RESUMO
AIMS/HYPOTHESIS: As obesity progresses, adipose tissue exhibits a hypoxic and inflammatory phenotype characterised by the infiltration of adipose tissue macrophages (ATMs). In this study, we examined how adipose tissue hypoxia is involved in the induction of the inflammatory M1 and anti-inflammatory M2 polarities of ATMs. METHODS: The hypoxic characteristics of ATMs were evaluated using flow cytometry after the injection of pimonidazole, a hypoxia probe, in normal-chow-fed or high-fat-fed mice. The expression of hypoxia-related and inflammation-related genes was then examined in M1/M2 ATMs and cultured macrophages. RESULTS: Pimonidazole uptake was greater in M1 ATMs than in M2 ATMs. This uptake was paralleled by the levels of inflammatory cytokines, such as TNF-α, IL-6 and IL-1ß. The expression level of hypoxia-related genes, as well as inflammation-related genes, was also higher in M1 ATMs than in M2 ATMs. The expression of Il6, Il1ß and Nos2 in cultured macrophages was increased by exposure to hypoxia in vitro but was markedly decreased by the gene deletion of Hif1a. In contrast, the expression of Tnf, another inflammatory cytokine gene, was neither increased by exposure to hypoxia nor affected by Hif1a deficiency. These results suggest that hypoxia induces the inflammatory phenotypes of macrophages via Hif1a-dependent and -independent mechanisms. On the other hand, the expression of inflammatory genes in cultured M2 macrophages treated with IL-4 responded poorly to hypoxia. CONCLUSIONS/INTERPRETATION: Adipose tissue hypoxia induces an inflammatory phenotype via Hif1a-dependent and Hif1a-independent mechanisms in M1 ATMs but not in M2 ATMs.
Assuntos
Tecido Adiposo/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia , Macrófagos/metabolismo , Tecido Adiposo/citologia , Alelos , Animais , Células da Medula Óssea/citologia , Polaridade Celular , Citometria de Fluxo , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Nitroimidazóis/farmacocinética , FenótipoRESUMO
AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Nefropatias Diabéticas/fisiopatologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/fisiologia , Substituição de Aminoácidos , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Biomarcadores/urina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Cruzamentos Genéticos , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Progressão da Doença , Mesângio Glomerular/patologia , Células Secretoras de Insulina/metabolismo , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Masculino , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Mutantes/fisiologia , Estresse Oxidativo , Oxirredutases/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Transdução de SinaisRESUMO
Proinflammatory cytokines are well-known to inhibit insulin signaling to result in insulin resistance. IL-1alpha is also one of the proinflammatory cytokines, but the mechanism of how IL-1alpha induces insulin resistance remains unclear. We have now examined the effects of IL-1alpha on insulin signaling in 3T3-L1 adipocytes. Prolonged IL-1alpha treatment for 12 to 24 hours partially decreased the protein levels as well as the insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. mRNA for SOCS3, an endogenous inhibitor of insulin signaling, was dramatically augmented 4 hours after IL-1alpha treatment. Concomitantly, the level of IL-6 in the medium and STAT3 phosphorylation were increased by the prolonged IL-1alpha treatment. Addition of anti-IL-6 neutralizing antibody to the medium or overexpression of dominant-negative STAT3 decreased the IL-1alpha-stimulated STAT3 activation and SOCS3 induction, and ameliorated insulin signaling. These results suggest that the IL-1alpha-mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.
Assuntos
Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Insulina/metabolismo , Interleucina-1alfa/farmacologia , Interleucina-6/biossíntese , Transdução de Sinais/efeitos dos fármacos , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Células 3T3-L1 , Animais , Anticorpos/farmacologia , Genes Dominantes , Humanos , Camundongos , Testes de Neutralização , Fosforilação/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas , Fatores de TempoRESUMO
The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.
Assuntos
Arrestinas/fisiologia , Endotelina-1/farmacologia , Glucose/metabolismo , Proteínas Musculares , Proteínas Proto-Oncogênicas/metabolismo , Quinases da Família src/metabolismo , Células 3T3 , Animais , Arrestinas/metabolismo , Transporte Biológico , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Transportador de Glucose Tipo 4 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Camundongos , Microscopia de Fluorescência , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas Proto-Oncogênicas c-yes , Transdução de Sinais , beta-Arrestina 1 , beta-ArrestinasRESUMO
Osmotic shock induces GLUT4 translocation and glucose uptake through a mechanism independent of PI 3-kinase, but dependent on tyrosine phosphorylation of cellular proteins. To identify the tyrosine phosphorylated proteins required for osmotic shock-stimulated glucose uptake, we examined tyrosine phosphorylation of candidate proteins, and found that the 60-80kDa species including paxillin and the 120-130kDa species including p130Cas, PYK2, FAK and Gab1 were tyrosine-phosphorylated in response to osmotic shock. Inhibition of actin polymerization by cytochalasin D significantly decreased the tyrosine phosphorylation of paxillin, p130Cas, PYK2 and FAK but not Gab1, but had no effect on 2-deoxyglucose (DOG) uptake, suggesting a role for Gab1 in osmotic shock-induced glucose transport. Also, we found that osmotic shock increases the association of phospholipase C-gamma (PLC-gamma) with Gab1 and stimulates tyrosine phosphorylation of PLC-gamma itself. The PLC inhibitor, U73122, inhibited osmotic shock-induced 2-DOG uptake. These results suggest that tyrosine phosphorylation of Gab1 and subsequent recruitment and activation of PLC-gamma may play a role in osmotic shock-induced glucose transport.
Assuntos
Adipócitos/metabolismo , Glucose/metabolismo , Isoenzimas/fisiologia , Fosfoproteínas/fisiologia , Fosfolipases Tipo C/fisiologia , Células 3T3 , Proteínas Adaptadoras de Transdução de Sinal , Adipócitos/efeitos dos fármacos , Animais , Citocalasina D/farmacologia , Desoxiglucose/metabolismo , Immunoblotting , Camundongos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Pressão Osmótica , Fosfatidilinositol 3-Quinases/metabolismo , Fosfolipase C gama , Fosforilação , Testes de Precipitina , Tirosina/metabolismoRESUMO
Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Aorta/metabolismo , DNA/biossíntese , Glucosamina/farmacologia , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Azasserina/farmacologia , Bromodesoxiuridina/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2 , Glucose/farmacologia , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.
Assuntos
Acetilcisteína/análogos & derivados , Insulina/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Acetilcisteína/farmacologia , Proteínas Adaptadoras de Transdução de Sinal , Adenoviridae/genética , Aminoácidos/química , Animais , Transporte Biológico , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Cisteína Endopeptidases/metabolismo , Citosol/metabolismo , Desoxiglucose/farmacocinética , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Fatores de Iniciação em Eucariotos , Glucose/metabolismo , Humanos , Immunoblotting , Proteínas Substratos do Receptor de Insulina , Camundongos , Complexos Multienzimáticos/metabolismo , Fosforilação , Testes de Precipitina , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases S6 Ribossômicas/metabolismo , Serina/química , Transdução de Sinais , Sirolimo/farmacologia , Frações Subcelulares/metabolismo , Serina-Treonina Quinases TOR , Treonina/química , Fatores de Tempo , Tirosina/metabolismoRESUMO
Growth hormone (GH) is well known to induce in vivo insulin resistance. However, the molecular mechanism of GH-induced cellular insulin resistance is largely unknown. In this study, we demonstrated that chronic GH treatment of differentiated 3T3-L1 adipocytes reduces insulin-stimulated 2-deoxyglucose (DOG) uptake and activation of Akt (also known as protein kinase B), both of which are downstream effects of phosphatidylinositol (PI) 3-kinase, despite enhanced tyrosine phosphorylation of insulin receptor substrate (IRS)-1, association of IRS-1 with the p85 subunit of PI 3-kinase, and IRS-1-associated PI 3-kinase activity. In contrast, chronic GH treatment did not affect 2-DOG uptake and Akt activation induced by overexpression of a membrane-targeted form of the p110 subunit of PI 3-kinase (p110(CAAX)) or Akt activation stimulated by platelet-derived growth factor. Fractionation studies indicated that chronic GH treatment reduces insulin-stimulated translocation of Akt from the cytosol to the plasma membrane. Interestingly, chronic GH treatment increased insulin-stimulated association of IRS-1 with p85 and IRS-1-associated PI 3-kinase activity preferentially in the cytosol. These results indicate that cellular insulin resistance induced by chronic GH treatment in 3T3-L1 adipocytes is caused by uncoupling between activation of PI 3-kinase and its downstream signals, which is specific to the insulin-stimulated PI 3-kinase pathway. This effect of GH might result from the altered subcellular distribution of IRS-1-associated PI 3-kinase.
Assuntos
Adipócitos/fisiologia , Hormônio do Crescimento Humano/farmacologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/fisiologia , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Citosol/metabolismo , Desoxiglucose/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina , Fator de Crescimento Insulin-Like I/farmacologia , Janus Quinase 2 , Cinética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Microssomos/metabolismo , Fosfoproteínas/metabolismo , Fosfotirosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , TransfecçãoRESUMO
Serum amyloid P component (SAP) is a common protein constituent of all types of amyloid deposits. Using SAP-deficient mice generated through gene targeting, we and others have shown that SAP significantly promotes amyloid deposition. It has been speculated that SAP protects amyloid fibrils from degradation by coating their exterior surface. To assess potential ways of treating individuals with amyloidosis, we examined the persistence of splenic AA amyloid fibrils in SAP-deficient and wild-type mice. No enhancement in the rate of regression of splenic AA amyloid was observed in the SAP-deficient mice relative to wild-type mice. These results present, for the first time, evidence that lack of SAP in AA amyloid deposits does not enhance regression of the deposits in vivo and suggest that dissociation of bound SAP from AA amyloid deposits would not significantly accelerate regression of the deposits in vivo.
Assuntos
Amiloidose/metabolismo , Proteína Amiloide A Sérica/metabolismo , Componente Amiloide P Sérico/metabolismo , Baço/metabolismo , Amiloidose/patologia , Animais , Sítios de Ligação , Deleção de Genes , Genótipo , Camundongos , Proteína Amiloide A Sérica/efeitos dos fármacos , Componente Amiloide P Sérico/deficiência , Componente Amiloide P Sérico/farmacologia , Baço/patologiaRESUMO
Tumor necrosis factor (TNF)-alpha is one of the candidate mediators of insulin resistance associated with obesity, a major risk factor for the development of type 2 diabetes. The insulin resistance induced by TNF-alpha is antagonized by thiazolidinediones (TZDs), a new class of insulin-sensitizing drugs. The aim of the current study was to dissect the mechanism whereby pioglitazone, one of the TZDs, ameliorates TNF-alpha-induced insulin resistance in 3T3-L1 adipocytes. Pioglitazone restored insulin-stimulated 2-deoxyglucose (DOG) uptake, which was reduced by TNF-alpha, with concomitant restorations in tyrosine phosphorylation and protein levels of insulin receptor (IR) and insulin receptor substrate (IRS)-1, as well as association of the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase with IRS-1 and PI 3-kinase activity. Adenovirus-mediated gene transfer of either wild-type human peroxisome proliferator-activated receptor (PPAR)-gamma2 or a mutant carrying a replacement at the consensus mitogen-activated protein kinase phosphorylation site (hPPAR-gamma2-S112A) promoted adipogenesis of 3T3-L1 fibroblasts and restored TNF-alpha-induced decrease of triglyceride in adipocytes as effectively as pioglitazone. Overexpression of the PPAR-gamma proteins in TNF-alpha-treated adipocytes restored protein levels of IR/IRS-1, but did not improve insulin-stimulated tyrosine phosphorylation of IR/IRS-1 or insulin-stimulated 2-DOG uptake. These results indicate that the ability of pioglitazone to restore insulin-stimulated tyrosine phosphorylation of IR/IRS-1, which is necessary for amelioration of TNF-alpha-induced insulin resistance, may be independent of the adipogenic activity of PPAR-gamma that regulates protein levels of IR/IRS-1.
Assuntos
Adipócitos/fisiologia , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Tiazóis/farmacologia , Tiazolidinedionas , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador alfa/farmacologia , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Diferenciação Celular , Linhagem Celular , Desoxiglucose/metabolismo , Humanos , Insulina/farmacologia , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Fosfotirosina/metabolismo , Pioglitazona , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Transfecção , Fator de Crescimento Transformador alfa/antagonistas & inibidoresRESUMO
In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells.
Assuntos
Adipócitos/citologia , Diferenciação Celular , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Quinases , Proteína do Retinoblastoma/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Células 3T3 , Adipócitos/efeitos dos fármacos , Adipócitos/enzimologia , Adipócitos/metabolismo , Androstadienos/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/enzimologia , Células Clonais/metabolismo , Dexametasona/farmacologia , Flavonoides/farmacologia , Insulina/farmacologia , Antagonistas da Insulina/farmacologia , MAP Quinase Quinase 1 , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Serina-Treonina Quinases TOR , WortmaninaRESUMO
Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.
Assuntos
Adipócitos/metabolismo , Desoxiglucose/metabolismo , Glucose/metabolismo , Ácido Palmítico/farmacologia , Células 3T3 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Diferenciação Celular , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Cinética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotirosina/metabolismo , Ratos , Receptor de Insulina/metabolismoRESUMO
To examine the molecular mechanism of insulin receptor trafficking, we investigated the intracellular signaling molecules that regulate this process in Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirect immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin receptor localization to perinuclear compartment. This insulin-induced insulin receptor trafficking was not affected by treatment of the cells with PI3-kinase inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated protein (MAP) kinase-Erk kinase] inhibitor (PD98059) partly inhibited the process in a dose-dependent manner. Interestingly, treatment with both wortmannin and PD98059 almost completely inhibited insulin receptor trafficking. The functional importance of PI3-kinase and MAP kinase in the trafficking process was directly assessed by using single cell microinjection analysis. Microinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein gave the same results as treatment with wortmannin and PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down-regulation from the cell surface and insulin receptor degradation, after prolonged incubation with insulin, were markedly impaired by the treatment. These results suggest that PI3-kinase and MAP kinase pathways synergistically regulate insulin receptor trafficking at a step subsequent to the receptor internalization.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Receptor de Insulina/fisiologia , Transdução de Sinais/fisiologia , Androstadienos/farmacologia , Animais , Linhagem Celular , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Humanos , Insulina/metabolismo , Insulina/farmacologia , Cinética , Ratos , Receptor de Insulina/efeitos dos fármacos , Receptor de Insulina/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , WortmaninaRESUMO
Wear of yttria-zirconia (zirconia) in the femoral head was investigated in mature mongrel dogs weighing 10 to 13 kg. Two dogs, which were used as a control group, were sacrificed 18 months after implantation of the uncemented modular hip system with an alumina ceramic (alumina) femoral head. A zirconia femoral head was implanted in five dogs: one was sacrificed 12 months after implantation, two at 18 months, and two at 24 months. In each femoral head and polyethylene (PE) socket, the surface was observed by means of scanning electron microscopy (SEM); the mean articulation surface roughness on the femoral head and PE socket and the thickness of the PE socket were measured. Wear was not seen on the surface of either the zirconia or the alumina heads. In both groups, minute white spots on the smooth surface of the PE socket were visible by SEM. In the alumina and zirconia groups the mean roughness was 0.1 microm. The mean thickness of the PE socket was reduced by 0.2 mm in the alumina group. In the zirconia group it was reduced by 0.2 to 0.3 mm. However, the mechanical strength of zirconia is known to be greater than that of alumina and it may be possible to reduce the diameter of the femoral head. The smaller zirconia head may contribute to the reduction of the wear of the PE socket in an uncemented modular total hip system.
Assuntos
Artroplastia de Quadril , Materiais Biocompatíveis , Prótese de Quadril , Ligas Metalo-Cerâmicas , Polietilenos , Zircônio , Animais , Cerâmica , CãesRESUMO
Shc is phosphorylated on Tyr-239/240 and/or Tyr-317, which serves as a docking site for Grb2. To clarify the relative involvement of Shc Tyr-239/240 and Tyr-317 in insulin-induced mitogenesis, we generated expression vectors for Y317F (1F)-Shc, Y239/240F (2F)-Shc, and Y239/240/317F (3F)-Shc, and stably transfected them into Rat1 fibroblasts expressing insulin receptors (HIRc). Insulin-induced Shc phosphorylation and subsequent association with Grb2 was enhanced in wild-type (WT)-Shc cell. In contrast, insulin-stimulated Shc phosphorylation and Shc.Grb2 association were significantly decreased in 1F-Shc and 3F-Shc cells, while these were only slightly affected and almost comparable in 2F cells compared with those in parental HIRc cells. The kinetics of MAP kinase activation closely paralleled the kinetics of Shc phosphorylation and Shc.Grb2 association. Thus, insulin stimulation of MAP kinase activation occurred more rapidly in WT-Shc cells, and the activation was delayed in 1F-Shc and 3F-Shc cells, while it was comparable in 2F-Shc cells compared with that in HIRc cells. Furthermore, WT-Shc cells displayed enhanced sensitivity to insulin stimulation of thymidine incorporation. Importantly, the sensitivity was significantly decreased in 1F-Shc and 3F-Shc cells, while it was almost comparable in 2F-Shc cells compared with that in HIRc cells. These results indicate that Shc Tyr-317 is more predominant insulin-induced phosphorylation site than Tyr-239/240 for coupling with Grb2 leading to MAP kinase activation and mitogenesis in Rat1 fibroblasts.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Insulina/farmacologia , Proteínas/metabolismo , Receptor de Insulina/fisiologia , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Clonagem Molecular , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/metabolismo , Proteínas/química , Ratos , Receptor de Insulina/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transfecção , Tirosina , Domínios de Homologia de srcRESUMO
We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Complexos Multienzimáticos/metabolismo , Receptor de Insulina/metabolismo , Animais , Anticorpos/administração & dosagem , Anticorpos/imunologia , Células COS , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Hidrólise , Imuno-Histoquímica , Microinjeções , Mutação , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Receptor de Insulina/genéticaRESUMO
We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Fibroblastos/metabolismo , Insulina/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases , Animais , Linhagem Celular , Proteína Substrato Associada a Crk , Proteínas do Citoesqueleto/metabolismo , Eletroforese em Gel de Poliacrilamida , Fatores de Troca do Nucleotídeo Guanina , Humanos , Proteínas de Membrana/metabolismo , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Proto-Oncogênicas c-cbl , Proteínas Proto-Oncogênicas c-crk , Ratos , Proteína p130 Retinoblastoma-Like , Proteínas Son Of Sevenless , SuínosRESUMO
Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated nonesterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1-4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu4:Tyr1), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7% that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Palmitatos/farmacologia , Receptor de Insulina/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Transportador de Glucose Tipo 1 , Humanos , Soros Imunes/imunologia , Immunoblotting , Insulina/análise , Insulina/farmacologia , Proteínas Substratos do Receptor de Insulina , Radioisótopos do Iodo , Camundongos , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/imunologia , Fosfatidilinositol 3-Quinases/análise , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/análise , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Testes de Precipitina , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/metabolismo , Proteínas/análise , Proteínas/efeitos dos fármacos , Proteínas/imunologia , Proteínas/metabolismo , Coelhos , Receptor de Insulina/análise , Receptor de Insulina/efeitos dos fármacos , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fatores de TempoRESUMO
The role of stress proteins on the function of insulin receptor is not well understood. In the rat-1 fibroblasts overexpressing human insulin receptors, heat shock protein (Hsp) 90 was co-immunoprecipitated with insulin receptors and the association was not affected by insulin stimulation. A GST-fusion protein containing the intracellular insulin receptor beta subunit was associated with Hsp 90 in vitro, suggesting the direct interaction of this protein with insulin receptor beta-subunit. Furthermore, microinjection of anti-Hsp 90 antibody into these cells completely inhibited insulin-stimulated mitogenesis. However, neither epidermal growth factor-stimulated nor serum-stimulated mitogenic signal in the cells was affected by the antibody microinjection. These results suggest that Hsp 90 constitutively binds to insulin receptor beta-subunit, which may be necessary for insulin signaling in mitogenesis.