RESUMO
Cystinuria is a classic heritable aminoaciduria that involves the defective transepithelial transport of cystine and dibasic amino acids in the kidney and intestine. Six missense mutations in the human rBAT gene, which is involved in high-affinity transport of cystine and dibasic amino acids in kidney and intestine, segregate with cystinuria. These mutations account for 30% of the cystinuria chromosomes studied. Homozygosity for the most common mutation (M467T) was detected in three cystinuric siblings. Mutation M467T nearly abolished the amino acid transport activity induced by rBAT in Xenopus oocytes. These results establish rBAT as a cystinuria gene.
Assuntos
Sistemas de Transporte de Aminoácidos Básicos , Proteínas de Transporte/genética , Cromossomos Humanos Par 2 , Cistina/metabolismo , Cistinúria/genética , Genes Recessivos , Glicoproteínas de Membrana/genética , Adolescente , Adulto , Sequência de Bases , Transporte Biológico , Criança , Mapeamento Cromossômico , Cistinúria/metabolismo , Análise Mutacional de DNA , Primers do DNA , Feminino , Genes , Homozigoto , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/ultraestrutura , Túbulos Renais/metabolismo , Túbulos Renais/ultraestrutura , Masculino , Microvilosidades/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Tecido Adiposo/efeitos dos fármacos , Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/farmacologia , Dioxóis/farmacologia , Transportador de Glucose Tipo 4/biossíntese , Glucose/metabolismo , Monoaminoxidase/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Tecido Adiposo/patologia , Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Cobaias , Insulina/fisiologia , Gordura Intra-Abdominal/metabolismo , Masculino , Ratos , Gordura Subcutânea/metabolismoRESUMO
The present study was designed to investigate the effect of phospholipase C and compounds known to promote synthesis of cAMP on System A transport activity under basal and insulin-stimulated conditions in the incubated muscle. In parallel, we also examined the effect of these agents on muscle glucose transport activity. Phospholipase C caused marked stimulation of alpha-(methyl)-aminoisobutyric acid (MeAIB--a System-A-specific analogue) uptake uptake and that of 3-O-methylglucose by the incubated muscle. In contrast, the activatory effect of insulin on System A was largely inhibited by phospholipase C. The effects of phospholipase C on transport processes differed from the effects provoked by phorbol esters (TPA), indicating that they are not just a consequence of TPA-sensitive protein kinase C activation. Agents such as isoproterenol, cholera toxin or forskolin, known cAMP inducers, caused glycogen depletion and stimulation of lactate production in the incubated muscle. However, these agents did not alter basal or insulin-stimulated MeAIB uptake. Isoproterenol and cholera toxin did not affect maximal stimulation of 3-O-methylglucose uptake caused by insulin. Our data indicate that System A transport is activated by phospholipase C in skeletal muscle, and that this effect is not due simply to activation of TPA-sensitive isoforms of protein kinase C. The effect of insulin on System A is reduced by either phospholipase C or TPA, which suggests the mediation of protein kinase C. On the basis of the lack of effect of cAMP-inducing agents on insulin-stimulated System A and glucose transport activities, we conclude that cAMP-dependent protein kinase does not cause any generalized blockade of insulin action in skeletal muscle, in contrast to what has been reported in other cell types.
Assuntos
Aminoácidos/metabolismo , AMP Cíclico/biossíntese , Insulina/metabolismo , Músculos/efeitos dos fármacos , Fosfolipases Tipo C/farmacologia , 3-O-Metilglucose , Animais , Transporte Biológico , Toxina da Cólera/farmacologia , Dantroleno/farmacologia , Glucose/metabolismo , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Metilglucosídeos/metabolismo , Músculos/metabolismo , Ratos , Ratos Wistar , Verapamil/farmacologia , beta-Alanina/análogos & derivados , beta-Alanina/metabolismoRESUMO
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.
Assuntos
Endotélio Vascular/metabolismo , Glucose/farmacologia , Transporte Biológico , Linhagem Celular , Cicloeximida , Glucose/metabolismo , Hexoquinase/metabolismo , Humanos , Fosforilação , Biossíntese de Proteínas , Fatores de TempoRESUMO
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.
Assuntos
Benzilaminas/uso terapêutico , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Intolerância à Glucose/tratamento farmacológico , Intolerância à Glucose/etiologia , Hiperglicemia/tratamento farmacológico , Proteínas Musculares , Vanadatos/administração & dosagem , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Benzilaminas/administração & dosagem , Benzilaminas/antagonistas & inibidores , Transporte Biológico/efeitos dos fármacos , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Relação Dose-Resposta a Droga , Esquema de Medicação , Quimioterapia Combinada , Glucose/fisiologia , Transportador de Glucose Tipo 4 , Masculino , Proteínas de Transporte de Monossacarídeos/metabolismo , Ratos , Ratos Wistar , Semicarbazidas/farmacologia , Vanadatos/antagonistas & inibidores , Vanadatos/uso terapêuticoRESUMO
Insulin rapidly stimulates glucose transport in muscle fiber. This process controls the utilization of glucose in skeletal muscle, and it is deficient in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. The effect of insulin on muscle glucose transport is mainly due to the recruitment of GLUT4 glucose carriers to the cell surface of the muscle fiber. There is increasing evidence that the recruitment of GLUT4 carriers triggered by insulin affects selective domains of sarcolemma and transverse tubules. In contrast, GLUT1 is located mainly in sarcolemma and is absent in transverse tubules, and insulin does not alter its cellular distribution in muscle fiber. The differential distribution of GLUT1 and GLUT4 in the cell surface raises new questions regarding the precise endocytic and exocytic pathways that are functional in the muscle fiber. The current view of insulin-induced GLUT4 translocation is based mainly on studies performed in adipocytes. These studies have proposed the existence of intracellular compartments of GLUT4 that respond to insulin in a highly homogeneous manner. However, studies performed in skeletal muscle have identified insulin-sensitive as well as insulin-insensitive intracellular GLUT4-containing membranes. These data open a new perspective on the dynamics of intracellular GLUT4 compartments in insulin-sensitive cells.
Assuntos
Caveolinas , Glucose/metabolismo , Glicoproteínas , Insulina/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Músculos/metabolismo , Animais , Transporte Biológico , Caveolina 1 , Compartimento Celular/efeitos dos fármacos , Fracionamento Celular , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Complexo de Golgi/metabolismo , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Músculos/ultraestrutura , Ratos , Receptor de Insulina/metabolismo , Sarcolema/metabolismoRESUMO
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.
Assuntos
Proteínas Musculares , Músculo Esquelético/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Transdução de Sinais , Somatomedinas/fisiologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/genética , Cromonas/farmacologia , Transportador de Glucose Tipo 4 , Isoenzimas/biossíntese , Microtúbulos/genética , Microtúbulos/fisiologia , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/genética , Morfolinas/farmacologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miogenina/biossíntese , Miogenina/genética , Fosfatidilinositol 3-Quinases/biossíntese , Fosfatidilinositol 3-Quinases/genética , Ratos , Transdução de Sinais/genética , Somatomedinas/genéticaRESUMO
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.
Assuntos
Adipócitos/efeitos dos fármacos , Agonistas Adrenérgicos beta/farmacologia , Amina Oxidase (contendo Cobre)/metabolismo , Moléculas de Adesão Celular/metabolismo , Isoproterenol/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Células 3T3-L1 , Adipócitos/enzimologia , Adipócitos/metabolismo , Amina Oxidase (contendo Cobre)/análise , Animais , Western Blotting , Técnicas de Cultura de Células , Fracionamento Celular , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Camundongos , SolubilidadeRESUMO
Insulin action in skeletal muscle is markedly depressed at late pregnancy. The purpose of this study was to investigate whether insulin resistance of skeletal muscle during pregnancy is associated to intrinsic alterations in the biological activities of insulin receptor. To that end, insulin receptors from mixed, red and white skeletal muscle from control and 19-20 days pregnant rats were partially purified and insulin binding and tyrosine kinase activities were evaluated. Muscle insulin receptors from diabetic rats were also studied provided that changes in receptor number and tyrosine kinase activities had been clearly substantiated. Total high affinity insulin binding sites expressed either per gram of tissue or per milligram of protein were similar in muscles from control and pregnant rats, in contrast to diabetic rats in which an increased high affinity receptor number was observed. No differences in affinity were detected for high affinity binding sites in any of the groups investigated. The integrity of the partially purified insulin receptors from control and pregnant groups was identical as determined by affinity cross-linking of [125I-TyrB26]insulin to the receptor and by beta-subunit phosphorylation. Autophosphorylation of the beta-subunit and the pattern of phosphopeptides obtained after digestion of phosphorylated beta-subunit with trypsin, elastase, and staphylococcal V8 protease were indistinguishable in control and pregnant groups. Tyrosine receptor kinase was also similar in receptor preparations from control and pregnant muscle. This is in contrast to diabetes in which a defective tyrosine kinase was confirmed. In order to detect possible differences due to the fiber type, further sets of experiments were performed in receptor preparations from red and white muscle. In keeping with previous data, tyrosine kinase activity of the insulin receptor was 2.5-fold greater in red muscle than white muscle; however, under these conditions, receptor kinase activity was unmodified in preparations from pregnant rats in red and white muscle fibers. Recent evidence has revealed the existence of an insulin binding inhibitor in muscle extracts. We detected the presence of such an inhibitor in the flow-through fraction after WGA chromatography. This inhibitory activity was found to be greater in muscle extracts obtained from pregnant rats as compared to fractions from control rats. We conclude that insulin resistance of skeletal muscle at late pregnancy is not explained by intrinsic modifications of insulin receptor binding or kinase activities.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Resistência à Insulina/fisiologia , Músculos/fisiologia , Fosfotransferases/metabolismo , Prenhez/fisiologia , Receptor de Insulina/metabolismo , Animais , Feminino , Insulina/metabolismo , Músculos/metabolismo , Gravidez , Ratos , Ratos Endogâmicos , Receptor de Insulina/ultraestruturaRESUMO
During lactation, the mammary gland shows high metabolic activity, which is dependent at least in part on the availability of glucose. We have studied the regulation of glucose transporter expression in different types of cell in rat mammary gland during the reproductive cycle. Glucose transporter expression varied markedly in the mammary gland during ontogeny. Thus, GLUT1 protein expression increased progressively during pregnancy, reaching the highest levels during lactation. A peak of lactation, GLUT1 protein content, expressed per g issue, was greater in mammary gland than in GLUT1-enriched tissues such as rat brain. In contrast, GLUT4 showed a marked decrease during pregnancy and practically disappeared during lactation. Regardless of the developmental stage, GLUT4 was expressed in adipocytes but not in epithelial cells from mammary gland. On the other hand, GLUT1 was expressed in both cell types. The overall pattern of GLUT1 and GLUT4 expression during the reproductive cycle reflects differences in the cell composition of the mammary gland. Thus, whereas adipocytes predominate before pregnancy, epithelial cells are the main cell type in the mammary gland during late pregnancy and lactation. Immunofluorescence and immunoelectron microscopy indicated that GLUT1 was essentially localized to the basolateral domain of epithelial cells in the mammary gland at peak of lactation, and hardly any labeling was found in the luminal membrane of epithelial cells. GLUT1 expression was acutely regulated in epithelial cells from mammary gland. Thus, GLUT1 protein markedly decreased in lactating rats 24 h after abrupt weaning in the presence of a moderate decrease in GLUT1 mRNA levels. The effect of weaning on GLUT1 protein content was not due to the fall in the plasma concentration of PRL. Thus, bromocriptine treatment for 24 h decreased GLUT1 mRNA levels in the mammary gland, but did not alter the content of GLUT1 protein. Our results demonstrate 1) the existence of a high and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland, and 2) that GLUT1 expression is acutely regulated at a posttranslational level by weaning; this is not mimicked by bromocriptine treatment, which rules out PRL as the regulatory factor involved in the effect.
Assuntos
Lactação/metabolismo , Glândulas Mamárias Animais/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Prenhez/metabolismo , Animais , Northern Blotting , Membrana Celular/metabolismo , Eletroforese em Gel de Poliacrilamida , Células Epiteliais , Epitélio/metabolismo , Epitélio/ultraestrutura , Feminino , Imunofluorescência , Expressão Gênica , Transportador de Glucose Tipo 1 , Immunoblotting , Imuno-Histoquímica , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/ultraestrutura , Microscopia Imunoeletrônica , Proteínas de Transporte de Monossacarídeos/análise , Gravidez , Ratos , Ratos Wistar , DesmameRESUMO
The expression of GLUT-1 (erythroid/Hep G2) and GLUT-4 (muscle/fat) glucose transporters was assessed during development in rat heart, skeletal muscle, and brown adipose tissue. GLUT-4 protein expression was detectable in fetal heart by day 21 of pregnancy; it increased progressively after birth, attaining levels close to those of adults at day 15 post natal. In contrast, GLUT-4 messenger RNA (mRNA) was already present in hearts from 17 day-old fetuses. GLUT-4 mRNA stayed low during early postnatal life in heart and brown adipose tissue and only increased after day 10 post natal. The expression pattern for GLUT-4 protein in skeletal muscle during development was comparable to that observed in heart. In contrast to heart and skeletal muscle, GLUT-4 protein in brown adipose tissue was detected in high levels (30% of adult) during late fetal life. During fetal life, GLUT-1 presented a very high expression level in brown adipose tissue, heart, and skeletal muscle. Soon after birth, GLUT-1 protein diminished progressively, attaining adult levels at day 10 in heart and skeletal muscle. GLUT-1 mRNA levels in heart followed a similar pattern to the GLUT-1 protein, being very high during fetal life and decreasing early in post natal life. GLUT-1 protein showed a complex pattern in brown adipose tissue: fetal levels were high, decreased after birth, and increased subsequently in post natal life, reaching a peak by day 9. Progesterone-induced postmaturity protected against the decrease in GLUT-1 protein associated with post natal life in skeletal muscle and brown adipose tissue. However, GLUT-4 induction was not blocked by postmaturity in any of the tissues subjected to study. These results indicate that: 1) during fetal and early post natal life, GLUT-1 is a predominant glucose transporter isotype expressed in heart, skeletal muscle, and brown adipose tissue; 2) during early post natal life there is a generalized GLUT-1 repression; 3) during development, there is a close correlation between protein and mRNA levels for GLUT-1, and therefore regulation at a pretranslational level plays a major regulatory role; 4) the onset of GLUT-4 protein induction occurs between days 20-21 of fetal life; based on data obtained in rat heart and brown adipose tissue, there is a dissociation during development between mRNA and protein levels for GLUT-4, suggesting modifications at translational or posttranslational steps; and 5) postmaturity blocks the decrease in GLUT-1 expression but not the induction of GLUT-4, observed soon after birth.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Tecido Adiposo Marrom/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Proteínas de Transporte de Monossacarídeos/metabolismo , Desenvolvimento Muscular , RNA Mensageiro/metabolismo , Tecido Adiposo Marrom/embriologia , Tecido Adiposo Marrom/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Northern Blotting , Desenvolvimento Embrionário e Fetal , Feminino , Regulação da Expressão Gênica , Idade Gestacional , Coração/embriologia , Proteínas de Transporte de Monossacarídeos/genética , Músculos/embriologia , Músculos/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos , Gravidez , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/genética , Ratos , Ratos EndogâmicosRESUMO
We have previously reported that innervation-dependent basal contractile activity regulates in an inverse manner the expression of GLUT1 and GLUT4 glucose transporters in skeletal muscle. Based on the facts that muscle innervation decreases and muscle denervation increases cAMP levels, we investigated whether cAMP might mediate the effects of innervation/denervation on glucose transporter expression. Treatment of L6E9 myotubes with 8-bromo-cAMP, forskolin, or monobutyryl-8-bromo-cAMP led to a marked decrease in GLUT4 protein levels; 8-bromo-cAMP also diminished GLUT4 messenger RNA (mRNA), suggesting pretranslational repression. In contrast, L6E9 myoblasts and myotubes responded to 8-bromo-cAMP or forskolin by increasing the cell content of GLUT1 protein. Induction of GLUT1 protein was a consequence of the activation of different mechanisms in myoblast and myotube cells; whereas 8-bromo-cAMP treatment caused a substantial increase in GLUT1 mRNA in myoblasts, no change in GLUT1 mRNA was detected in myotubes. The increase in GLUT1 mRNA in L6E9 myoblasts induced by 8-bromo-cAMP was the result of transcriptional activation, as concluded from transfection analysis of 2.1 kilobases of the rat GLUT1 gene promoter fused to the bacterial chloramphenicol acetyltransferase gene. Furthermore, the stimulatory effect of 8-bromo-cAMP on the transcriptional activity of the GLUT1 promoter required a 33-bp sequence lying 5' upstream of the transcription start site. In all, cAMP inversely regulates GLUT4 and GLUT1 glucose transporter expression in muscle cells. Furthermore, our results suggest that down-regulation of GLUT4 expression and up-regulation of GLUT1 expression in muscle associated with denervation are partly attributable to cAMP.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , AMP Cíclico/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas Musculares , Músculo Esquelético/metabolismo , Regiões Promotoras Genéticas , Transcrição Gênica/fisiologia , Animais , Bucladesina/análogos & derivados , Bucladesina/farmacologia , Linhagem Celular , Membrana Celular/metabolismo , Cloranfenicol O-Acetiltransferase , Colforsina/farmacologia , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Cinética , Proteínas de Transporte de Monossacarídeos/genética , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Transcrição Gênica/efeitos dos fármacos , TransfecçãoRESUMO
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.
Assuntos
Fator de Crescimento Insulin-Like II/farmacologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Fenômenos Fisiológicos Sanguíneos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Cromonas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Morfolinas/farmacologia , Proteínas Musculares/metabolismo , Músculo Esquelético/fisiologia , Ratos , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Sirolimo/farmacologiaRESUMO
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.
Assuntos
Insulina/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares , Músculo Esquelético/metabolismo , Proteínas de Transporte Vesicular , Animais , Transporte Biológico , Transportador de Glucose Tipo 4 , Masculino , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Miocárdio/citologia , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas R-SNARE , Ratos , Ratos Wistar , Proteínas SNARE , Sintaxina 1 , Distribuição Tecidual , Proteína 3 Associada à Membrana da VesículaRESUMO
We have studied the activity of system A transport in skeletal muscle during experimental diabetes. Five days after streptozotocin injection, rats showed a marked hyperglycemia and a substantial decrease in the content of GLUT-4 protein in skeletal muscle and adipose tissue. Under these conditions, basal uptake of 2-(methyl)aminoisobutyric acid (MeAIB), an index of system A transport activity, was enhanced in extensor digitorum longus (EDL) muscles from diabetic rats compared to controls. Furthermore, insulin-stimulated MeAIB uptake by the incubated EDL and soleus muscles was markedly greater in diabetic than in control rats. The derepressive phase of adaptive regulation was partially blocked in the diabetic muscle, and incubation of muscles for 3 h in the absence of amino acids led to a lower stimulation of system A transport activity in muscles from diabetic groups compared to controls. We propose that the activated system A might participate in the enhanced alanine release from muscle cells that occurs in diabetes.
Assuntos
Aminoácidos/metabolismo , Diabetes Mellitus Experimental/metabolismo , Músculos/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Insulina/farmacologia , Masculino , Proteínas de Transporte de Monossacarídeos/metabolismo , Ratos , Ratos Endogâmicos , EstreptozocinaRESUMO
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.
Assuntos
Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Animais , Transporte Biológico Ativo , Diabetes Mellitus Experimental/metabolismo , Jejum/metabolismo , Regulação da Expressão Gênica , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Miocárdio/citologia , RNA Mensageiro/análise , Ratos , Hormônios Tireóideos/fisiologiaRESUMO
In this study we explored the expression of GLUT4 glucose carriers in muscle and adipose tissues from streptozotocin-induced diabetic and benfluorex-treated rats. In nondiabetic rats, benfluorex treatment decreased GLUT4 protein content in muscle and brown adipose tissue, with no change in GLUT4 mRNA. This effect occurred in the presence of normal circulating levels of insulin and glucose. Seventeen days after streptozotocin injection, diabetic rats showed a decreased GLUT4 protein content in adipose tissues and in both red and white skeletal muscle. Diabetic rats showed decreased GLUT4 mRNA levels in white and brown adipose tissue, whereas messenger concentrations remained unaltered in red and white fibers of skeletal muscle. The interaction of benfluorex and diabetes on GLUT4 protein expression showed a tissue-specific pattern. Benfluorex treatment to some extent prevented the decrease in GLUT4 protein in white and brown adipose tissue and in white muscle associated with diabetes. In contrast, diabetes and benfluorex caused an additive decrease in GLUT4 expression in red skeletal muscle. The effects of benfluorex on GLUT4 content in tissues from diabetic rats occurred in the absence of alterations in GLUT4 mRNA levels, suggesting a modification of translational or posttranslational steps. Benfluorex did not ameliorate the hyperglycemia of diabetic rats. Our results indicate that red and white skeletal muscle respond to diabetes and benfluorex in a heterogeneous manner, which suggests the existence of differences in the mechanisms that regulate GLUT4 expression. Furthermore, our data indicate that GLUT4 expression in muscle and adipose tissue can be regulated by modification of translational or posttranslational steps.
Assuntos
Tecido Adiposo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Fenfluramina/análogos & derivados , Hipoglicemiantes/farmacologia , Proteínas de Transporte de Monossacarídeos/análise , Proteínas Musculares , Músculos/metabolismo , Animais , Fenfluramina/farmacologia , Transportador de Glucose Tipo 4 , Masculino , Proteínas de Transporte de Monossacarídeos/genética , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , EstreptozocinaRESUMO
Benzyl succinate inhibited insulin binding and tyrosine receptor kinase in a concentration-dependent manner in the partially purified insulin receptor preparation from rat skeletal muscle. Benzyl succinate lowered the apparent number of high-affinity insulin binding sites. We have made use of the inhibitory effect of benzyl succinate to investigate the possible presence of spare high-affinity insulin receptors in muscle. Benzyl succinate inhibited the effect of a supramaximal concentration of insulin on 3-O-methylglucose uptake, 2-(methylamino)isobutyric acid uptake and lactate production by the incubated muscle. Furthermore, the inhibitory effect of benzyl succinate on insulin binding in vitro closely correlated with its inhibitory effect on insulin action in vivo. These findings suggest the absence of spare high-affinity insulin receptors in skeletal muscle. In contrast to data obtained in skeletal muscle, benzyl succinate did not affect the maximally insulin-stimulated glucose transport, although it caused a marked decrease in insulin sensitivity in isolated rat adipocytes, for which the existence of spare insulin receptors is well documented.
Assuntos
Antagonistas da Insulina/farmacologia , Insulina/metabolismo , Músculos/metabolismo , Receptor de Insulina/fisiologia , Succinatos/farmacologia , 3-O-Metilglucose , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animais , Insulina/farmacologia , Lactatos/biossíntese , Ácido Láctico , Masculino , Metilglucosídeos/metabolismo , Músculos/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Wistar , Receptor de Insulina/efeitos dos fármacosRESUMO
To study the role of undernourishment in the negative effects of ethanol during pregnancy and to determine whether maternal ethanol intake modifies metabolic response to starvation at late gestation, female rats receiving ethanol in their drinking water before and during pregnancy (ethanol group) were compared with animals that received the same amount of solid diet as the ethanol group rats (pair-fed group) and with normal rats fed ad libitum (control group). All animals were killed on the 21st day of gestation, either in the fed state or after 24-hours fasting. The body weight of ethanol rats was lower than that of controls but higher than that of pair-fed rats. When compared with controls, ethanol and pair-fed rats had reduced fetal body weights, whereas fetal body length was reduced only in the former. In the fed state, blood glucose concentration was lower in the ethanol and pair-fed rats and fetuses than in controls. Twenty-four-hour starvation caused a reduction in this parameter only in control and ethanol mothers. In the fed state, maternal liver glycogen concentration was lower in ethanol and higher in pair-fed mothers than in controls. Blood beta-hydroxybutyrate levels were higher in ethanol-treated mothers than in the others, and 24-hour starvation increased this parameter in ethanol and control rats to a greater extent than in the pair-fed ones. Liver triacylglyceride concentration was higher in ethanol-treated mothers than in the other two groups, and starvation caused this concentration to increase in ethanol and control groups but not in the pair-fed group.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Alcoolismo/fisiopatologia , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Distúrbios Nutricionais/fisiopatologia , Complicações na Gravidez/fisiopatologia , Inanição , Alcoolismo/complicações , Animais , Glicemia/análise , Peso Corporal , Modelos Animais de Doenças , Feminino , Feto/fisiologia , Lipídeos/sangue , Fígado/patologia , Glicogênio Hepático/análise , Distúrbios Nutricionais/complicações , Tamanho do Órgão , Gravidez , Ratos , Ratos Endogâmicos , Valores de ReferênciaRESUMO
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.