-1 ligada a la membrana, no se modificó por el isoproterenol. Además, observamos dos maneras diferentes de regulación de la liberación de SSAO/VAP-1 por adipocitos 3T3-L1 a través de metabolitos implicados en diabetes y fisiopatología del tejido adiposo. Nuestro trabajo permite un mejor entendimiento de estos niveles plasmáticos aumentados de SSAO/VAP-1 observados en diabetes
Asunto(s)
Animales , Ratones , Adipocitos , Agonistas Adrenérgicos beta/farmacocinética , Amina Oxidasa (conteniendo Cobre) , Moléculas de Adhesión Celular , Isoproterenol/farmacología , Western Blotting , Moléculas de Adhesión Celular/metabolismo , Fraccionamiento Celular , Células 3T3-L1 , Adipocitos/enzimología , Adipocitos/metabolismo , Amina Oxidasa (conteniendo Cobre)/análisis , Amina Oxidasa (conteniendo Cobre)/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , SolubilidadRESUMEN
Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO such as benzylamine in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in mouse 3T3-L1 adipocytes and in isolated rat adipocytes. Here we examined whether this combination of molecules also stimulates glucose transport in adipocytes from streptozotocin-induced diabetic rats and from Goto-Kakizaki diabetic rats. As previously reported, adipocytes obtained from streptozotocin-induced diabetic rats, showed a reduced stimulation of glucose transport in response to insulin. Under these conditions, the combination of benzylamine and vanadate caused a marked stimulation of glucose transport that was similar to the stimulation detected in control adipocytes. Adipocytes isolated from Goto-Kakizaki diabetic rats also showed a defective response to insulin; however, acute incubation in the presence of benzylamine and vanadate stimulated glucose transport in these cells to the same extent than in adipocytes from non-diabetic rats. These data indicate that adipocytes obtained from two different models of animal diabetes do not show resistance to the activation of glucose transport by SSAO activity, which is in contrast to the well reported resistance to insulin action. It seems to suggest that SSAO activity in combination with vanadate triggers a glucose transport-activating intracellular pathway that remains intact in the diabetic state. Further, our data support the view that the combination of benzylamine and vanadate could be an effective therapy in diabetes.
Asunto(s)
Adipocitos/metabolismo , Amina Oxidasa (conteniendo Cobre)/metabolismo , Bencilaminas/farmacología , Desoxiglucosa/metabolismo , Diabetes Mellitus Experimental/metabolismo , Vanadatos/farmacología , Adipocitos/enzimología , Animales , Transporte Biológico/efectos de los fármacos , Diabetes Mellitus Experimental/enzimología , Insulina/farmacología , Masculino , Ratas , Ratas Mutantes , Ratas WistarRESUMEN
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.
Asunto(s)
Tejido Adiposo/citología , Tejido Adiposo/efectos de los fármacos , Bencilaminas/farmacología , Insulina/farmacología , Tiramina/farmacología , 1-Metil-3-Isobutilxantina/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Células 3T3 , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Amina Oxidasa (conteniendo Cobre)/antagonistas & inhibidores , Amina Oxidasa (conteniendo Cobre)/metabolismo , Animales , Bencilaminas/antagonistas & inhibidores , Bencilaminas/metabolismo , Materiales Biomiméticos/metabolismo , Materiales Biomiméticos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Dexametasona/metabolismo , Dexametasona/farmacología , Insulina/metabolismo , Ratones , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Triglicéridos/química , Triglicéridos/metabolismo , Tiramina/antagonistas & inhibidores , Tiramina/metabolismoRESUMEN
Semicarbazide-sensitive amine oxidase (SSAO) is highly expressed in adipose cells, and substrates of SSAO, such as benzylamine, in combination with low concentrations of vanadate strongly stimulate glucose transport and GLUT4 recruitment in 3T3-L1 and rat adipocytes. Here we examined whether acute and chronic administration of benzylamine and vanadate in vivo enhances glucose tolerance and reduces hyperglycemia in diabetic rats. Acute intravenous administration of these drugs enhanced glucose tolerance in nondiabetic rats and in streptozotocin (STZ)-induced diabetic rats. This occurred in the absence of changes in plasma insulin concentrations. However, the administration of benzylamine or vanadate alone did not improve glucose tolerance. The improvement caused by benzylamine plus vanadate was abolished when rats were pretreated with the SSAO-inhibitor semicarbazide. Chronic administration of benzylamine and vanadate exerted potent antidiabetic effects in STZ-induced diabetic rats. Although daily administration of vanadate alone (50 and 25 micromol x kg(-1) x day(-1) i.p.) for 2 weeks had little or no effect on glycemia, vanadate plus benzylamine reduced hyperglycemia in diabetic rats, enhanced basal and insulin-stimulated glucose transport, and upregulated GLUT4 expression in isolated adipocytes. In all, our results substantiated that acute and chronic administration of benzylamine with low dosages of vanadate have potent antidiabetic effects in rats.
Asunto(s)
Bencilaminas/uso terapéutico , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/complicaciones , Intolerancia a la Glucosa/tratamiento farmacológico , Intolerancia a la Glucosa/etiología , Hiperglucemia/tratamiento farmacológico , Proteínas Musculares , Vanadatos/administración & dosificación , Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Animales , Bencilaminas/administración & dosificación , Bencilaminas/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Quimioterapia Combinada , Glucosa/fisiología , Transportador de Glucosa de Tipo 4 , Masculino , Proteínas de Transporte de Monosacáridos/metabolismo , Ratas , Ratas Wistar , Semicarbacidas/farmacología , Vanadatos/antagonistas & inhibidores , Vanadatos/uso terapéuticoRESUMEN
We have previously reported that substrates of monoamine oxidase (MAO) and semicarbazide-sensitive amine oxidase (SSAO) exert short-term insulin-like effects in rat adipocytes, such as stimulation of glucose transport. In the present work, we studied whether these substrates could also mimic long-term actions of insulin. Adipose differentiation of 3T3 F442A cells, which is highly insulin-dependent, served as a model to test the effects of sustained administration of amine oxidase substrates. Daily treatment of confluent cells with 0.75 mM tyramine (a substrate of MAO and SSAO) or benzylamine (a substrate of SSAO) over 1 week caused the acquisition of typical adipocyte morphology. The stimulation of protein synthesis and triacylglycerol accumulation caused by tyramine or benzylamine reached one half of that promoted by insulin. This effect was insensitive to pargyline (an MAO inhibitor), but was inhibited by semicarbazide (an SSAO inhibitor) and by N-acetylcysteine (an antioxidant agent), suggesting the involvement of the H(2)O(2) generated during SSAO-dependent amine oxidation. Chronic administration of amine oxidase substrates also induced the emergence of adipose conversion markers, such as aP2, glycerol-3-phosphate dehydrogenase, the glucose transporter GLUT4, and SSAO itself. Moreover, cells treated with amines acquired the same insulin sensitivity regarding glucose transport as adipocytes classically differentiated with insulin. In all, most of the adipogenic effects of amines were additive to insulin. Our data reveal that amine oxidase substrates partially mimic the adipogenic effect of insulin in cultured preadipocytes. Furthermore, they suggest that SSAO not only represents a novel late marker of adipogenesis, but could also be directly involved in the triggering of terminal adipocyte differentiation.
Asunto(s)
Adipocitos/efectos de los fármacos , Amina Oxidasa (conteniendo Cobre)/metabolismo , Bencilaminas/farmacología , Diferenciación Celular/efectos de los fármacos , Insulina/farmacología , Tiramina/farmacología , Células 3T3 , Acetilcisteína/farmacología , Adipocitos/citología , Animales , Antioxidantes/farmacología , Bencilaminas/metabolismo , Glucosa/metabolismo , Ratones , Imitación Molecular , Especificidad por Sustrato , Tiramina/metabolismoRESUMEN
It has been shown that the combination of benzylamine or tyramine and low concentrations of vanadate markedly stimulates glucose transport in rat adipocytes by a mechanism that requires semicarbazide-sensitive amine oxidase (SSAO) activity and H(2)O(2) formation. Here we have further analysed the insulin-like effects of the combination of SSAO substrates and vanadate and we have studied the signal-transduction pathway activated in rat adipocytes. We found that several SSAO substrates (benzylamine, tyramine, methylamine, n-decylamine, histamine, tryptamine or beta-phenylethylamine), in combination with low concentrations of vanadate, stimulate glucose transport in isolated rat adipocytes. Furthermore, SSAO substrates together with vanadate stimulated the recruitment of GLUT4 to the cell surface in isolated rat adipocytes. Benzylamine plus vanadate also stimulated glucose transport and GLUT4 translocation in 3T3-L1 adipocytes. Benzylamine or tyramine in combination with vanadate potently stimulated the tyrosine phosphorylation of both insulin receptor substrate (IRS)-1 and IRS-3. In contrast, benzylamine and vanadate caused only a weak stimulation of insulin receptor kinase. Benzylamine or tyramine in combination with vanadate also stimulated phosphoinositide 3-kinase activity; wortmannin abolished the stimulatory effect of benzylamine and vanadate on glucose transport in adipose cells. Furthermore, the administration of benzylamine and vanadate in vivo caused a rapid lowering of plasma glucose levels, which took place in the absence of alterations in plasma insulin. On the basis of these results we propose that SSAO activity regulates glucose transport in adipocytes. SSAO oxidative activity stimulates glucose transport via the translocation of GLUT4 carriers to the cell surface, resulting from a potent tyrosine phosphorylation of IRS-1 and IRS-3 and phosphoinositide 3-kinase activation. Our results also indicate that substrates of SSAO might regulate glucose disposal in vivo.
Asunto(s)
Adipocitos/efectos de los fármacos , Amina Oxidasa (conteniendo Cobre)/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Tirosina/metabolismo , Vanadatos/farmacología , Células 3T3 , Adipocitos/enzimología , Adipocitos/metabolismo , Animales , Bencilaminas/administración & dosificación , Bencilaminas/farmacología , Transportador de Glucosa de Tipo 4 , Proteínas Sustrato del Receptor de Insulina , Masculino , Ratones , Fosforilación , Ratas , Ratas Wistar , Especificidad por Sustrato , Vanadatos/administración & dosificaciónRESUMEN
Glucose uptake is autoregulated in a variety of cell types and it is thought that glucose transport is the major step that is subjected to control by sugar availability. Here, we examined the effect of high glucose concentrations on the rate of glucose uptake by human ECV-304 umbilical vein-derived endothelial cells. A rise in the glucose concentration in the medium led a dose-dependent decrease in the rate of 2-deoxyglucose uptake. The effect of high glucose was independent of protein synthesis and the time-course analysis indicated that it was relatively slow. The effect was not due to inhibition of glucose transport since neither the expression nor the subcellular distribution of the major glucose transporter GLUT1, nor the rate of 3-O-methylglucose uptake was affected. The total in vitro assayed hexokinase activity and the expression of hexokinase-I were similar in cells treated or not with high concentrations of glucose. In contrast, exposure of cells to a high glucose concentration caused a marked decrease in phosphorylated 2-deoxyglucose/free 2-deoxyglucose ratio. This suggests the existence of alterations in the rate of in vivo glucose phosphorylation in response to high glucose. In summary, we conclude that ECV304 human endothelial cells reduce glucose utilization in response to enhanced levels of glucose in the medium by inhibiting the rate of glucose phosphorylation, rather than by blocking glucose transport. This suggests a novel metabolic effect of high glucose on cellular glucose utilization.
Asunto(s)
Endotelio Vascular/metabolismo , Glucosa/farmacología , Transporte Biológico , Línea Celular , Cicloheximida , Glucosa/metabolismo , Hexoquinasa/metabolismo , Humanos , Fosforilación , Biosíntesis de Proteínas , Factores de TiempoRESUMEN
Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.
Asunto(s)
Proteínas I-kappa B , Factor II del Crecimiento Similar a la Insulina/farmacología , Músculo Esquelético/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Arginina/farmacología , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas Musculares/metabolismo , Músculo Esquelético/enzimología , Inhibidor NF-kappaB alfa , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II , Nitroarginina/farmacología , Prolina/análogos & derivados , Prolina/farmacología , Ratas , Transducción de Señal , Tiocarbamatos/farmacologíaRESUMEN
Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal.
Asunto(s)
Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Miocardio/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Células Cultivadas , Proteínas de Unión al ADN/análisis , Regulación del Desarrollo de la Expresión Génica , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Corazón/embriología , Corazón/crecimiento & desarrollo , Proteínas Nucleares/análisis , Regiones Promotoras Genéticas , ARN Mensajero/genética , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Factor de Transcripción Sp1/genética , Transcripción GenéticaRESUMEN
It has been reported that benfluorex ameliorates the insulin resistance induced by high-fat feeding when its administration is initiated at the same time as the change in diet. Here we have examined whether benfluorex reverses insulin resistance when this is established in middle-aged rats chronically maintained on a high-fat diet. Untreated 12-month-old rats that had been subjected to a high-fat diet for the last 6 months showed markedly lower insulin-induced stimulation of 2-deoxyglucose uptake by strips of soleus muscle and a reduced expression of GLUT4 glucose carriers in skeletal muscle. However, animals subjected to the same protocol but treated with benfluorex during the last month of high-fat feeding showed marked improvement in insulin-stimulated glucose transport by soleus muscle. Benfluorex treatment caused a substantial increase in the content of GLUT4 protein in white muscle; however, GLUT4 levels in red muscle remained low. Our results indicate: (i) that benfluorex treatment in middle-aged rats reverses the insulin resistance induced by high-fat feeding in soleus muscle; (ii) benfluorex is active even when it is administered once the insulin-resistant state is already established; (iii) reversion of muscle insulin resistance by benfluorex can occur independently of modifications in GLUT4 protein expression.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Fenfluramina/análogos & derivados , Resistencia a la Insulina/fisiología , Proteínas Musculares , Músculo Esquelético/efectos de los fármacos , Envejecimiento , Animales , Transporte Biológico/efectos de los fármacos , Glucemia/metabolismo , Northern Blotting , Peso Corporal , Desoxiglucosa/metabolismo , Ingestión de Energía , Fenfluramina/farmacología , Transportador de Glucosa de Tipo 4 , Hipolipemiantes/farmacología , Insulina/sangre , Insulina/farmacología , Masculino , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Insulin-like growth factors (IGFs) are potent stimulators of muscle differentiation, and phosphatidylinositol 3-kinase (PI 3-kinase) is an essential second messenger in this process. Little is known about the downstream effectors of the IGF/PI 3-kinase myogenic cascade, and contradictory observations have been reported concerning the involvement of p70 S6 kinase. In an attempt to clarify the role of p70 S6 kinase in myogenesis, here we have studied the effect of rapamycin on rat, mouse, and human skeletal muscle cell differentiation. Both insulin and IGF-II activated p70 S6 kinase in rat L6E9 and mouse Sol8 myoblasts, which was markedly inhibited at 1 ng/ml rapamycin concentrations. Consistent with previous observations in a variety of cell lines, rapamycin exerted a potent inhibitory effect on L6E9 and Sol8 serum-induced myoblast proliferation. In contrast, even at high concentrations (20 ng/ml), rapamycin had no effect on IGF-II-induced proliferation or differentiation. Indeed, neither the morphological differentiation, as assessed by myotube formation, nor the expression of muscle-specific markers such as myogenin, myosin heavy chain, or GLUT4 (glucose transporter-4) glucose carriers was altered by rapamycin. Moreover, here we extended our studies on IGF-II-induced myogenesis to human myoblasts derived from skeletal muscle biopsies. We show that, as observed for rat and mouse muscle cells, human myoblasts can be induced to form multinucleated myotubes in the presence of exogenous IGF-II. Moreover, IGF-II-induced human myotube formation was totally blocked by LY294002, a specific PI 3-kinase inhibitor, but remained unaffected in the presence of rapamycin.
Asunto(s)
Factor II del Crecimiento Similar a la Insulina/farmacología , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Proteínas Quinasas S6 Ribosómicas/metabolismo , Animales , Fenómenos Fisiológicos Sanguíneos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Cromonas/farmacología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Morfolinas/farmacología , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiología , Ratas , Proteínas Quinasas S6 Ribosómicas/antagonistas & inhibidores , Sirolimus/farmacologíaRESUMEN
4F2hc is an almost ubiquitous transmembrane protein in mammalian cells; upon expression in Xenopus laevis oocytes, it induces amino acid transport with characteristics of system y+L. Indirect evidence fostered speculation that function requires the association of 4F2hc with another protein endogenous to oocytes and native tissues. We show that expression of system y+L-like amino acid transport activity by 4F2hc in oocytes is limited by an endogenous factor and that direct covalent modification of external cysteine residue(s) of an oocyte membrane protein blocks system y+L/4F2hc transport activity, based on the following. 1) Induction of system y+L-like activity saturates at very low doses of human 4F2hc cRNA (0.1 ng/oocyte). This saturation occurs with very low expression of 4F2hc at the oocyte surface, and further increased expression of the protein at the cell surface does not result in higher induction of system y+L-like activity. 2) Human 4F2hc contains only two cysteine residues (C109 and C330). We mutated these residues, singly and in combination, to serine (C109S; CS1, C330S; CS2 and C109S-C330S, Cys-less). Mutation CS2 had no effect on the expressed system y+L-like transport activity, whereas C109S-containing mutants (CS1 and Cys-less) retained only partial y+L-like transport activity (30 to 50% of wild type). 3) Hg2+, the organic mercury compounds pCMB, and the membrane-impermeant pCMBS almost completely inactivated system y+L-like induced by human 4F2hc wild type and all the mutants studied. This was reversed by ss-mercaptoethanol, indicating that external cysteine residue(s) are the target of this inactivation. 4) Sensitivity to Hg2+ inactivation is increased by pretreatment of oocytes with ss-mercaptoethanol or in the C109S-containing mutants (CS1 and Cys-less). The increased Hg2+ reactivity of C109S-containing mutants supports the possibility that C109 may be linked by a disulfide bond to the Hg2+-targeted cysteine residue of the associated protein. These results indicate that 4F2hc is intimately associated with a membrane oocyte protein for the expression of system y+L amino acid transport activity. To our knowledge, this is the first direct evidence for a heteromultimeric protein structure of an organic solute carrier in mammals.
Asunto(s)
Aminoácidos/metabolismo , Antígenos CD/química , Proteínas Portadoras/química , 4-Cloromercuribencenosulfonato/farmacología , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Transporte Biológico/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cisteína/química , Cisteína/metabolismo , Proteína-1 Reguladora de Fusión , Regulación de la Expresión Génica , Humanos , Sustancias Macromoleculares , Mercaptoetanol/farmacología , Cloruro de Mercurio/farmacología , Mutagénesis Sitio-Dirigida , Oocitos , Conformación Proteica , Procesamiento Proteico-Postraduccional , ARN/genética , Proteínas Recombinantes de Fusión/química , Xenopus laevis , Ácido p-Cloromercuribenzoico/farmacologíaRESUMEN
The previous characterization of an abundant population of non-adrenergic imidazoline-I2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle. Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.
Asunto(s)
Adipocitos/metabolismo , Amina Oxidasa (conteniendo Cobre) , Carcinógenos/farmacología , Membrana Celular/metabolismo , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Semicarbacidas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Transportador de Glucosa de Tipo 4 , Masculino , Microsomas/metabolismo , Ratas , Ratas WistarRESUMEN
Nonadrenergic imidazoline I2-binding sites colocalize with monoamine oxidase (MAO) in various tissues. As white adipocytes from various species have been reported to be very rich in I2-sites, the authors consider whether these cells show a substantial MAO activity and explore its functional role. Oxidation of [14C]tyramine by rat adipocyte membranes was dependent on both MAO and semicarbazide-sensitive amine oxidase (SSAO). Tyramine oxidation was identical in membranes and in intact adipocytes (Vmax: 11-12 nmol/min/mg protein). A similar effect of MAO and SSAO inhibitors was obtained in both the intact cells and the membranes: half of the activity was sensitive to semicarbazide and the other half more easily inhibited by MAO-A than by MAO-B inhibitors. As the reaction catalyzed by amine oxidases generates H2O2, which mimicks certain insulin effects in adipocytes, we tested whether tyramine oxidation influences glucose transport in adipocytes. One mM tyramine weakly stimulated glucose transport. A clear potentiation of tyramine effect occurred in the presence of 0.1 mM vanadate, ineffective by itself, reaching half-maximal insulin stimulation. This stimulation was sensitive to MAO and SSAO inhibitors and to catalase. The 5-fold activation of glucose transport was accompanied by translocation of GLUT4 transporters to the plasma membrane. This shows that tyramine is readily oxidized by adipocytes and potentiates the effects of vanadium on glucose transport through release of hydrogen peroxide. The role of the amine oxidases, which are highly expressed in adipocytes, allows them to be considered as more than mere scavengers of circulating amines.
Asunto(s)
Adipocitos/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Amina Oxidasa (conteniendo Cobre) , Glucosa/metabolismo , Proteínas Musculares , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Tiramina/farmacología , Vanadatos/farmacología , Agonistas alfa-Adrenérgicos/metabolismo , Animales , Transporte Biológico , Sinergismo Farmacológico , Transportador de Glucosa de Tipo 4 , Peróxido de Hidrógeno/metabolismo , Masculino , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Proteínas de Transporte de Monosacáridos/metabolismo , Ratas , Ratas Wistar , Tiramina/metabolismo , Vanadatos/metabolismoRESUMEN
Phosphatidylinositol 3 (PI 3)-kinases are potently inhibited by two structurally unrelated membrane-permeant reagents: wortmannin and LY294002. By using these two inhibitors we first suggested the involvement of a PI 3-kinase activity in muscle cell differentiation. However, several reports have described that these compounds are not as selective for PI 3-kinase activity as assumed. Here we show that LY294002 blocks the myogenic pathway elicited by insulin-like growth factors (IGFs), and we confirm the specific involvement of PI 3-kinase in IGF-induced myogenesis by overexpressing in L6E9 myoblasts a dominant negative p85 PI 3-kinase-regulatory subunit (L6E9-delta p85). IGF-I, des(1-3)IGF-I, or IGF-II induced L6E9 skeletal muscle cell differentiation as measured by myotube formation, myogenin gene expression, and GLUT4 glucose carrier induction. The addition of LY294002 to the differentiation medium totally inhibited these IGF-induced myogenic events without altering the expression of a non-muscle-specific protein, beta1-integrin. Independent clones of L6E9 myoblasts expressing a dominant negative mutant of the p85-regulatory subunit (delta p85) showed markedly impaired glucose transport activity and formation of p85/p110 complexes in response to insulin, consistent with the inhibition of PI 3-kinase activity. IGF-induced myogenic parameters in L6E9-delta p85 cells, ie. cell fusion and myogenin gene and GLUT4 expression, were severely impaired compared with parental cells or L6E9 cells expressing wild-type p85. In all, data presented here indicate that PI 3-kinase is essential for IGF-induced muscle differentiation and that the specific PI 3-kinase subclass involved in myogenesis is the heterodimeric p85-p110 enzyme.
Asunto(s)
Proteínas Musculares , Músculo Esquelético/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal , Somatomedinas/fisiología , Animales , Diferenciación Celular/genética , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/genética , Cromonas/farmacología , Transportador de Glucosa de Tipo 4 , Isoenzimas/biosíntesis , Microtúbulos/genética , Microtúbulos/fisiología , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas de Transporte de Monosacáridos/genética , Morfolinas/farmacología , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Miogenina/biosíntesis , Miogenina/genética , Fosfatidilinositol 3-Quinasas/biosíntesis , Fosfatidilinositol 3-Quinasas/genética , Ratas , Transducción de Señal/genética , Somatomedinas/genéticaRESUMEN
Cardiac muscle is characterized by a high rate of glucose consumption. In the absence of insulin, glucose transport into cardiomyocytes limits the rate of glucose utilization and therefore it is important to understand the regulation of glucose transporters. Cardiac muscle cells express 2 distinct glucose transporters, GLUT4 and GLUT1; although GLUT4 is quantitatively the more important glucose transporter expressed in heart, GLUT1 is also expressed at a substantial level. In isolated rat cardiomyocytes, insulin acutely stimulates glucose transport and translocates both GLUT4 and GLUT1 from an intracellular site to the cell surface. Recent evidence indicates the existence of at least 2 distinct intracellular membrane populations enriched in GLUT4 with a different protein composition. Elucidation of the intracellular location of these 2 GLUT4 vesicle pools in cardiac myocytes, their role in GLUT4 trafficking, and their relation to insulin-induced GLUT4 translocation needs to be addressed.
Asunto(s)
Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Miocardio/metabolismo , Animales , Transporte Biológico Activo , Diabetes Mellitus Experimental/metabolismo , Ayuno/metabolismo , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Miocardio/citología , ARN Mensajero/análisis , Ratas , Hormonas Tiroideas/fisiologíaRESUMEN
A major objective for the understanding of muscle glucose disposal is the elucidation of the intracellular trafficking pathway of GLUT4 glucose carriers in the muscle fiber. In this report, we provide functional and biochemical characterization of two distinct intracellular GLUT4 vesicle pools obtained from rat skeletal muscle. The two pools showed a differential response to insulin; thus, one showed a marked decrease in GLUT4 levels but the other did not. They also showed a markedly different protein composition as detected by quantitative vesicle immunoisolation analysis. The GLUT4 pool showing no response to insulin contained SCAMP proteins and the vSNARE proteins VAMP2 and cellubrevin, whereas only VAMP2 was found in the insulin-recruitable GLUT4 pool. SDS-PAGE and further silver staining of the immunoprecipitates revealed discrete polypeptide bands associated to the insulin-sensitive pool, and all these polypeptide bands were found in the insulin-insensitive population. Furthermore, some polypeptide bands were exclusive to the insulin-insensitive population. The presence of cellubrevin and SCAMP proteins, endosomal markers, suggest that the insulin-insensitive GLUT4 membrane population belongs to an endosomal compartment. In addition, we favor the view that the insulin-sensitive GLUT4 membrane pool is segregated from the endosomal GLUT4 population and is undergoes exocytosis to the cell surface in response to insulin. Intracellular GLUT4 membranes obtained from skeletal muscle contain cellubrevin, and VAMP2 and GLUT4-vesicles from cardiomyocytes also contain cellubrevin. This suggests that vSNARE proteins are key constituents of GLUT4 vesicles. The presence of the tSNARE protein SNAP25 in skeletal muscle membranes and SNAP25 and syntaxin 1A and syntaxin 1B in cardiomyocyte plasma membranes further suggest a role of the SNAREs in GLUT4 trafficking in muscle.
Asunto(s)
Insulina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Transportador de Glucosa de Tipo 4 , Masculino , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Miocardio/citología , Miocardio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas R-SNARE , Ratas , Ratas Wistar , Proteínas SNARE , Sintaxina 1 , Distribución Tisular , Proteína 3 de Membrana Asociada a VesículasRESUMEN
The interaction of middle-aging and chronic high-fat feeding on glucose transport in skeletal muscle and adipose tissue was examined. To this end, we studied the effects of 6 month treatment with a high-fat diet in 12-month old rats. Chronic high-fat feeding led to a substantial reduction in GLUT4 glucose transporter expression both in adipose tissue and in skeletal muscle, which was additive to the reduction in GLUT4 protein content detected in aged rats. In parallel, the high-fat diet led to a reduced insulin-stimulated glucose transport in the incubated soleus muscle and isolated adipocytes; insulin resistance induced by high-fat feeding was superimposed to the decreased insulin response detected in aged rats. Different mechanisms were responsible for GLUT4 repression in response to high-fat feeding or aging in skeletal muscles and adipose tissue.