RESUMEN
OBJECTIVES: Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS: The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS: Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION: Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.
Asunto(s)
Apoptosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Hipoxia , Ketamina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Supervivencia Celular , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Estrés Oxidativo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Venas UmbilicalesRESUMEN
OBJECTIVES: Hypoxia leads to endothelial cell inflammation, apoptosis, and damage, which plays an important role in the complications associated with ischemic cardiovascular disease. As an oxidoreductase, p66Shc plays an important role in the regulation of reactive oxygen species (ROS) production and apoptosis. Ketamine is widely used in clinics. This study was designed to assess the potential protective effect of ketamine against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs). Moreover, we explored the potential mechanism by which ketamine protected against hypoxia-induced endothelial injury. METHODS: The protective effects of ketamine against hypoxia-induced injury was assessed using cell viability and adhesion assays, quantitative polymerase chain reaction, and western blotting. RESULTS: Our data showed that hypoxia reduced HUVEC viability, increased the adhesion between HUVECs and monocytes, and upregulated the expression of endothelial adhesion molecules at the protein and mRNA levels. Moreover, hypoxia increased ROS accumulation and upregulated p66Shc expression. Furthermore, hypoxia downregulated sirt1 expression in HUVECs. Alternatively, ketamine was shown to reverse the hypoxia-mediated reduction of cell viability and increase in the adhesion between HUVECs and monocytes, ameliorate hypoxia-induced ROS accumulation, and suppress p66Shc expression. Moreover, EX527, a sirt1 inhibitor, reversed the protective effects of ketamine against the hypoxia-mediated reduction of cell viability and increase in adhesion between HUVECs and monocytes. CONCLUSION: Ketamine reduces hypoxia-induced p66Shc expression and attenuates ROS accumulation via upregulating sirt1 in HUVECs, thus attenuating hypoxia-induced endothelial cell inflammation and apoptosis.
Asunto(s)
Humanos , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Ketamina/farmacología , Hipoxia , Venas Umbilicales , Supervivencia Celular , Estrés Oxidativo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Programmed and damage aging theories have traditionally been conceived as stand-alone schools of thought. However, the p66Shc adaptor protein has demonstrated that aging-regulating genes and reactive oxygen species (ROS) are closely interconnected, since its absence modifies metabolic homeostasis by providing oxidative stress resistance and promoting longevity. p66Shc(-/-) mice are a unique opportunity to further comprehend the bidirectional relationship between redox homeostasis and the imbalance of mitochondrial biogenesis and dynamics during aging. This study shows that brain mitochondria of p66Shc(-/-) aged mice exhibit a reduced alteration of redox balance with a decrease in both ROS generation and its detoxification activity. We also demonstrate a strong link between reactive nitrogen species (RNS) and mitochondrial function, morphology, and biogenesis, where low levels of ONOO- formation present in aged p66Shc(-/-) mouse brain prevent protein nitration, delaying the loss of biological functions characteristic of the aging process. Sirt3 modulates age-associated mitochondrial biology and function via lysine deacetylation of target proteins, and we show that its regulation depends on its nitration status and is benefited by the improved NAD+/NADH ratio in aged p66Shc(-/-) brain mitochondria. Low levels of protein nitration and acetylation could cause the metabolic homeostasis maintenance observed during aging in this group, thus increasing its lifespan.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Mitocondrias/metabolismo , Especies de Nitrógeno Reactivo/metabolismo , Sirtuina 3/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Animales , Homeostasis , Ratones , Ratones NoqueadosRESUMEN
Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.
Asunto(s)
Antioxidantes/farmacología , Portadores de Fármacos/farmacología , Embrión de Mamíferos/efectos de los fármacos , Melatonina/farmacología , Poliésteres/química , Ácidos Polimetacrílicos/química , Animales , Antioxidantes/química , Apoptosis/efectos de los fármacos , Caspasa 3/genética , Caspasa 3/metabolismo , Catalasa/genética , Catalasa/metabolismo , Bovinos , Medios de Cultivo/química , Portadores de Fármacos/química , Composición de Medicamentos , Embrión de Mamíferos/fisiología , Desarrollo Embrionario/efectos de los fármacos , Femenino , Fertilización In Vitro , Regulación del Desarrollo de la Expresión Génica , Masculino , Melatonina/química , Nanocápsulas/química , Embarazo , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Accumulating evidence indicates that apoptosis is activated in the aged skeletal muscle, contributing to sarcopenia. We have previously demonstrated that testosterone protects against hydrogen peroxide (H2O2)-induced apoptosis in C2C12 muscle cells, at different levels: morphological, physiological, biochemical and molecular. In the present study we observed that H2O2 induces the mitochondrial permeability transition pore (mPTP) opening and exerts p53 activation in a time-dependent way, with a maximum response after 1-2h of treatment. Testosterone treatment, prior to H2O2, reduces not only p53 phosphorylation but also p66Shc expression, activation and its mitochondrial localization, at the same time that it prevents the mPTP opening. Furthermore, testosterone diminishes JNK and PKCßI phosphorylation induced by H2O2 and probably contributing thus, to reduce the activation of p66Shc. Thus, the mPTP opening, p53, JNK and PKCßI activation, as well as p66Shc mRNA increase, induced by oxidative stress, were reduced by testosterone pretreatment. The data presented in this work show some of the components upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents.
Asunto(s)
Estrés Oxidativo/efectos de los fármacos , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/metabolismo , Testosterona/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Ratones , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Músculo Esquelético/citología , Conformación Proteica/efectos de los fármacos , Proteína Quinasa C beta/metabolismo , Transporte de Proteínas/efectos de los fármacos , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src/genética , Factores de TiempoRESUMEN
17ß-Estradiol (E2) protects several non-reproductive tissues from apoptosis, including skeletal muscle. Previously, we showed that E2 at physiological concentrations prevented apoptosis induced by H2O2 in skeletal myoblasts. As we have also demonstrated a clear beneficial action of this hormone on skeletal muscle mitochondria, the present work further characterizes the signaling mechanisms modulated by E2 that are involved in mitochondria protection, which ultimately result in antiapoptosis. Here, we report that E2 through estrogen receptors (ERs) inhibited the H2O2-induced PKCδ and JNK activation, which results in the inhibition of phosphorylation and translocation to mitochondria of the adaptor protein p66Shc. In conjunction, the inhibition by the hormone of this H2O2-triggered signaling pathway results in protection of mitochondrial potential membrane. Our results provide basis for a putative mechanism by which E2 exerts beneficial effects on mitochondria, against oxidative stress, in skeletal muscle cells.
Asunto(s)
Estradiol/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Mioblastos Esqueléticos/efectos de los fármacos , Proteína Quinasa C-delta/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias Musculares/efectos de los fármacos , Fosforilación/efectos de los fármacos , Receptores de Estrógenos/metabolismo , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Mice lacking the 66 kDa isoform of the adapter molecule shcA (p66(shcA)) display increased resistance to oxidative stress and delayed aging. In cultured cell lines, p66 promotes formation of Reactive Oxygen Species (ROS) in mitochondria, and apoptotic cell death in response to a variety of pro-oxidant noxious stimuli. As mitochondrial ROS and oxidative cell damage are clearly involved in alcohol-induced pathology, we hypothesized that p66 may also have a role in ethanol. In vivo, changes observed in p66+/+ mice after 6-week exposure to ethanol in the drinking water, including elevated serum alanine aminotransferase (ALT), liver swelling and evident liver steatosis, were significantly attenuated in p66-/- mutant mice. Biochemical analysis of liver tissues revealed induction of the p66 protein by ethanol, whereas p66-deficient livers responded to alcohol with a significant upregulation of the mitochondrial antioxidant enzyme MnSOD, nearly absent in control mice. Evidence of an inverse correlation between expression level of p66 and protection from alcohol-induced oxidative stress was also confirmed in vitro in primary hepatocytes and in HepG2-E47 cells, an ethanol-responsive hepatoma cell line. In fact, MnSOD upregulation by exposure to ethanol in vitro was much more pronounced in p66KO versus wild-type isolated liver cells, and blunted in HepG2 cells overexpressing p66shc. p66 overexpression also prevented the activation of a luciferase reporter gene controlled by the SOD2 promoter, indicating that p66 repression of MnSOD operates at a transcriptional level. Finally, p66 generated ROS in HepG2 cells and potentiated oxidative stress and mitochondrial depolarization by ethanol. Taken together, the above observations clearly indicate a role for p66 in alcohol-induced cell damage, likely via a cell-autonomous mechanism involving reduced expression of antioxidant defenses and mitochondrial dysfunction.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Etanol/farmacología , Hígado/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Alanina Transaminasa/sangre , Animales , Carcinoma Hepatocelular , Línea Celular Tumoral , Hígado Graso Alcohólico/metabolismo , Hígado Graso Alcohólico/patología , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas , Masculino , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Especies Reactivas de Oxígeno/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Superóxido Dismutasa/metabolismoRESUMEN
Yeast soluble proteins were fractionated by calmodulin-agarose affinity chromatography and the Ca2+/calmodulin-binding proteins were analyzed by SDS-PAGE. One prominent protein of 66 kDa was excised from the gel, digested with trypsin and the masses of the resultant fragments were determined by MALDI/MS. Twenty-one of 38 monoisotopic peptide masses obtained after tryptic digestion were matched to the heat shock protein Ssb1/Hsp75, covering 37% of its sequence. Computational analysis of the primary structure of Ssb1/Hsp75 identified a unique potential amphipathic alpha-helix in its N-terminal ATPase domain with features of target regions for Ca2+/calmodulin binding. This region, which shares 89% similarity to the experimentally determined calmodulin-binding domain from mouse, Hsc70, is conserved in near half of the 113 members of the HSP70 family investigated, from yeast to plant and animals. Based on the sequence of this region, phylogenetic analysis grouped the HSP70s in three distinct branches. Two of them comprise the non-calmodulin binding Hsp70s BIP/GR78, a subfamily of eukaryotic HSP70 localized in the endoplasmic reticulum, and DnaK, a subfamily of prokaryotic HSP70. A third heterogeneous group is formed by eukaryotic cytosolic HSP70s containing the new calmodulin-binding motif and other cytosolic HSP70s whose sequences do not conform to those conserved motif, indicating that not all eukaryotic cytosolic Hsp70s are target for calmodulin regulation. Furthermore, the calmodulin-binding domain found in eukaryotic HSP70s is also the target for binding of Bag-1 - an enhancer of ADP/ATP exchange activity of Hsp70s. A model in which calmodulin displaces Bag-1 and modulates Ssb1/Hsp75 chaperone activity is discussed.
Asunto(s)
Calmodulina/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Saccharomyces cerevisiae/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Calmodulina/genética , Electroforesis en Gel de Poliacrilamida , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Espectrometría de Masas , Ratones , Filogenia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
BACKGROUND: Angiotensin II (AII) has been shown to contribute to the pathogenesis of hypertension and insulin resistance. In addition, the administration of selective AII type 1 receptor blockers has been shown to improve insulin sensitivity. However, only a few studies have addressed the molecular mechanisms involved in this association. Furthermore, in a previous study we illustrated that obese Zucker rats (OZR) present increased serine 994 (Ser994) phosphorylation of hepatic insulin receptor, and this event seems to be implicated in the regulation of the intrinsic IRK in this model of insulin resistance. OBJECTIVE AND DESIGN: We examined the effects of chronic treatment with irbesartan (50 mg/kg a day for 6 months) on the hepatic insulin signaling system of OZR. METHODS: The extent of phosphorylation of several components of the insulin signaling system was assessed by immunoprecipitation, followed by immunoblotting with phosphospecific antibodies. In addition, liver AII levels and fat deposits were determined by immunohistochemistry and Oil red O, respectively. RESULTS: OZR displayed a marked attenuation in the in-vivo phosphorylation of several components of the insulin signaling pathways in the liver, together with significantly higher hepatic AII levels and hepatic steatosis when compared with lean Zucker rats. We found that in the livers of OZR long-term administration of irbesartan is associated with: (i) increased insulin-stimulated insulin receptor tyrosine phosphorylation; (ii) decreased insulin receptor Ser994 phosphorylation; (iii) augmented insulin receptor substrate (IRS) 1 and 2 abundance and tyrosine phosphorylation; (iv) augmented association between IRS and the p85 regulatory subunit of phosphatidylinositol 3-kinase; (v) increased insulin-induced Akt phosphorylation; and (vi) decreased hepatic steatosis. CONCLUSION: The present study provides substantial information that demonstrates that long-term selective AII blockade by irbesartan improves insulin signaling and is associated with decreased insulin receptor Ser994 phosphorylation in the liver of a representative animal model of the human metabolic syndrome.
Asunto(s)
Bloqueadores del Receptor Tipo 1 de Angiotensina II/farmacología , Antihipertensivos/farmacología , Compuestos de Bifenilo/farmacología , Resistencia a la Insulina , Obesidad/metabolismo , Obesidad/fisiopatología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Tetrazoles/farmacología , Proteínas Adaptadoras Transductoras de Señales/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Análisis de Varianza , Angiotensina II/efectos de los fármacos , Angiotensina II/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/orina , Presión Sanguínea , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Hígado Graso/metabolismo , Hígado Graso/fisiopatología , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Irbesartán , Masculino , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/efectos de los fármacos , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Ratas , Ratas Zucker , Receptor de Insulina/efectos de los fármacos , Receptor de Insulina/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
PURPOSE: Ageing adversely affects the structure and function of lacrimal and salivary glands (LG and SG) and leads to marked insulin resistance that correlates with reduced insulin signal transduction. The aim of this study was to investigate whether ageing affects insulin signal transduction in LG and SG in vivo. METHODS: Male Wistar rats aged 20 months and 2 months (control group) were compared (n=8/group). Samples were removed under anaesthesia after i.v. injection of insulin, homogenized, immunoprecipitated with anti-insulin receptor (IR), Shc and STAT-1 antibodies and immunoblotted with antiphosphotyrosine antibody. RESULTS: The 20-month-old rats were significantly hyperinsulinaemic and presented a reduced rate of blood glucose disappearance in response to insulin, compared to the 2-month-old rats. The level of phosphorylation determined by densitometry in the older group of rats showed that ageing significantly reduced insulin-induced IR phosphorylation in LG and SG and STAT-1 phosphorylation in SG, compared to in the control group, but did not alter Shc phosphorylation. CONCLUSIONS: Ageing influences insulin signal transduction in the LG and SG of rats. Considering the major anabolic actions of insulin, these observations may help to explain the mechanisms of LG and SG dysfunctions observed in ageing.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Envejecimiento/fisiología , Aparato Lagrimal/fisiología , Glándulas Salivales/fisiología , Transducción de Señal/fisiología , Proteínas Adaptadoras del Transporte Vesicular , Animales , Glucemia/análisis , Proteínas de Unión al ADN/metabolismo , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Fosforilación , Ratas , Ratas Wistar , Receptor de Insulina/metabolismo , Factor de Transcripción STAT1 , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transactivadores/metabolismoRESUMEN
The structure and function of lacrimal and salivary glands present gender differences. Previous works have indicated a synergic action between insulin and androgens over lacrimal gland, and insulin-signaling pathways were recently described in lacrimal gland and salivary gland. Our present study investigates whether gender modulates the early steps of the insulin-signaling system in vivo. Eight-week-old male and female Wistar rats (n = 8/group) were compared to evaluate insulin serum levels and insulin tolerance tests by radioimmunoassay and glucose oxidase method, respectively. To assess insulin receptor (IR), Shc, STAT-1, ERK, and Akt phosphorylation in response to insulin in lacrimal gland and salivary gland, tissues from female and male rats (n = 5-8/group) were submitted to immunoprecipitation and immunoblotting or Western blotting protocol, and phosphorylation level was determined by densitometry. No difference was found in insulin serum levels or insulin tolerance tests comparing both groups. Nevertheless, lacrimal gland and salivary gland of female rats had a significantly lower insulin-induced IR phosphorylation compared with males. IR phosphorylation was not affected by the estrous cycle stage in either tissue. In addition, in females an apparent but not significant lower STAT and Akt phosphorylation in response to insulin was observed in the lacrimal gland, compared with males. Our findings suggest that alterations in insulin signal transduction may play a role in lacrimal gland and salivary gland gender differences.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Insulina/sangre , Aparato Lagrimal/metabolismo , Proteínas Serina-Treonina Quinasas , Glándulas Salivales/metabolismo , Caracteres Sexuales , Transducción de Señal/fisiología , Animales , Proteínas de Unión al ADN/metabolismo , Femenino , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Wistar , Receptor de Insulina/metabolismo , Factor de Transcripción STAT1 , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transactivadores/metabolismoRESUMEN
After receptor binding, growth hormone (GH) induces GH receptors (GHR) dimerization and JAK2 is activated after its association with a dimerized GHR, stimulating the tyrosyl phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2 and Shc proteins. G120K-PEG, a GH antagonist is produced by a mutation that blocks GH action by preventing the GHR dimerization. This study shows that the inhibitory effect of G120K-PEG was maximal with a GH:G120K-PEG ratio of 1:100, as no increase in JAK2 tyrosyl phosphorylation was observed with this dose of GH. When the dose of GH was increased and with a GH:G120K-PEG ratio of 1:10 some tyrosyl phosphorylation of JAK2 could be observed. Additionally, GH-induced IRS-1, IRS-2 and SHC tyrosyl phosphorylation was inhibited approximately 50% at equimolar concentrations of the antagonist of GH and almost abolished with a GH:G120K-PEG ratio of 1:100. The results clearly show that G120K-PEG inhibits GH signal transduction in mouse liver.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Antagonistas de Hormonas/farmacología , Hormona de Crecimiento Humana/antagonistas & inhibidores , Hígado/efectos de los fármacos , Polietilenglicoles/farmacología , Proteínas Proto-Oncogénicas , Transducción de Señal/efectos de los fármacos , Animales , Dimerización , Relación Dosis-Respuesta a Droga , Hormona de Crecimiento Humana/farmacología , Humanos , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 2 , Hígado/metabolismo , Masculino , Ratones , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/análisis , Prolactina/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Receptores de Somatotropina/química , Receptores de Somatotropina/efectos de los fármacos , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
Growth hormone (GH) and IGFs have a long distinguished history in diabetes, with possible participation in the development of renal complications. The implicated effect of GH in diabetic end-stage organ damage may be mediated by growth hormone receptor (GHR) or postreceptor events in GH signal transduction. The present study investigates the effects of diabetes induced by streptozotocin (STZ) on renal GH signaling. Our results demonstrate that JAK2, insulin receptor substrate (IRS)-1, Shc, ERKs, and Akt are widely distributed in the kidney, and after GH treatment, there is a significant increase in phosphorylation of these proteins in STZ-induced diabetic rats compared with controls. Moreover, the GH-induced association of IRS-1/phosphatidylinositol 3-kinase, IRS-1/growth factor receptor bound 2 (Grb2), and Shc/Grb2 are increased in diabetic rats as well. Immunohistochemical studies show that GH-induced p-Akt and p-ERK activation is apparently more pronounced in the kidneys of diabetic rats. Administration of G120K-PEG, a GH antagonist, in diabetic mice shows inhibitory effects on diabetic renal enlargement and reverses the alterations in GH signal transduction observed in diabetic animals. The present study demonstrates a role for GH signaling in the pathogenesis of early diabetic renal changes and suggests that specific GHR blockade may present a new concept in the treatment of diabetic kidney disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Diabetes Mellitus Experimental/fisiopatología , Hormona del Crecimiento/fisiología , Riñón/fisiopatología , Proteínas Proto-Oncogénicas , Receptores de Somatotropina/antagonistas & inhibidores , Transducción de Señal/fisiología , Sustitución de Aminoácidos , Animales , Glucemia/metabolismo , Peso Corporal , Diabetes Mellitus Experimental/patología , Proteína Adaptadora GRB2 , Hormona del Crecimiento/genética , Inmunohistoquímica , Proteínas Sustrato del Receptor de Insulina , Janus Quinasa 2 , Riñón/patología , Masculino , Tamaño de los Órganos , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Ratas , Ratas Wistar , Valores de Referencia , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Dominios Homologos srcRESUMEN
Shc protein phosphorylation has been extensively characterized as the initial step that activates a complex mitogenic pathway through its association with Grb2. In the present study, we investigated the adrenergic control of insulin-induced Shc phosphorylation and Shc-Grb2 association, and the modulating effect of streptozotocin-induced diabetes mellitus on Shc phosphorylation and Shc/Grb2 association. Acute treatment with epinephrine, which leads to a normoglycemic insulin-resistant state, does not affect insulin-induced Shc tyrosine phosphorylation or Shc-Grb2 association in liver, muscle, or fat. By contrast, a significant increase in insulin-induced Shc phosphorylation is observed in liver and muscle of rats treated with streptozotocin. The association of Shc/Grb2 is also increased in both tissues following insulin treatment. These data suggest that while epinephrine preserves the insulin-induced phosphorylation of Shc and the mitogenic pathway stimulated by Shc-Grb2 association, treatment with streptozotocin leads to a tissue-specific increase in the activity of the initial step that ultimately results in the activation of the Shc/Grb2 mitogenic pathway.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Tejido Adiposo/metabolismo , Agonistas Adrenérgicos/farmacología , Diabetes Mellitus Experimental/metabolismo , Epinefrina/farmacología , Insulina/fisiología , Hígado/metabolismo , Músculo Esquelético/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Dominios Homologos src/genética , Animales , Proteína Adaptadora GRB2 , Fosforilación , Ratas , Receptor de Insulina/metabolismo , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
PURPOSE: Insulin has been acknowledged as a mediator of several physiological events in lacrimal and salivary glands. We investigated the presence of insulin receptors and of insulin-induced autophosphorylation of the insulin receptor and activation of elements involved in the early steps of insulin signaling in lacrimal and salivary glands of rats. METHODS: Lacrimal and salivary glands of Wistar rats were removed and processed for immunohistochemistry using anti-insulin receptor and anti-IGF-1 receptor antibodies. The activation of insulin receptors following insulin treatment, and the involvement of insulin receptor substrates-1 and -2, Shc, JAK-2 and STAT-1, were analyzed by immunoprecipitation, followed by SDS-PAGE and immunoblotting of rat lacrimal and salivary glands after exposure to insulin. RESULTS: Insulin and IGF-1 receptors were present in rat lacrimal and salivary glands and were located predominantly in the cytoplasm and plasma membrane. Functional studies demonstrated that insulin induced a dose-dependent phosphorylation of the insulin receptor, IGF-1R, insulin receptor substrates-1 and -2, Shc, and STAT-1. In rats with streptozotocin-induced diabetes mellitus there was a significant reduction in insulin-induced insulin receptor and STAT-1 phosphorylation in the lacrimal gland but not in the salivary gland; there was no influence on Shc phosphorylation in either tissue. CONCLUSIONS: The present results indicate that insulin and IGF-1 receptors are expressed in lacrimal and salivary glands, and that insulin can induce the phosphorylation of its receptor and activate elements involved in the early steps of insulin signaling in both tissues.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Insulina/fisiología , Aparato Lagrimal/metabolismo , Proteínas Proto-Oncogénicas , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Glándulas Salivales/metabolismo , Transducción de Señal , Animales , Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Experimental/metabolismo , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 2 , Aparato Lagrimal/efectos de los fármacos , Masculino , Fosfoproteínas/metabolismo , Fosforilación , Pruebas de Precipitina , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción STAT1 , Glándulas Salivales/efectos de los fármacos , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Transactivadores/metabolismo , Tirosina/metabolismoRESUMEN
Insulin stimulates rapid tyrosine phosphorylation of the protein Shc, which subsequently binds to Grb2, resulting in the activation of a complex mitogenic signaling network. In this study, we examined the levels of Shc protein, its phosphorylation state and Shc-Grb2 association in liver, muscle and adipose tissue before and after insulin administration in three animal models of insulin resistance (chronic dexamethasone treatment, 72-h starvation and aging). There were no differences in Shc protein expression between tissues from control and insulin resistant animals. In fasted hypoinsulinemic rats, there was a decrease in insulin-induced Shc phosphorylation in liver and adipose tissue. However, a significant increase in Shc phosphorylation was observed in liver and muscle from dexamethasone-treated hyperinsulinemic rats and in liver, muscle and adipose tissue of hyperinsulinemic 20-month-old rats. Alterations in Shc phosphorylation correlated well with the level of Shc-Grb2 association. These results indicate that Shc tyrosyl phosphorylation and Shc-Grb2 association are regulated in the different types of insulin resistance and that this regulation is apparently related to the animals' plasma insulin levels. The Shc-Grb2 association is directly related to the insulin-induced tyrosyl phosphorylation of Shc.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Tejido Adiposo/metabolismo , Resistencia a la Insulina , Insulina/fisiología , Hígado/metabolismo , Músculo Esquelético/metabolismo , Proteínas/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Glucemia/metabolismo , Dexametasona/farmacología , Ingestión de Alimentos , Receptores ErbB/metabolismo , Ayuno , Proteína Adaptadora GRB2 , Hidrocortisona/sangre , Hiperinsulinismo/metabolismo , Insulina/sangre , Hígado/efectos de los fármacos , Masculino , Músculo Esquelético/efectos de los fármacos , Fosforilación , Ratas , Ratas Wistar , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Dominios Homologos srcRESUMEN
A growth-related branch of the insulin-signaling pathway was studied in the forebrain cortex and cerebellum of Wistar rats. Anesthetized rats received a bolus injection of saline or insulin through the cava vein after which fragments of cerebellum and forebrain cortex were excised and immediately homogenized. Insulin receptor and p46SHCA phosphorylation, and p46SHCA/GRB2 association were detected by immunoprecipitation and blotting with specific antibodies. Insulin stimulated the rapid phosphorylation of its receptor in cerebellum, followed by p46SHCA phosphorylation and GRB2 recruitment. The optimal insulin dose for the induction of p46SHCA/GRB2 binding was 60 microg, and time-course experiments showed that maximum phosphorylation/binding occurred 2-3 min after stimulation. Although insulin receptors and SHC were present in forebrain cortex, there was no increase in their phosphorylation, nor was there any recruitment of GRB2 following stimulation with insulin. Thus, although elements involved in the early intracellular response to insulin are present in the central nervous system, differences in their activation/regulation may account for the functional roles of insulin in these tissues.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Cerebelo/efectos de los fármacos , Hipoglucemiantes/farmacología , Insulina/farmacología , Prosencéfalo/efectos de los fármacos , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animales , Cerebelo/química , Proteína Adaptadora GRB2 , Masculino , Fosforilación , Prosencéfalo/química , Proteínas/análisis , Proteínas/metabolismo , Ratas , Ratas Wistar , Receptor de Insulina/análisis , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de SrcRESUMEN
GH stimulates the tyrosine phosphorylation of various cellular polypeptides, including the GH receptor itself, in an early part of the intracellular response. Some of these phosphorylations are catalyzed by a GH receptor-associated kinase identified as JAK2, a member of the Janus family of tyrosine kinases. In cultured cells, GH stimulates the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-2, and Shc. This study investigated whether GH could cause the tyrosine phosphorylation of IRSs and Shc proteins in fasted rat tissues in vivo. GH was administered to fasted Wistar rats via a portal vein, and extracts of different tissues were immunoprecipitated with specific antibodies. GH increased the tyrosine phosphorylation of IRS-1, IRS-2, JAK2, and Shc proteins in the liver, heart, kidney, muscle, and adipose tissue of rats. The roles of these substrates as signaling molecules for GH were further demonstrated by the finding that GH stimulated the association of IRS-1/2 with phosphatidylinositol 3-kinase, Grb2, and phosphotyrosine phosphatase and of Shc with Grb2. The correlation between JAK2 tyrosyl phosphorylation and IRS-1 tyrosyl phosphorylation in response to GH together with the results of the in vitro tyrosine kinase assay are consistent with the hypothesis that JAK2 may mediate GH-induced phosphorylation of IRS-1.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Adaptadoras del Transporte Vesicular , Hormona del Crecimiento/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogénicas , Receptor de Insulina/metabolismo , Tirosina/metabolismo , Animales , Proteína Adaptadora GRB2 , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 2 , Masculino , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6 , Proteínas Tirosina Fosfatasas/metabolismo , Ratas , Ratas Wistar , Proteínas Tirosina Fosfatasas con Dominio SH2 , Proteínas Adaptadoras de la Señalización Shc , Proteína Transformadora 1 que Contiene Dominios de Homología 2 de Src , Dominios Homologos srcRESUMEN
Shc is a novel type of tyrosine-phosphorylated protein activated in response to a wide variety of polypeptide ligands. In this study, we used immunoprecipitation and immunoblotting to examine the effect of insulin on Shc tyrosine phosphorylation and Shc/GRB2 association in insulin-sensitive tissues of the intact rat. Following an infusion of insulin, Shc was tyrosine-phosphorylated in the liver, skeletal muscle, and adipose tissue in a time- and dose-dependent fashion, which peaked 5 min after exposure to the hormone and, except in the case of adipose tissue, returned to basal values after 15 min. There was coimmunoprecipitation of Shc and the insulin receptor after stimulation with insulin. Receptor tyrosine kinase activity toward Shc was also observed. Following an infusion of insulin, Shc was found to associate with GRB2. These results demonstrate that after stimulation of rat tissues with insulin, Shc binds to the insulin receptor, is tyrosine-phosphorylated, and subsequently associated with GRB2.