RESUMO
Recent pharmacological findings regarding rimonabant, an anorectic and cannabinoid type 1 receptor (CB1R) antagonist, strongly suggest that some of its effects on the metabolic parameters and energy balance in rats are not related to the centrally mediated reduction in caloric intake. Instead, they may be associated with acute induction of glycogenolysis in the liver, in combination with transient increase in glucose oxidation and persistent increase in fat oxidation. It is possible that rimonabant produced direct short- or long-term stimulatory effect on these processes in primary and cultured rat cells. Rimonabant slightly stimulated ß-oxidation of long-chain fatty acids in cultured rat myocytes overexpressing glucose transporter isoform 4, as well as activated phosphorylation of adenosine monophosphate-dependent protein kinase (AMPK) in primary rat hepatocytes upon long-term incubation. However, short-term action of rimonabant failed to stimulate ß-oxidation in myocytes, myotubes, and hepatocytes, as well as to upregulate AMPK phosphorylation, glycogenolysis, and cAMP levels in hepatocytes. As a consequence, the acute effects of rimonabant on hepatic glycogen content (reduction) and total energy expenditure (increase) in rats fed with a standard diet cannot be explained by direct stimulation of glycogenolysis and fatty acid oxidation in muscles and liver. Rather, these effects seem to be centrally mediated.
Assuntos
Ácidos Graxos/metabolismo , Glicogenólise/efeitos dos fármacos , Fígado/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Oxirredução/efeitos dos fármacos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Rimonabanto/farmacologia , Adenilato Quinase/metabolismo , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The insulin-independent inhibition of lipolysis by palmitate, the anti-diabetic sulphonylurea glimepiride and H2O2 in rat adipocytes involves stimulation of the glycosylphosphatidylinositol (GPI)-specific phospholipase-C (GPI-PLC) and subsequent translocation of the GPI-anchored membrane ectoproteins (GPI-proteins), Gce1 and cluster of differentiation antigen (CD73), from specialized plasma membrane microdomains (DIGs) to cytosolic lipid droplets (LDs). This results in cAMP degradation at the LD surface and failure to activate hormone-sensitive lipase. Reactive oxygen species (ROS) may trigger this sequence of events in response to palmitate and glimepiride. EXPERIMENTAL APPROACH: The effects of various inhibitors of ROS production on the release of H2O2, GPI anchor cleavage and translocation of the photoaffinity-labelled or metabolically labelled Gce1 and CD73 from DIGs to LD and inhibition of lipolysis by different fatty acids and sulphonylureas were studied with primary rat adipocytes. KEY RESULTS: Glimepiride and palmitate induced the production of H2O2 via the plasma membrane NADPH oxidase and mitochondrial complexes I and III, respectively. Inhibition of ROS production was accompanied by the loss of (i) GPI-PLC activation, (ii) Gce1 and CD73 translocation and (iii) lipolysis inhibition in response to palmitate and glimepiride. Non-metabolizable fatty acids and the sulphonylurea drug tolbutamide were inactive. NADPH oxidase and GPI-PLC activities colocalized at DIGs were stimulated by glimepiride but not tolbutamide. CONCLUSIONS AND IMPLICATIONS: The data suggest that ROS mediate GPI-PLC activation at DIGs and subsequent GPI-protein translocation from DIGs to LD in adipocytes which leads to inhibition of lipolysis by palmitate and glimepiride. This insulin-independent anti-lipolytic mechanism may be engaged by future anti-diabetic drugs.
Assuntos
Glicosilfosfatidilinositol Diacilglicerol-Liase/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Peróxido de Hidrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , 5'-Nucleotidase/metabolismo , Adipócitos , Animais , AMP Cíclico/metabolismo , Ácidos Graxos/farmacologia , Lipólise/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , NADPH Oxidases/metabolismo , Transporte Proteico , Ratos , Espécies Reativas de Oxigênio/antagonistas & inibidores , Compostos de Sulfonilureia/farmacologiaRESUMO
Signaling molecules downstream from the insulin receptor, such as the insulin receptor substrate protein 1 (IRS-1), are also activated by other receptor tyrosine kinases. Here we demonstrate that the non-receptor tyrosine kinases, focal adhesion kinase pp125(FAK) and Src-class kinase pp59(Lyn), after insulin-independent activation by phosphoinositolglycans (PIG), can cross talk to metabolic insulin signaling in rat and 3T3-L1 adipocytes. Introduction by electroporation of neutralizing antibodies against pp59(Lyn) and pp125(FAK) into isolated rat adipocytes blocked IRS-1 tyrosine phosphorylation in response to PIG but not insulin. Introduction of peptides encompassing either the major autophosphorylation site of pp125(FAK), tyrosine 397, or its regulatory loop with the twin tyrosines 576 and 577 inhibited PIG-induced IRS-1 tyrosine phosphorylation and glucose transport. PIG-induced pp59(Lyn) kinase activation and pp125(FAK) tyrosine phosphorylation were impaired by the former and latter peptide, respectively. Up-regulation of pp125(FAK) by integrin clustering diminished PIG-induced IRS-1 tyrosine phosphorylation and glucose transport in nonadherent but not adherent adipocytes. In conclusion, PIG induced IRS-1 tyrosine phosphorylation by causing (integrin antagonized) recruitment of IRS-1 and pp59(Lyn) to the common signaling platform molecule pp125(FAK), where cross talk of PIG-like structures and extracellular matrix proteins to metabolic insulin signaling may converge, possibly for the integration of the demands of glucose metabolism and cell architecture.
Assuntos
Adipócitos/metabolismo , Insulina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacologia , Sequência de Carboidratos , Adesão Celular/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Proteínas Substratos do Receptor de Insulina , Integrinas/metabolismo , Masculino , Mimetismo Molecular , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Fosfatidilinositóis/química , Fosfatidilinositóis/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas Tirosina Quinases/imunologia , Ratos , Ratos Wistar , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismo , Quinases da Família src/genéticaRESUMO
Transfer of spheroplasts from the yeast Saccharomyces cerevisiae to glucose leads to the activation of an endogenous (glycosyl)-phosphatidylinositol-specific phospholipase C ([G]PI-PLC), which cleaves the anchor of at least one glycosyl-phosphatidylinositol (GPI)-anchored protein, the cyclic AMP (cAMP)-binding ectoprotein Gce1p (G. Müller and W. Bandlow, J. Cell Biol. 122:325-336, 1993). Analyses of the turnover of two constituents of the anchor, myo-inositol and ethanolamine, relative to the protein label as well as separation of the two differently processed versions of Gce1p by isoelectric focusing in spheroplasts demonstrate the glucose-induced conversion of amphiphilic Gce1p first into a lipolytically cleaved hydrophilic intermediate, which is then processed into another hydrophilic version lacking both myo-inositol and ethanolamine. When incubated with unlabeled spheroplasts, the lipolytically cleaved intermediate prepared in vitro is converted into the version lacking all anchor constituents, whereby the anchor glycan is apparently removed as a whole. The secondary cleavage ensues independently of the carbon source, attributing the key role in glucose-induced anchor processing to the endogenous (G)PI-PLC. The secondary processing of the lipolytically cleaved intermediate of Gce1p at the plasma membrane is correlated with the emergence of a covalently linked high-molecular-weight form of a cAMP-binding protein at the cell wall. This protein lacks anchor components, and its protein moiety appears to be identical with double-processed Gce1p detectable at the plasma membrane in spheroplasts. The data suggest that glucose-induced double processing of GPI anchors represents part of a mechanism of regulated cell wall expression of proteins in yeast cells.
Assuntos
Proteínas Fúngicas/metabolismo , Glucose/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte , Parede Celular/metabolismo , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Etanolamina , Etanolaminas/metabolismo , Proteínas Fúngicas/química , Inositol/metabolismo , Ponto Isoelétrico , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Esferoplastos/metabolismoRESUMO
Caveolae and caveolin-containing detergent-insoluble glycolipid-enriched rafts (DIG) have been implicated to function as plasma membrane microcompartments or domains for the preassembly of signaling complexes, keeping them in the basal inactive state. So far, only limited in vivo evidence is available for the regulation of the interaction between caveolae-DIG and signaling components in response to extracellular stimuli. Here, we demonstrate that in isolated rat adipocytes, synthetic intracellular caveolin binding domain (CBD) peptide derived from caveolin-associated pp59(Lyn) (10 to 100 microM) or exogenous phosphoinositolglycan derived from glycosyl-phosphatidylinositol (GPI) membrane protein anchor (PIG; 1 to 10 microM) triggers the concentration-dependent release of caveolar components and the GPI-anchored protein Gce1, as well as the nonreceptor tyrosine kinases pp59(Lyn) and pp125(Fak), from interaction with caveolin (up to 45 to 85%). This dissociation, which parallels redistribution of the components from DIG to non-DIG areas of the adipocyte plasma membrane (up to 30 to 75%), is accompanied by tyrosine phosphorylation and activation of pp59(Lyn) and pp125(Fak) (up to 8- and 11-fold) but not of the insulin receptor. This correlates well to increased tyrosine phosphorylation of caveolin and the insulin receptor substrate protein 1 (up to 6- and 15-fold), as well as elevated phosphatidylinositol-3' kinase activity and glucose transport (to up to 7- and 13-fold). Insulin-mimetic signaling by both CBD peptide and PIG as well as redistribution induced by CBD peptide, but not by PIG, was blocked by synthetic intracellular caveolin scaffolding domain (CSD) peptide. These data suggest that in adipocytes a subset of signaling components is concentrated at caveolae-DIG via the interaction between their CBD and the CSD of caveolin. These inhibitory interactions are relieved by PIG. Thus, caveolae-DIG may operate as signalosomes for insulin-independent positive cross talk to metabolic insulin signaling downstream of the insulin receptor based on redistribution and accompanying activation of nonreceptor tyrosine kinases.
Assuntos
Adipócitos/metabolismo , Caveolinas/metabolismo , Glicolipídeos/metabolismo , Microdomínios da Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Quinases da Família src/metabolismo , Adipócitos/citologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Caveolina 1 , Células Cultivadas , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glucose/metabolismo , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina , Masculino , Dados de Sequência Molecular , Fosfatidilinositóis/metabolismo , Fosforilação , Ratos , Ratos Wistar , Tirosina/metabolismoRESUMO
Sulfonylurea drugs are widely used in the therapy of NIDDM. The improvement of glucose tolerance after long-term treatment of NIDDM patients with the drug can be explained by stimulation of glucose utilization in peripheral tissues that are characterized by insulin resistance in these patients. We studied whether the novel sulfonylurea drug, glimepiride, stimulates glucose transport into isolated insulin-resistant rat adipocytes. After long-term incubation of the cells in primary culture with high concentrations of glucose, glutamine, and insulin, stimulation of glucose transport by insulin was significantly reduced both with respect to maximal responsiveness (65% decrease of Vmax) and sensitivity (2.6-fold increase of ED50) compared with adipocytes cultured in medium containing a low concentration of glucose and no insulin. This reflects insulin resistance of glucose transport. In contrast, both responsiveness and sensitivity of glucose transport toward stimulation by glimepiride were only marginally reduced in insulin-resistant adipocytes (15% decrease of Vmax; 1.2-fold increase of ED50) versus control cells. Glimepiride, in combination with glucose and glutamine during the primary culture, caused desensitization of the glucose transport system toward stimulation by insulin, but to a lesser degree than insulin itself (50% reduction of Vmax; ninefold increase of ED50). Again, the maximal responsiveness and sensitivity of glucose transport toward stimulation by glimepiride were only slightly diminished. The presence of glimepiride during primary culture did not antagonize the induction of insulin resistance of glucose transport. The stimulation of glucose transport in insulin-resistant adipocytes by glimepiride is caused by translocation of glucose transporters from low-density microsomes to plasma membranes as demonstrated by subcellular fractionation and immunoblotting with anti-GLUT1 and anti-GLUT4 antibodies. Immunoprecipitation of GLUT4 from 32Pi- and [35S]methionine-labeled adipocytes revealed that the insulin resistance of GLUT4 translocation is accompanied by increased (three- to fourfold) phosphorylation of GLUT4 in both low-density microsomes and plasma membranes. Short-term treatment of desensitized adipocytes with glimepiride or insulin reduced GLUT4 phosphorylation by approximately 70 and 25%, respectively, in both fractions. We conclude that glimepiride activates glucose transport by stimulation of GLUT1 and GLUT4 translocation in rat adipocytes via interference at a site downstream of the putative molecular defect in the signaling cascade between the insulin receptor and the glucose transport system induced by high concentrations of glucose and insulin. The molecular site of glimepiride action is related to GLUT4 phosphorylation/dephosphorylation, which may regulate glucose transporter activity and translocation.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Musculares , Compostos de Sulfonilureia/farmacologia , Adipócitos/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Epididimo , Transportador de Glucose Tipo 1 , Transportador de Glucose Tipo 4 , Cinética , Masculino , Metionina/metabolismo , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/isolamento & purificação , Fosfatos/metabolismo , Radioisótopos de Fósforo , Fosforilação , Ratos , Ratos Wistar , Radioisótopos de EnxofreRESUMO
Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types. However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects. In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane protein, Gce1p, from the yeast Saccharomyces cerevisiae. The structure of the resulting PIG-P, NH2-Tyr-Cys-Asn-ethanolamine-PO4-6(Man1-2)Man1-2Man1-+ ++6Man1-4GlcNH(2)1-6myo-inositol-1,2-cyclicPO4, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors. PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes. Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms. The concentration-dependent effects of the PIG-P reached 70-90% of the maximal insulin activity with EC50-values of 0.5-5 microM. Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity. The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.
Assuntos
Adipócitos/metabolismo , Diafragma/metabolismo , Glicosilfosfatidilinositóis/farmacologia , Insulina/metabolismo , Proteínas Musculares , Miocárdio/metabolismo , Saccharomyces cerevisiae/química , Agonistas Adrenérgicos beta/farmacologia , Animais , Cromatografia por Troca Iônica , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Glucose/metabolismo , Transportador de Glucose Tipo 4 , Glicerol-3-Fosfato O-Aciltransferase/fisiologia , Glicogênio Sintase/fisiologia , Glicosilfosfatidilinositóis/análise , Glicosilfosfatidilinositóis/química , Isomerismo , Isoproterenol/farmacologia , Metabolismo dos Lipídeos , Masculino , Proteínas de Transporte de Monossacarídeos/metabolismo , Miocárdio/citologia , Ratos , Ratos WistarRESUMO
The long-term hypoglycemic activity of sulphonylurea drugs has been attributed, in part at least, to the stimulation of glucose utilization in extra-pancreatic tissues. The novel sulphonylurea, glimepiride, gives rise to a longer lasting reduction in the blood sugar level in dogs and rabbits compared to glibenclamide (Geisen K, Drug Res 38: 1120-1130, 1988). This cannot be explained adequately by elevated plasma insulin levels. This study investigated whether this prolonged hypoglycemic phase was based on the drug's abilities to stimulate glucose utilization and affect the underlying regulatory mechanisms in insulin-sensitive cells in vitro. It was found that in the absence of added insulin, glimepiride and glibenclamide (1-50 microM) stimulated lipogenesis (3T3 adipocytes) and glycogenesis (isolated rat diaphragm) approximately 4.5- and 2.5-fold, respectively, and reduced the isoproterenol-stimulated lipolysis (rat adipocytes) up to 40-60%. The increased glucose utilization was correlated with a 3-4-fold higher 2-deoxyglucose transport rate and amount of GLUT4 at the plasma membrane, as well as with increased activities of key metabolic enzymes (glycerol-3-phosphate acyltransferase, glycogen synthase) within the same concentration range. Furthermore, the low Km cAMP-specific phosphodiesterase was activated 1.8-fold, whereas the cytosolic cAMP level and protein kinase A activity ratios were significantly lowered after incubation of isoproterenol-stimulated rat adipocytes with the sulphonylureas. In many of the aspects studied the novel sulphonylurea, glimepride, exhibited slightly lower ED50-values than glibenclamide. This study demonstrates correlations existing between drug-induced stimulation of glucose transport/metabolism and cAMP degradation/protein kinase A inhibition as well as between the relative efficiencies of glimepiride and glibenclamide in inducing these extra-pancreatic processes. Therefore, it is suggested that the stimulation of glucose utilization by sulphonylureas is mediated by a decrease of cAMP-dependent phosphorylation of GLUT4 and glucose metabolizing enzymes. The therapeutic relevance of extra-pancreatic effects of sulphonylureas, in general, and of the differences between glimepiride and glibenclamide as observed in vitro in this work, in particular, remain to be elucidated.
Assuntos
Adipócitos/efeitos dos fármacos , AMP Cíclico/metabolismo , Diafragma/efeitos dos fármacos , Glucose/metabolismo , Glibureto/farmacologia , Compostos de Sulfonilureia/farmacologia , Células 3T3 , Adipócitos/metabolismo , Animais , Transporte Biológico , Diafragma/metabolismo , Enzimas/metabolismo , Glicogênio/biossíntese , Lipídeos/biossíntese , Masculino , Camundongos , Ratos , Ratos WistarRESUMO
BACKGROUND AND PURPOSE: In addition to predominant localization at detergent-insoluble, glycolipid-enriched plasma membrane microdomains (DIGs), glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-proteins) have been found associated with lipid droplets (LDs) and adiposomes. Adiposomes are vesicles that are released from adipocytes in response to anti-lipolytic and lipogenic signals, such as H(2)O(2), palmitate and the antidiabetic sulfonylurea drug, glimepiride, and harbour (c)AMP-degrading GPI-proteins, among them the 5-nucleotidase CD73. Here the role of adiposomes in GPI-protein-mediated information transfer was studied. EXPERIMENTAL APPROACH: Adiposomes were incubated with isolated rat adipocytes under various conditions. Trafficking of CD73 and lipid synthesis were analysed. KEY RESULTS: Upon blockade of GPI-protein trafficking, CD73 specifically associated with DIGs of small, and to a lower degree, large, adipocytes. On reversal of the blockade, CD73 appeared at cytosolic LD in time- adiposome concentration- and signal (H(2)O(2) > glimepiride > palmitate)-dependent fashion. The salt- and carbonate-resistant association of CD73 with structurally intact DIGs and LD was dependent on its intact GPI anchor. Upon incubation with small and to a lower degree, large adipocytes, adiposomes increased lipid synthesis in the absence or presence of H(2)O(2), glimepiride and palmitate and improved the sensitivity toward these signals. Upregulation of lipid synthesis by adiposomes was dependent on the translocation of CD73 with intact GPI anchors from DIGs to LD. CONCLUSIONS: The signal-induced transfer of GPI-anchored CD73 from adiposomes via DIGs to LD of adipocytes mediates paracrine upregulation of lipid synthesis within the adipose tissue.
Assuntos
5'-Nucleotidase/metabolismo , Adipócitos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Metabolismo dos Lipídeos , Vesículas Transportadoras/metabolismo , 5'-Nucleotidase/biossíntese , 5'-Nucleotidase/química , Adipócitos/citologia , Animais , Tamanho Celular , Células Cultivadas , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Proteínas Ligadas por GPI , Glicosilfosfatidilinositóis/química , Peróxido de Hidrogênio/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Ácido Palmítico/farmacologia , Comunicação Parácrina , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Compostos de Sulfonilureia/farmacologia , Fatores de Tempo , Vesículas Transportadoras/química , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND AND PURPOSE: Adipocytes release membrane vesicles called adiposomes, which harbor the glycosylphosphatidylinositol-anchored proteins (GPI proteins), Gce1 and CD73, after induction with palmitate, H(2)O(2) and the sulphonylurea drug glimepiride. The role of lipid droplets (LD) in trafficking of GPI proteins from detergent-insoluble, glycolipid-enriched, plasma membrane microdomains (DIGs) to adiposomes in rat adipocytes was studied. EXPERIMENTAL APPROACH: Redistribution of Gce1 and CD73 was followed by pulse-chase and long-term labelling, western blot analysis and activity determinations with subcellular fractions and cell-free systems exposed to palmitate, H(2)O(2) and glimepiride. KEY RESULTS: In response to these signals, Gce1 and CD73 disappeared from DIGs, then transiently appeared in LD and finally were released into adiposomes from small, and, more efficiently, large adipocytes. From DIGs to LD, Gce1 and CD73 were accompanied by cholesterol. Cholesterol depletion from DIGs or LD caused accumulation at DIGs or accelerated loss from LD and release into adiposomes, respectively, of the GPI proteins. Blockade of translocation of Gce1, CD73, caveolin-1 and perilipin-A from DIGs to LD blocked LD biogenesis and long term-accumulation of LD interfered with induced release of the GPI proteins into adiposomes. GPI protein release was up-regulated upon long term-depletion of LD. Adiposomes were released by a DIGs-based cell-free system, but only in presence of LD. CONCLUSIONS: GPI proteins are translocated from DIGs to LD prior to their release into adiposomes, which is regulated by cholesterol, LD content and LD biogenesis. This detour may serve to transfer information about the LD content and to control lipolysis/esterification between large and small adipocytes via GPI protein-harbouring adiposomes.
Assuntos
5'-Nucleotidase/metabolismo , Adipócitos/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Microdomínios da Membrana/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Adipócitos/ultraestrutura , Animais , Proteínas de Transporte , Caveolina 1/metabolismo , Células Cultivadas , Colesterol/metabolismo , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Peróxido de Hidrogênio/farmacologia , Masculino , Palmitatos/farmacologia , Perilipina-1 , Fosfoproteínas/metabolismo , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Compostos de Sulfonilureia/farmacologiaRESUMO
Previously, we have studied the lipolytic cleavage of a glycosyl-phosphatidylinositol (GPI)-anchored plasma membrane protein in yeast in response to a physiologically relevant external signal, i.e., transfer of spehroplasts from lactate to glucose medium (cf. Müller and Bandlow (1993) J. Cell. Biol. 122, 325-336). In the present study the glucose-induced lipolytic processing of myo-[14C]inositol-labeled total GPI proteins of the plasma membrane and in particular of two such proteins, Gas1p and Gce1p, was examined in yeast spheroplasts. It was found that a small number of GPI proteins, among them Gce1p, are readily cleaved, whereas Gas1p and the majority of the GPI proteins are relatively little affected. Glucose-induced processing of Gce1pcould be demonstrated also in intact cells. Increased GPI cleavage after exposure of cells or spheroplasts to glucose is not due to stimulation of cell surface expression of Gce1p, as the amount of total GPI-anchored Gce1p bound to plasma membranes is comparable in cells grown in glucose or lactate. In agreement with this, Brefeldin A added together with the label blocks transport of newly made Gce1p to the cell surface and, in the consequence, cleavage of labeled Gce1p in spheroplasted yeast cells. (The drug is ineffective in intact cells). On the other hand, Brefeldin A does not significantly affect glucose-induced processing of inositol-labeled Gce1p at the plasma membrane when present during the period of nutritional upshift. We discuss that addition of glucose to the cells leads to the activation of a GPI-specific phospholipase which accepts only a subset of GPI proteins as substrates. This interpretation is additionally corroborated by the finding that purified [14C]inositol-labeled Gcep1p is lipolytically cleaved when incubated with Triton X-100-insoluble membrane complexes isolated from glucose-induced but not from uninduced spheroplasts. It is concluded that the phospholipase is present in these complexes and its state of activity is preserved during the preparation. GPI anchor cleavage in yeast appears to resemble strikingly the situation in insulin-responsive adipocytes subsequently to stimulation by insulin of glucose transport into these cells.
Assuntos
Glucose/farmacologia , Glicosilfosfatidilinositóis/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Brefeldina A , Proteínas de Transporte/química , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Ciclopentanos/farmacologia , Glicosilfosfatidilinositóis/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/efeitos dos fármacos , Fosfolipases/metabolismo , Conformação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/efeitos dos fármacos , Transdução de Sinais , Esferoplastos/metabolismoRESUMO
The insulin signal transduction cascade provides a number of sites downstream of the insulin receptor (IR) for cross-talk from other signaling pathways. Tyrosine phosphorylation of the IR substrates IRS-1/2 and metabolic insulin-mimetic activity in insulin-responsive cells can be provoked by soluble phosphoinositolglycans (PIG), which trigger redistribution from detergent-insoluble glycolipid-enriched raft domains (DIGs) to other areas of the plasma membrane and thereby activation of nonreceptor tyrosine kinases (NRTK) [Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol. Cell. Biol. 21, 4553-4567]. Here we describe that stimulation of glucose transport in isolated rat adipocytes by a different stimulus, the sulfonylurea glimepiride, is also based on IRS-1/2 tyrosine phosphorylation and downstream insulin-mimetic signaling involving activation of the NRTK, pp59(Lyn), and pp125(Fak), as well as tyrosine phosphoryation of the DIGs component caveolin. As is the case for PIG 41, glimepiride causes the concentration-dependent dissociation of pp59(Lyn) from caveolin and release of this NRTK and the glycosyl-phosphatidylinositol-anchored (GPI) proteins, Gce1 and 5'-nucleotidase, from total and anti-caveolin-immunoisolated DIGs. This results in their movement to detergent-insoluble raft domains of higher buoyant density (non-DIGs areas). IRS-1/2 tyrosine phosphorylation and glucose transport activation by both glimepiride and PIG are blocked by introduction into adipocytes of the caveolin scaffolding domain peptide which mimicks the negative effect of caveolin on pp59(Lyn) activity. Tyrosine phosphorylation of the NRTK, IRS-1/2, and caveolin as well as release of the NRTK and GPI proteins from DIGs and their redistribution into non-DIGs areas in response to PIG is also inhibited by treatment of intact adipocytes with either trypsin plus salt or N-ethylmaleimide (NEM). In contrast, the putative trypsin/salt/NEM-sensitive cell surface component (CIR) is not required for glimepiride-induced glucose transport, IRS-1/2 tyrosine phosphorylation, and redistribution of GPI proteins and NRTK. The data suggest that CIR is involved in concentrating signaling molecules at DIGs vs detergent-insoluble non-DIGs areas. These inhibitory interactions are relieved in response to putative physiological (PIG) or pharmacological (sulfonylurea) stimuli via different molecular mechanisms (dependent on or independent of CIR, respectively) thereby inducing IR-independent positive cross-talk to metabolic insulin signaling.
Assuntos
Adipócitos/efeitos dos fármacos , Glicosilfosfatidilinositóis/farmacologia , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Metabolismo dos Lipídeos , Transdução de Sinais , Compostos de Sulfonilureia/farmacologia , Adipócitos/metabolismo , Animais , Transporte Biológico , Caveolinas/metabolismo , Células Cultivadas , Etilmaleimida/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismo , Quinases da Família src/metabolismoRESUMO
A novel phosphoinositolglycan-peptide (PIG-P) from the yeast Saccharomyces cerevisiae potently mimicks insulin action on glucose transport and metabolism in rat muscle and adipose tissue. The aim of the present study was to elucidate the cellular signalling pathways of this insulin-mimetic compound. Rapid onset and reversibility of PIG-P action on glucose transport were observed in isolated adipocytes with a half-time of transport stimulation of 6-8 min (insulin less than 5 min). Combined treatment with PIG-P and insulin indicated additive stimulation of glucose transport at submaximal concentrations and non-additive action of both agents at maximal doses. The tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) was markedly increased in response to PIG-P in rat cardiomyocytes without any effect on the tyrosine phosphorylation of the insulin receptor beta-subunit. PIG-P action in these cells was accompanied by phosphorylation/dephosphorylation of several proteins with molecular masses of 15-30 kDa, a response not detected with insulin. Downstream signalling of IRS-1 was then analysed by monitoring IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in cardiomyocytes. A stable (2 and 15 min incubation with PIG-P) 7-fold stimulation corresponding to about 50% of insulin action could be detected. Increased tyrosine phosphorylation of IRS-1 and enhanced PI 3-kinase activity in response to PIG-P independent of the insulin receptor was also observed in isolated adipocytes. Involvement of PI 3-kinase in PIG-P action was subsequently confirmed by the dose-dependent inhibition of PIG-P-activated glucose transport in rat diaphragm and adipocytes by the PI 3-kinase inhibitors wortmannin and LY294002. These data suggest divergent upstream signalling by insulin and PIG-P involving phosphoproteins not affected by insulin. However, PIG-P and insulin action converge at the level of IRS-1 inducing insulin-independent PI 3-kinase-mediated signalling to glucose transport.
Assuntos
Tecido Adiposo/fisiologia , Glicosilfosfatidilinositóis/metabolismo , Insulina/fisiologia , Músculos/fisiologia , Animais , Transporte Biológico , Eletroforese em Gel Bidimensional , Glucose/metabolismo , Proteínas Substratos do Receptor de Insulina , Masculino , Miocárdio/metabolismo , Fosfoproteínas/metabolismo , Fosforilação , Fosfotirosina , Ratos , Ratos Wistar , Receptor de Insulina/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismoRESUMO
A set of synthetic phosphoinositolglycan (PIG) compounds has been demonstrated to exert insulin-mimetic activity on glucose and lipid metabolism in rat adipocytes differing considerably in potency [compound 41>37>45>>7>1; W. Frick, A. Bauer, J. Bauer, S. Wied and G. Müller, G. (1998) Biochemistry 37, 13421-13436]. In the present study we examine whether these differences are based on the capability of the PIG compounds to stimulate signalling components which are thought to mediate metabolic insulin action. Studies using a tyrosine kinase inhibitor and introduction into adipocytes of anti-phosphotyrosine or inhibitory anti-insulin receptor beta-subunit antibodies demonstrated dependence on tyrosine phosphorylation but independence of insulin receptor kinase activation of the insulin-mimetic signalling and metabolic activity of the PIG compounds. The five compounds elicited in rat adipocytes a significant increase in tyrosine phosphorylation of both insulin receptor substrate 1 (IRS-1) and IRS-3 and, to a minor degree, IRS-2, in IRS-1/3-associated phosphatidylinositol 3-kinase (PI 3-K) protein as well as activity, and in protein kinase B (PKB) activity as well as phosphorylation. This was most pronounced for compound 41, approaching 65-95% of the maximal insulin response (MIR) at 20 microM, and declined in the order of compounds 37, 45, 7 and 1. The same ranking was true for the maximal inhibition of glycogen synthase kinase 3 activity (GSK-3) (41, 75% of MIR; compound 37, 65%; compound 7, 25%; compound 1, 10%) and GSK-3 autophosphorylation. The half-maximal concentrations effective for signalling (compound 41, 2-5 microM; compound 37, 10-20 microM) corresponded well to those stimulating glucose and lipid metabolism. Interestingly, compounds 37 and 41 stimulated mitogen-activated protein kinase (MAPK) and protein synthesis in rat adipocytes to only about 20-30% (at 50 microM) of MIR. We conclude that in rat adipocytes: (i) the potency of PIG compounds to regulate glucose/lipid metabolism depends on the activation of PI 3-K and PKB and inhibition of GSK-3; (ii) initiation of tyrosine phosphorylation of IRS-1/3 is sufficient and activation of the PI 3-K cascade is required for insulin-mimetic metabolic signalling; and (iii) PIG compounds are quite selective for the PI 3-K compared to the MAPK cascade, (iv) PIG compounds seem to use the same signalling components downstream of PI 3-K (including Rab4) for stimulation of glucose transport as does insulin. Thus the early signalling step(s) used by PIG, but not by insulin, may represent a target for the treatment of insulin-resistant states.
Assuntos
Adipócitos/efeitos dos fármacos , Glicosilfosfatidilinositóis/metabolismo , Insulina/farmacologia , Mimetismo Molecular , Transdução de Sinais/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Transporte Biológico , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ativação Enzimática , Proteínas de Ligação ao GTP/metabolismo , Glucose/metabolismo , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Insulina/química , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Ratos , Ratos Wistar , Receptor de Insulina/metabolismo , Proteínas rab4 de Ligação ao GTPRESUMO
Phosphoinositolglycan molecules isolated from insulin-sensitive mammalian tissues have been demonstrated in numerous in vitro studies to exert partial insulin-mimetic activity on glucose and lipid metabolism in insulin-sensitive cells. However, their ill-defined structures, heterogeneous nature, and limited availability have prohibited the analysis of the underlying molecular mechanism. Phosphoinositolglycan-peptide (PIG-P) of defined and homogeneous structure prepared in large scale from the core glycan of a glycosyl-phosphatidylinositol-anchored membrane protein from Saccharomyces cerevisiae has recently been shown to stimulate glucose transport as well as a number of glucose-metabolizing enzymes and pathways to up to 90% (at 2 to 10 microns) of the maximal insulin effect in isolated rat adipocytes, cardiomyocytes, and diaphragms (G. Müller et al., 1997, Endocrinology 138: 3459-3476). Consequently, we used this PIG-P for the present study in which we compare its intracellular signaling with that of insulin. The activation of glucose transport by both PIG-P and insulin in isolated rat adipocytes and diaphragms was found to require stimulation of phosphatidylinositol (PI) 3-kinase but to be independent of functional p70S6kinase and mitogen-activated protein kinase. The increase in glycerol-3-phosphate acyltransferase activity in rat adipocytes in response to PIG-P and insulin was dependent on both PI 3-kinase and p70S6kinase. This suggest that the signaling pathways for PIG-P and insulin to glucose transport and metabolism converage at the level of PI 3-kinase. A component of the PIG-P signaling pathway located up-stream of PI 3-kinase was identified by desensitization of isolated rat adipocytes for PIG-P action by combined treatment with trypsin and NaCl under conditions that preserved cell viability and the insulin-mimetic activity of sodium vanadate but completely blunted the insulin response. Incubation of the cells with either trypsin or NaCl alone was ineffective. The desensitized adipocytes were reconstituted for stimulation of lipogenesis by PIG-P by addition of the concentrated trypsin/salt extract. The reconstituted adipocytes exhibited 65-75% of the maximal PIG-P response and similar EC50 values for PIG-P (2 to 5 microns) compared with control cells. A proteinaceous N-ethylmaleimide (NEM)-sensitive component contained in the trypsin/salt extract was demonstrated to bind in a functional manner to the adipocyte plasma membrane of desensitized adipocytes via bipolar interactions. An excess of trypsin/salt extract inhibited PIG-P action in untreated adipocytes in a competitive fashion compatible with a receptor function for PIG-P of this protein. The presence of the putative PIG-P receptor protein in detergent-insoluble complexes prepared from isolated rat adipocytes suggests that caveolae/detergent-insoluble complexes of the plasma membrane may play a role in insulin-mimetic signaling by PIG-P. Furthermore, treatment of isolated rat diaphragms and adipocytes with PIG-P as well as with other agents exerting partially insulin-mimetic activity, such as PI-specific phospholipase C (PLC) and the sulfonylurea glimepiride, triggered tyrosine phosphorylation of the caveolar marker protein caveolin, which was apparently correlated with stimulation of lipogenesis. Strikingly, in adipocytes subjected to combined trypsin/salt treatment, PIG-P, PI-specific PLC, and glimepiride failed completely to provoke insulin-mimetic effects. A working model is presented for a signaling pathway in insulin-sensitive cells used by PIG(-P) molecules which involves GPI structures, the trypsin/salt- and NEM-sensitive receptor protein for PIG-P, and additional proteins located in caveolae/detergent-insoluble complexes.
Assuntos
Caveolinas , Glicosilfosfatidilinositóis/fisiologia , Insulina/fisiologia , Transdução de Sinais/fisiologia , Adipócitos/metabolismo , Animais , Proteínas de Transporte/metabolismo , Caveolina 1 , Extratos Celulares/química , Glicosilfosfatidilinositóis/metabolismo , Proteínas Substratos do Receptor de Insulina , Masculino , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fosfoproteínas/fisiologia , Fosforilação , Ratos , Receptor de Insulina/fisiologia , Saccharomyces cerevisiae , Tripsina , Tirosina/metabolismoRESUMO
Phosphoinositolglycan (PIG) molecules have been implicated to stimulate glucose and lipid metabolism in insulin-sensitive cells and tissues in vitro and in vivo. The structural requirements for this partial insulin-mimetic activity remained unclear so far. For establishment of a first structure-activity relationship, a number of PIG compounds were synthesized consisting of the complete or shortened/mutated glycan moiety derived from the structure of the glycosylphosphatidylinositol (GPI) anchor of the GPI-anchored protein, Gce1p, from yeast. The PIG compounds were divided into four classes according to their insulin-mimetic activity in vitro with the typical representatives: compound 41, HO-SO2-O-6Manalpha1(Manalpha1-2)-2Manalpha1 (6-HSO3)- -6Manalpha1-4GluNb eta1-6(D)inositol-1,2-(cyclic)-phosphate; compound 37, HO-PO(H)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manal pha1-4GluNbeta1-6( D)inositol-1,2-(cyclic)-phosphate; compound 7, HO-PO(H)O-6Manalpha1-4GluN(1-6(L)inositol-1,2-(cyclic)-ph osp hate; and compound 1, HO-PO(H)O-6Manalpha1-4GluN(1-6(L)inositol. Compounds 41 and 37 stimulated lipogenesis up to 90% (at 20 microM) of the maximal insulin response but with differing concentrations required for 50% activation (EC50 values 2.5 +/- 0.9 vs 4.9 +/- 1.7 microM) as well as glycogen synthase (4.7 +/- 1 vs 9.5 +/- 1.5 microM) and glycerol-3-phosphate acyltransferase (3.5 +/- 0.8 vs 8.0 +/- 1.1 microM). Compound 7 was clearly less potent (20% of the maximal insulin response at 100 microM), whereas compound 1 was almost inactive. This relative ranking in the insulin-mimetic potency between members of the PIG classes (e.g., 41 > 37 >> 7 > 1) was also observed for the (i) activation of glucose transport and glucose transporter isoform 4 translocation in isolated normal and insulin-resistant adipocytes, (ii) inhibition of lipolysis in adipocytes, (iii) stimulation of glucose transport and glycogen synthesis in isolated normal and insulin-resistant diaphragms, and (iv) induction of tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) in diaphragms. The complete glycan core structure (Man3-GluN) of typical GPI anchors including a mannose side chain and the inositolphosphate moiety was required for maximal insulin-mimetic activity of the PIG compounds with some variations possible with respect to the type of residues coupled to the terminal mannose/inositol as well as the type of linkages involved. These data argue for the potency and specificity of the interaction of PIG molecules with putative signaling component(s) (presumably at the level of the IRS proteins) in adipose and muscle cells which finally lead to insulin-mimetic metabolic activity even in insulin-resistant states.