RESUMO
BACKGROUND: The repair of burns in diabetic patients is a clinical problem. It is relevant to study alternative therapies that can improve the healing process. Our aim was to investigate the effects of Solidago chilensis associated or not with laser on burns in diabetic rats. METHODS: The animals were divided in four groups (n = 30): C- without treatment; S- S. chilensis extract; L-laser irradiated; LS- laser and S. chilensis. In 7, 14 and 21 days samples were collected after the injury to structural, morphometric and molecular analysis. RESULTS: Our results demonstrate the association of S. chilensis and laser reduced the inflammatory infiltrate and favored the angiogenesis. In the groups treated only with laser or with the plant extract showed higher levels of VEGF. The low-level laser therapy (LLLT) promoted higher collagen I and reduction of collagen III. It was also observed higher MMP-2 activation and a decreasing of the active isoform of MMP-9 in the S, L and LS groups. CONCLUSIONS: The treatments improved the repair of burns in diabetic rats, since it reduced the inflammatory infiltrate and favored the collagen organization presenting similar effects in the burn repair of the diabetics.
Assuntos
Queimaduras , Diabetes Mellitus Experimental , Solidago , Animais , Queimaduras/terapia , Humanos , Lasers , Ratos , Ratos Wistar , Solidago/química , CicatrizaçãoRESUMO
In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.
Assuntos
Citocinas/metabolismo , Ácidos Graxos/administração & dosagem , Hipotálamo/metabolismo , Obesidade/induzido quimicamente , Obesidade/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Animais , Anticorpos/administração & dosagem , Peso Corporal/efeitos dos fármacos , Citocinas/classificação , Citocinas/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipotálamo/efeitos dos fármacos , Imunoprecipitação , Indóis , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Microglia/efeitos dos fármacos , Mutação , Obesidade/imunologia , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologiaRESUMO
Acting in the hypothalamus, tumor necrosis factor-alpha (TNF-alpha) produces a potent anorexigenic effect. However, the molecular mechanisms involved in this phenomenon are poorly characterized. In this study, we investigate the capacity of TNF-alpha to activate signal transduction in the hypothalamus through elements of the pathways employed by the anorexigenic hormones insulin and leptin. High dose TNF-alpha promotes a reduction of 25% in 12h food intake, which is an inhibitory effect that is marginally inferior to that produced by insulin and leptin. In addition, high dose TNF-alpha increases body temperature and respiratory quotient, effects not reproduced by insulin or leptin. TNF-alpha, predominantly at the high dose, is also capable of activating canonical pro-inflammatory signal transduction in the hypothalamus, inducing JNK, p38, and NFkappaB, which results in the transcription of early responsive genes and expression of proteins of the SOCS family. Also, TNF-alpha activates signal transduction through JAK-2 and STAT-3, but does not activate signal transduction through early and intermediary elements of the insulin/leptin signaling pathways such as IRS-2, Akt, ERK and FOXO1. When co-injected with insulin or leptin, TNF-alpha, at both high and low doses, partially impairs signal transduction through IRS-2, Akt, ERK and FOXO1 but not through JAK-2 and STAT-3. This effect is accompanied by the partial inhibition of the anorexigenic effects of insulin and leptin, when the low, but not the high dose of TNF-alpha is employed. In conclusion, TNF-alpha, on a dose-dependent way, modulates insulin and leptin signaling and action in the hypothalamus.
Assuntos
Respiração Celular/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Insulina/metabolismo , Leptina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Relação Dose-Resposta a Droga , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Hipotálamo/metabolismo , Immunoblotting , Imunoprecipitação , Insulina/administração & dosagem , Insulina/farmacologia , Janus Quinase 2/metabolismo , Leptina/administração & dosagem , Leptina/farmacologia , Masculino , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic beta-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Proteínas SNARE/genética , Sinaptotagminas/genética , Animais , Animais Recém-Nascidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imunoquímica , Insulina/genética , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/embriologia , Microscopia Confocal , Gravidez , RNA Mensageiro/análise , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas SNARE/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Proteína 25 Associada a Sinaptossoma/metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 2 Associada à Membrana da Vesícula/metabolismoRESUMO
TNF-alpha acts on the hypothalamus modulating food intake and energy expenditure through mechanisms incompletely elucidated. Here, we explore the hypothesis that, to modulate insulin-induced anorexigenic signaling in hypothalamus, TNF-alpha requires the synthesis of NO. TNF-alpha activates signal transduction through JNK and p38 in hypothalamus, peaking at 10(-8) M. This is accompanied by the induction of expression of the inducible and neuronal forms of NOS, in both cases peaking at 10(-12) M. In addition, TNF-alpha stimulates NOS catalytic activity. Pre-treatment with TNF-alpha at a low dose (10(-12) M) inhibits insulin-dependent anorexigenic signaling, and this effect is abolished in iNOS but not in nNOS knockout mice.
Assuntos
Comportamento Alimentar/fisiologia , Hipotálamo/efeitos dos fármacos , Insulina/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Relação Dose-Resposta a Droga , Hipotálamo/fisiologia , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/administração & dosagemRESUMO
The cytokine-like hormone leptin is known to exert important functions on the modulation of immune responses. Some of these effects are dependent on the property of leptin to modulate the apoptosis of thymic cells. In the present study, we used Wistar rats to investigate the molecular mechanisms involved in leptin-dependent control of apoptosis in thymus. Apoptosis was evaluated by flow cytometry and ELISA for nucleosome determination, whereas signal transduction was evaluated by immunoprecipitation, immunoblot, and confocal microscopy. The Ob receptor (ObR) was expressed in most thymic cells and its relative amount reduced progressively during thymocyte maturation. ObR expression was colocalized with Janus kinase (JAK)-2 and signal transducer and activator of transcription-3, and an acute, in vivo, injection of leptin promoted the tyrosine phosphorylation of JAK-2 and the engagement of signal transducer and activator of transcription-3. The treatment with leptin also led to the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and serine phosphorylation of Akt. Chronic treatment with leptin reduced thymic apoptosis, an effect that was not inhibited by the JAK inhibitor AG(490) but was significantly inhibited by the phosphatidylinositol 3-kinase inhibitor LY(294002) and an antisense oligonucleotide to IRS-1. Thus, leptin inhibits the apoptosis of thymic cells through a mechanism that is independent of the activation of JAK-2 but depends on the engagement of the IRS-1/phosphatidylinositol 3-kinase pathway.
Assuntos
Apoptose/efeitos dos fármacos , Janus Quinase 2/fisiologia , Leptina/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Timo/efeitos dos fármacos , Animais , Proteínas Substratos do Receptor de Insulina , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Ratos Wistar , Receptores de Superfície Celular/análise , Receptores para Leptina , Fator de Transcrição STAT3/fisiologia , Transdução de Sinais/efeitos dos fármacos , Timo/citologiaRESUMO
Tumor necrosis factor-alpha (TNF-alpha) is known to participate in the wastage syndrome that accompanies cancer and severe infectious diseases. More recently, a role for TNF-alpha in the pathogenesis of type 2 diabetes mellitus and obesity has been shown. Much of the regulatory action exerted by TNF-alpha upon the control of energy stores depends on its action on the hypothalamus. In this study, we show that TNF-alpha activates canonical pro-inflammatory signal transduction pathways in the hypothalamus of rats. These signaling events lead to the transcriptional activation of an early responsive gene and to the induction of expression of cytokines and a cytokine responsive protein such as interleukin-1beta, interleukin-6, interleukin-10 and suppressor of cytokine signalling-3, respectively. In addition, TNF-alpha induces the expression of neurotransmitters involved in the control of feeding and thermogenesis. Thus, TNF-alpha may act directly in the hypothalamus inducing a pro-inflammatory response and the modulation of expression of neurotransmitters involved in energy homeostasis.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Neurotransmissores/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/administração & dosagem , Animais , Comportamento Animal , Western Blotting/métodos , Citocinas/genética , Interações Medicamentosas/fisiologia , Ingestão de Alimentos/efeitos dos fármacos , Genes Reporter/fisiologia , Hipotálamo/metabolismo , Injeções Intraventriculares/métodos , Insulina/farmacologia , Masculino , Neurotransmissores/genética , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transfecção/métodosRESUMO
The adaptation of pancreatic islets to pregnancy includes increased beta cell proliferation, expansion of islet mass, and increased insulin synthesis and secretion. Most of these adaptations are induced by prolactin (PRL). We have previously described that in vitro PRL treatment increases ERK3 expression in isolated rat pancreatic islets. This study shows that ERK3 is also upregulated during pregnancy. Islets from pregnant rats treated with antisense oligonucleotide targeted to the PRL receptor displayed a significant reduction in ERK3 expression. Immunohistochemical double-staining showed that ERK3 expression is restricted to pancreatic beta cells. Transfection with antisense oligonucleotide targeted to ERK3 abolished the insulin secretion stimulated by glucose in rat islets and by PMA in RINm5F cells. Therefore, we examined the participation of ERK3 in the activation of a cellular target involved in secretory events, the microtubule associated protein MAP2. PMA induced ERK3 phosphorylation that was companied by an increase in ERK3/MAP2 association and MAP2 phosphorylation. These observations provide evidence that ERK3 is involved in the regulation of stimulus-secretion coupling in pancreatic beta cells.
Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/biossíntese , Receptores da Prolactina/metabolismo , Animais , Células Cultivadas , Feminino , Glucose/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Modelos Animais , Oligonucleotídeos Antissenso , Fosforilação , Gravidez , Ratos , Ratos Wistar , Acetato de Tetradecanoilforbol/farmacologia , Regulação para CimaRESUMO
During pregnancy and the perinatal period of life, prolactin (PRL) and other lactogenic substances induce adaptation and maturation of the stimulus-secretion coupling system in pancreatic â-cells. Since the SNARE molecules, SNAP-25, syntaxin 1, VAMP-2, and synaptotagmins participate in insulin secretion, we investigated whether the improved secretory response to glucose during these periods involves alteration in the expression of these proteins. mRNA was extracted from neonatal rat islets cultured for 5 days in the presence of PRL and from pregnant rats (17th-18th days of pregnancy) and reverse transcribed. The expression of genes was analyzed by semi-quantitative RT-PCR assay. The expression of proteins was analyzed by Western blotting and confocal microscopy. Transcription and expression of all SNARE genes and proteins were increased in islets from pregnant and PRL-treated neonatal rats when compared with controls. The only exception was VAMP-2 production in islets from pregnant rats. Increased mRNA and protein expression of synaptotagmin IV, but not the isoform I, also was observed in islets from pregnant and PRL-treated rats. This effect was not inhibited by wortmannin or PD098059, inhibitors of the PI3-kinase and MAPK pathways, respectively. As revealed by confocal laser microscopy, both syntaxin 1A and synaptotagmin IV were immunolocated in islet cells, including the insulin-containing cells. These results indicate that PRL modulates the final steps of insulin secretion by increasing the expression of proteins involved in membrane fusion.
Assuntos
Animais , Feminino , Gravidez , Ratos , Regulação da Expressão Gênica no Desenvolvimento/genética , Insulina , Ilhotas Pancreáticas , Prolactina/farmacologia , Proteínas SNARE/genética , Sinaptotagminas/genética , Animais Recém-Nascidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Immunoblotting , Imunoquímica , Insulina/genética , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/embriologia , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , RNA Mensageiro/análise , Proteínas SNARE/metabolismo , /genética , /metabolismo , Sinaptotagminas/metabolismo , Sintaxina 1/genética , Sintaxina 1/metabolismo , /genética , /metabolismoRESUMO
OBJECTIVE: To investigate whether insulin and leptin share common intracellular signal transduction pathways and to determine whether these hormonal signaling systems modulate each other's action in rat hypothalamus. RESEARCH METHODS AND PROCEDURES: Male Wistar rats were studied after chronic implantation of an intracerebroventricular catheter into the third ventricle. Immunoprecipitation and immunoblotting were used to examine the activation of insulin and leptin signaling molecules in the rat hypothalamus. RESULTS: Insulin alone is able to produce molecular activation of insulin receptor substrates (IRSs)/phosphatidylinositol 3-kinase (PI 3-kinase)/Akt and mitogen-activated protein (MAP) kinase signaling pathways in hypothalamus, whereas leptin alone activates MAP kinase and IRSs/PI 3-kinase signaling with no effect on Akt. Combined infusion of leptin and insulin provokes a dual action. There was no quantitative potentialization of any single hormone's action on the elements of the insulin signaling pathway, IRSs/PI 3-kinase/Akt, and MAP kinase. Conversely, leptin plus insulin leads to quantitative potentialization of molecular signaling through the Janus kinase/signal transducer and activator of transcription pathway. DISCUSSION: We provide evidence for a convergence of leptin and insulin signaling at the level of IRSs-PI 3-kinase and a divergence at the level of Akt. Moreover, our results indicate a direct and positive cross-talk between insulin and leptin at the level of Janus kinase 2 and signal transducer and activator of transcription 3 tyrosine phosphorylation. This mechanism may serve to potentiate the activity of both insulin and leptin pathways and to increase stimulation in physiological processes such as the control of food intake and body weight, which are under the combined control of insulin and leptin.
Assuntos
Hipotálamo/fisiologia , Insulina/fisiologia , Leptina/fisiologia , Animais , Western Blotting , Proteínas de Ligação a DNA/fisiologia , Ingestão de Alimentos/fisiologia , Injeções Intraventriculares , Proteínas Substratos do Receptor de Insulina , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Fosfoproteínas/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Fator de Transcrição STAT3 , Transdução de Sinais/fisiologia , Transativadores/fisiologiaRESUMO
Prolactin (PRL) exerts its biological effects mainly by activating the Janus kinase/signal transducer and activator of transcription 5 (JAK/STAT5) signaling pathway. We have recently demonstrated that PRL also stimulates the insulin receptor substrates/phosphatidylinositol 3-kinase (IRSs/PI3K) and SH2-plekstrin homology domain (SHC)/ERK pathways in islets of neonatal rats. In the present study, we investigated the involvement of the PI3K and MAP kinase (MAPK) cascades in islet development and growth in pregnant rats. The protein expression of AKT1, p70S6K and SHC was higher in islets from pregnant compared with control rats. Higher basal levels of tyrosine phosphorylation were found in classic transducers of insulin cell signaling (IRS1, IRS2 and SHC). Increased levels of threonine/tyrosine phosphorylation of ERK1/2 and serine phosphorylation of AKT and p70S6K were also detected. To assess the participation of PRL in these phenomena, pregnant and control rats were treated with an antisense oligonucleotide to reduce the expression of the PRL receptor (PRLR). Phosphorylation of AKT was reduced in islets from pregnant and control rats, whereas p70S6K protein levels were reduced only in islets from treated pregnant rats. Finally, glucose-induced insulin secretion was reduced in islets from pregnant but not from control rats treated with the PRLR antisense oligonucleotide. In conclusion, downstream proteins of the PI3K (AKT and p70S6K) and MAPK (SHC and ERK1/2) cascades are regulated by PRL signaling in islets from pregnant rats. These findings indicate that these pathways participate in the increase in islet mass and the sensitivity to glucose during pregnancy.
Assuntos
Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Receptores da Prolactina/metabolismo , Animais , Células Cultivadas , Feminino , Glucose/farmacologia , Insulina/metabolismo , Secreção de Insulina , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Gravidez , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Ratos , Ratos Wistar , Receptores da Prolactina/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismoRESUMO
The effects of prolactin (PRL) on transcript profile expression in 24h cultured pancreatic adult rat islets were investigated by cDNA expression array analysis to identify possible candidate mRNA species that encode proteins involved in the maturation and growth of the endocrine pancreas. The expression of 54 out of 588 genes was altered by treatment with PRL. The differentially expressed transcripts identified were distributed in six main categories involved in cell proliferation and differentiation, namely, cell cycle regulation, signal transduction, transcription factors and coactivators, translational machinery, Ca(2+)-mediated exocytosis, and immuno-response. Treatment with PRL also reduced the expression of genes related to apoptosis. Several genes, whose expression was previously not known to be modulated by PRL were also identified including macrophage migration inhibitory factor and Ca(2+)/calmodulin-dependent protein kinase IV. These genes have recently been shown to play a crucial role in insulin secretion and insulin gene expression, respectively. Treatment with PRL also modified the expression of AKT2 and bone morphogenetic protein receptor 1A that control glucose homeostasis and directly affect the behavior of endocrine pancreas and/or the sensitivity of target tissues to insulin. In conclusion, PRL induces several patterns of gene expression in pancreatic islet cells. The analysis of these different patterns will be useful for understanding the complex mechanism of action of PRL in the maturation and differentiation of pancreatic islets.
Assuntos
Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Prolactina/farmacologia , Animais , Western Blotting , Células Cultivadas , Feminino , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Short-term muscle denervation is a reproducible model of tissue-specific insulin resistance. To investigate the molecular basis of insulin resistance in denervated muscle, the downstream signaling molecules of the insulin-signaling pathway were examined in intact and denervated soleus muscle of rats. Short-term denervation induced a significant fall in glucose clearance rates (62% of control, P < 0.05) as detected by euglycemic hyperinsulinemic clamp and was associated with a significant decrease in insulin-stimulated tyrosine phosphorylation of the insulin receptor (IR; 73% of control, P < 0.05), IR substrate 1 (IRS1; 69% of control, P < 0.05), and IRS2 (82% of control, P < 0.05) and serine phosphorylation of Akt (39% of control, P < 0.05). Moreover, denervation reduced insulin-induced association between IRS1/IRS2 and p85/phosphatidylinositol (PI) 3-kinase. Nevertheless, denervation caused an increase in PI 3-kinase activity associated with IRS1 (275%, P < 0.05) and IRS2 (180%, P < 0.05), but the contents of phosphorylated PI detected by HPLC were significantly reduced in lipid fractions. In the face of the apparent discrepancy, we evaluated the expression and activity of the 5-inositol, lipid phosphatase SH2 domain-containing inositol phosphatase (SHIP2), and the serine phosphorylation of p85/PI 3-kinase. No major differences in SHIP2 expression were detected between intact and denervated muscle. However, serine phosphorylation of p85/PI 3-kinase was reduced in denervated muscle, whereas the blockade of SHIP2 expression by antisense oligonucleotide treatment led to partial restoration of phosphorylated PI contents and to improved glucose uptake. Thus modulation of the functional status of SHIP2 may be a major mechanism of insulin resistance induced by denervation.
Assuntos
Resistência à Insulina , Músculo Esquelético/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Glicemia/metabolismo , Modelos Animais de Doenças , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Masculino , Denervação Muscular , Músculo Esquelético/inervação , Oligonucleotídeos Antissenso/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Fosfatidilinositóis/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Tirosina/metabolismoRESUMO
Several neural, hormonal and biochemical inputs actively participate in the balance of insulin secretion induced by blood glucose fluctuations. The exact role of insulin as an autocrine and paracrine participant in the control of its own secretion remains to be determined, mostly due to insufficient knowledge about the molecular phenomena that govern insulin signaling in pancreatic islets. In the present experiments we demonstrate that higher insulin receptor and insulin receptor substrates-1 and -2 (IRS1 and IRS2) concentrations are predominantly encountered in cells of the periphery of rat pancreatic islets, as compared to centrally located cells, and that partial blockade of IRS1 protein expression by antisense oligonucleotide treatment leads to improved insulin secretion induced by glucose overload, which is accompanied by lower steady-state glucagon secretion and blunted glucose-induced glucagon fall. These data reinforce the inhibitory role of insulin upon its own secretion in isolated, undisrupted pancreatic islets.
Assuntos
Glucose/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Fosfoproteínas/antagonistas & inibidores , Animais , Sequência de Bases , Primers do DNA , Imuno-Histoquímica , Proteínas Substratos do Receptor de Insulina , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Ratos , Ratos WistarRESUMO
We examined the effect of three daily foot-shock stress sessions on glucose homeostasis, insulin secretion by isolated pancreatic islets, insulin sensitivity of white adipocytes, and glycogen stores in the liver and soleus muscle of rats. Stressed rats had plasma glucose (128.3 +/- 22.9 mg/dL) and insulin (1.09 +/- 0.33 ng/mL) levels higher than the controls (glucose, 73.8 +/- 3.5 mg/dL; insulin, 0.53 +/- 0.11 ng/mL, ANOVA plus Fisher's test; p < 0.05). After a glucose overload, the plasma glucose, but not insulin, levels remained higher (area under the curve 8.19 +/- 1.03 vs. 4.84 +/- 1.33 g/dL 30 min and 102.7 +/- 12.2 vs. 93.2 +/- 16.1 ng/mL 30 min, respectively). Although, the area under the insulin curve was higher in stressed (72.8 +/- 9.8 ng/mL) rats than in control rats (34.9 +/- 6.9 ng/mL) in the initial 10 min after glucose overload. The insulin release stimulated by glucose in pancreatic islets was not modified after stress. Adipocytes basal lipolysis was higher (stressed, 1.03 +/- 0.14; control, 0.69 +/- 0.11 micromol of glycerol in 60 min/100 mg of total lipids) but maximal lipolysis stimulated by norepinephrine was not different (stressed, 1.82 +/- 0.35; control, 1.46 +/- 0.09 micromol of glycerol in 60 min/100 mg of total lipids) after stress. Insulin dose-dependently inhibited the lipolytic response to norepinephrine by up to 35% in adipocytes from control rats but had no effect on adipocytes from stressed rats. The liver glycogen content was unaltered by stress, but was lower in soleus muscle from stressed rats than in control rats (0.45 +/- 0.04 vs. 0.35 +/- 0.04 mg/100 mg of wet tissue). These results suggest that rats submitted to foot-shock stress develop hyperglycemia along with hyperinsulinemia as a consequence of insulin subsensitivity in adipose tissue, with no alteration in the pancreatic sensitivity to glucose. Foot-shock stress may therefore provide a useful short-term model of insulin subsensitivity.