ABSTRACT
Introduction: The pathophysiology of nonalcoholic steatohepatitis (NAS) includes, basically, insulin resistance, inflammation and oxidative stress. Thus, a study of immunostaining for liver insulin, adiponectin, tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) receptors was conducted. Objective: To expand the knowledge about the pathophysiological and molecular mechanisms underlying the experimental model of steatohepatitis in rats fed a high-fat diet. Method: Twenty Wistar rats were divided into two groups: G1 (control, fed a standard diet), and G2 (fed a high-fat diet containing 58% of energy derived from fat, 18% from protein and 24% from carbohydrate). After eight weeks the animals were sacrificed. Blood glucose, insulin, total cholesterol, high-density lipoprotein (HDL), the very low-density lipoproteins (VLDL), triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were determined. The liver tissue was submitted to histopathological analysis, using a NAS score. In immunohistochemistry, we studied the expression of the insulin receptor, adiponectin, TNF-α and iNOS by tissue microarray method. Results and conclusion: There was marked cytoplasmic immunostaining for TNF-α and iNOS mediators in the group on a fat diet. Regarding insulin and adiponectin molecular markers, a reduction of cytoplasmic immunoreactivity of these antigens was observed in the group on a fat diet, reflecting, respectively, the state of hepatocellular inflammation (steatohepatitis) and insulin resistance in this experimental model of fat liver disease...
Introdução: Os mecanismos fisiopatológicos da esteato-hepatite não alcoólica incluem basicamente resistência insulínica, processo inflamatório e estresse oxidativo. Desta forma, um estudo sobre o padrão de imunoexpressão hepática para receptores de insulina, adiponectina, fator de necrose tumoral alfa (TNF-α) e sintase indutível do óxido nítrico (iNOS) foi conduzido. Objetivo: Ampliar os conhecimentos sobre os mecanismos moleculares subjacentes, em modelo experimental de esteato-hepatite. Método: Vinte ratos Wistar com dois meses de idade, pesando de 250 a 300 mg foram subdivididos em dois grupos: G1 (controle normal, submetido à dieta padrão) e G2 (grupo-controle, submetido à dieta hiperlipídica contendo 58% de energia derivada de gorduras, 18% de proteínas e 24% de carboidratos). Após oito semanas, os animais foram sacrificados; o sangue, submetido à análise bioquímica; e o fígado, removido e fixado em formalina tamponada e emblocado em parafina para estudo histopatológico. Para estudo imuno-histoquímico, foi utilizada a técnica de microarranjo de tecido. As lâminas obtidas foram submetidas à incubação com os anticorpos contra adiponectina, receptor de insulina, TNF-α e iNOS. Resultados e conclusão: Observou-se marcada imunoexpressão citoplasmática para os mediadores TNF-α e iNOS no grupo submetido à dieta hiperlipídica. No que diz respeito aos marcadores moleculares insulina e adiponectina, observou-se uma redução da imunoexpressão citoplasmática desses anticorpos no grupo submetido à dieta hiperlipídica, traduzindo, respectivamente, o estado de inflamação hepatocelular (esteato-hepatite) e resistência insulínica, desenvolvidos nesse modelo experimental de doença hepática gordurosa...
Subject(s)
Animals , Rats , Adiponectin/analysis , Fatty Liver/physiopathology , Insulin Resistance , Immunohistochemistry/methods , Disease Models, Animal , Tumor Necrosis Factor-alpha/analysis , Fatty Liver/metabolism , Nitric Oxide Synthase/analysis , Rats, Wistar , Receptor, Insulin/analysisABSTRACT
A insulina é um hormônio anabólico com efeitos metabólicos potentes. Os eventos que ocorrem após a ligação da insulina são específicos e estritamente regulados. Definir as etapas que levam à especificidade desse sinal representa um desafio para as pesquisas bioquímicas, todavia podem resultar no desenvolvimento de novas abordagens terapêuticas para pacientes que sofrem de estados de resistência à insulina, especialmente a síndrome metabólica, que compreende o diabetes do tipo 2. O receptor de insulina pertence a uma família de receptores de fatores de crescimento que têm atividade tirosina quinase intrínseca. Após a ligação da insulina, o receptor sofre autofosforilação em múltiplos resíduos de tirosina. Isso resulta na ativação da quinase do receptor e na conseqüente fosforilação em tirosina de uma família de substratos do receptor de insulina (IAS). De forma similar a outros fatores de crescimento, a insulina usa fosforilação e interações proteína-proteína como ferramentas essenciais para transmitir o sinal. Dessa forma, o sinal é transmitido do receptor ao efetor final, promovendo a translocação de vesículas contendo transportadores de glicose (GLUT4) do conteúdo intracelular para a membrana plasmática, a ativação da síntese de glicogênio e de proteínas, e a transcrição de genes específicos.
Subject(s)
Humans , Male , Female , Protein-Tyrosine Kinases , Receptor, Insulin/analysis , Insulin Resistance/physiology , Diabetes Mellitus/physiopathology , Obesity/complications , Obesity/diagnosis , Metabolic Syndrome/complications , Metabolic Syndrome/diagnosisABSTRACT
Insulin stimulates the tyrosine kinase activity of its receptor resulting in the tyrosine phosphorylation of pp185, which contains insulin receptor substrates IRS-1 and IRS-2. These early steps in insulin action are essential for the metabolic effects of insulin. Feeding animals a high-fructose diet results in insulin resistance. However, the exact molecular mechanism underlying this effect is unknown. In the present study, we determined the levels and phosphorylation status of the insulin receptor and pp185 (IRS-1/2) in liver and muscle of rats submitted to a high-fructose diet evaluated by immunoblotting with specific antibodies. Feeding fructose (28 days) induced a discrete insulin resistance, as demonstrated by the insulin tolerance test. Plasma glucose and serum insulin and cholesterol levels of the two groups of rats, fructose-fed and control, were similar, whereas plasma triacylglycerol concentration was significantly increased in the rats submitted to the fructose diet (P<0.05). There were no changes in insulin receptor concentration in the liver or muscle of either group. However, insulin-stimulated receptor autophosphorylation was reduced to 72 + or - 4 percent (P<0.05) in the liver of high-fructose rats. The IRS-1 protein levels were similar in both liver and muscle of the two groups of rats. In contrast, there was a significant decrease in insulin-induced pp185 (IRS-1/2) phosphorylation, to 83 + or - 5 percent (P<0.05) in liver and to 77 + or - 4 percent (P<0.05) in muscle of the high-fructose rats. These data suggest that changes in the early steps of insulin signal transduction may have an important role in the insulin resistance induced by high-fructose feeding
Subject(s)
Animals , Rats , Male , Fructose/adverse effects , Liver/drug effects , Muscles/drug effects , Receptor, Insulin/analysis , Disease Models, Animal , Glucose Intolerance/metabolism , Insulin Resistance , Phosphorylation , Rats, WistarABSTRACT
Colon carcinoma is the most common tumor of the gastrointestinal tract. According to some investigators, insulin, epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) may be involved in the neoplastic proliferation. Insulin-binding and receptor tyrosine kinase activity were investigated in colon carcinomas and in normal colons. The insulin receptor concentration, as shown by binding assays, was 17.4 ñ 4.3 fmol/mug in normal colon and 29.69 ñ 9.4 fmol/mug in colon carcinoma. Nevertheless, the insulin affinity of the receptor was similar in both groups (Kd ( 1 nM). Both normal and neoplastic colon showed phosphorylation of the insulin receptor. The electrophoretic migration of the Beta-subunit of the insulin receptors purified from colon carcinomas was similar to that of normal colon and both tissues demonstrated an insulin-dependent autophosphorylation. The receptor tyrosine kinase activity was measured by the incorporation of [gamma32P]ATP into the Beta-subunit. The basal and the insulin-stimulated tyrosine kinase activities were significantly higher in colon carcinomas compared to normal colon tissues (2.2 and 1.6 times, respectively). Understanding the metabolism of neoplastic cells may contribute to the development of prevention strategies as well as new therapies. It is now necessary to study other steps of the insulin signal transduction pathway, such as insulin receptor substrate 1 phosphorylation.
Subject(s)
Humans , Colonic Neoplasms/chemistry , In Vitro Techniques , Receptor, Insulin/analysis , Wheat Germ AgglutininsABSTRACT
Se estudian los receptores de insulina en macrófagos peritoneales de ratón, inducidos con medio tioglicolato y en macrófagos residentes cultivados in vitro durante distintos tiempos. Los macrófagos inducidos presentan una disminución del número de receptores de insulina (1,5 x 10 4/célula con respecto a las residencias (5,2 x 10 4/célula) El número de receptores aumenta en los macrófagos peritoneales de 3,7 x 10 4/célula hasta 2,1 x 10 5/célula durante 72 horas de cultivo in vitro
Subject(s)
Mice , Animals , Male , Female , Macrophages/analysis , Receptor, Insulin/analysis , ThioglycolatesABSTRACT
Se realiza la cuantificación y caracterización de los receptores de insulina en la superficie de macrófagos peritoneales de ratón. Se detectaron 2 400 receptores por célula, los cuales presentan heterogenicidad aparente. La constante de afinidad para los receptores libres (ke) es de 2,82 x 108M-1 y para los receotores ocupados (kf) de 2,39 x 108M-1
Subject(s)
Mice , Animals , Macrophages/analysis , Receptor, Insulin/analysisABSTRACT
Con objeto de investigar el estado de los receptores celulares de insulina en la diabetes mellitus postpancreatitis, se estudiaron siete pacientes con diabetes consecutiva a pancreatitis y se compararon con diez voluntarios sanos. En todos se determinó la capacidad de unión a la insulina de los eritrocitos circulantes. La capacidad máxima de unión específica fue mayor en el testigo (23.0 ñ 3.5 vs 14.6 ñ 1.9% P <0.01), lo que se atribuyó a un aumento en la afinidad de los receptores. Esta se encontró en cuatro diabéticos con deficit ponderal quienes además tenían disminución en el número de los receptores por eritrocito; en los otros tres diabéticos que tenían peso normal la afinidad y el número de receptores estaban en límites normales. El estado de los receptores eritrocitarios en la diabetes postpancreatitis es variable y entre otros factores, puede modificarse por el estado nutricional
Subject(s)
Adolescent , Adult , Middle Aged , Humans , Male , Female , Diabetes Mellitus/physiopathology , Insulin/blood , Receptor, Insulin/analysis , Iodine Radioisotopes , Pancreatitis/complicationsABSTRACT
En timocitos de ratas adrenoprivas (Ax) y adrenoprivas-diabéticas (AxD) se estudió la presencia de receptores de insulina y la acción de la hormona "in vitro" e "in vivo" sobre el transporte de ácido alfa -aminoisobutírico (AIB). En ambos grupos la insulina "in vivo" incrementó el transporte dee AIB, mientras que "in vitro" no tuvo ningún efecto. El hecho de que los timocitos carecieran de receptores de insulina se correlaciona con la falta de acción "in vitro", mientras que el efecto "in vivo" sugeriría que la hormona ejercería sobre los timocitos de animales adultos un efecto indirecto a través de alteraciones metabólicas