RESUMO
The metabolic syndrome describes a group of signs that increase the likelihood for developing type 2 diabetes mellitus, cardiovascular diseases and some types of cancer. The action of insulin depends on its binding to membrane receptors on its target cells. We wonder if blood insulin could travel bound to proteins and if, in the presence of hyperinsulinemia, a soluble insulin receptor might be generated. We used young adult Wistar rats (which have no predisposition to obesity or diabetes), whose drinking water was added 20 % of sugar and that were fed a standard diet ad libitum for two and six months. They were compared with control rats under the same conditions, but that had running water for consumption. At two months, the rats developed central obesity, moderate hypertension, high triglyceride levels, hyperinsulinemia, glucose intolerance and insulin resistance, i.e. metabolic syndrome. Electrophoresis of the rats' plasma proteins was performed, followed by Western Blot (WB) for insulin and for the outer portion of the insulin receptor. The bands corresponding to insulin and to the receptor external part were at the same molecular weight level, 25-fold higher than that of free insulin. We demonstrated that insulin, both in control animals and in those with hyperinsulinemia, travels bound to the receptor outer portion (ectodomain), which we called soluble insulin receptor, and that is released al higher amounts in response to plasma insulin increase; in rats with metabolic syndrome and hyperinsulinemia, plasma levels are much higher than in controls. Soluble insulin receptor increase in blood might be an early sign of metabolic syndrome.
El síndrome metabólico es un conjunto de signos que aumentan la probabilidad de desarrollar diabetes mellitus tipo 2, enfermedades cardiovasculares y algunos tipos de cáncer. La acción de la insulina depende de su unión a los receptores en la membrana de sus células diana. Para responder a la pregunta de si la insulina en la sangre podría viajar unida a proteínas y si en presencia de hiperinsulinemia podría generarse un receptor soluble de insulina, utilizamos ratas wistar (no tienen predisposición a la obesidad ni a la diabetes), adultas jóvenes, a cuya agua de consumo se adicionó 20 % de azúcar y a las que se les administró dieta estándar ad libitum, durante dos y seis meses; fueron comparadas con ratas control que tuvieron las mismas condiciones, pero con agua corriente para consumo. A los dos meses, las ratas desarrollaron obesidad central, hipertensión moderada, triglicéridos altos, hiperinsulinemia, intolerancia a la glucosa y resistencia a la insulina, es decir, síndrome metabólico. Se realizó electroforesis de las proteínas del plasma de las ratas, seguida de Western Blot para insulina y para la porción externa del receptor de insulina. Las bandas correspondientes a la insulina y la parte externa del receptor estaban al mismo nivel de peso molecular, 25 veces mayor que el de la insulina libre. Demostramos que la insulina, tanto en animales testigo como en aquellos con hiperinsulinemia, viaja unida a la porción externa del receptor (ectodominio), al cual denominamos receptor soluble de insulina, que se libera en mayor cantidad en respuesta al incremento en la insulina plasmática; en las ratas con síndrome metabólico e hiperinsulinemia, los niveles en plasma son mucho mayores que en los controles. El incremento del receptor soluble de insulina en sangre podría ser un dato temprano de síndrome metabólico.
Assuntos
Antígenos CD/sangue , Insulina/sangue , Síndrome Metabólica/sangue , Receptor de Insulina/sangue , Animais , Antígenos CD/fisiologia , Western Blotting , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Eletroforese , Hiperinsulinismo/sangue , Insulina/fisiologia , Resistência à Insulina , Síndrome Metabólica/etiologia , Ratos , Ratos Wistar , Receptor de Insulina/fisiologiaRESUMO
The metabolic syndrome describes a group of signs that increase the likelihood for developing type 2 diabetes mellitus, cardiovascular diseases and some types of cancer. The action of insulin depends on its binding to membrane receptors on its target cells. We wonder if blood insulin could travel bound to proteins and if, in the presence of hyperinsulinemia, a soluble insulin receptor might be generated. We used young adult Wistar rats (which have no predisposition to obesity or diabetes), whose drinking water was added 20 % of sugar and that were fed a standard diet ad libitum for two and six months. They were compared with control rats under the same conditions, but that had running water for consumption. At two months, the rats developed central obesity, moderate hypertension, high triglyceride levels, hyperinsulinemia, glucose intolerance and insulin resistance, i.e., metabolic syndrome. Electrophoresis of the rats' plasma proteins was performed, followed by Western Blot (WB) for insulin and for the outer portion of the insulin receptor. The bands corresponding to insulin and to the receptor external part were at the same molecular weight level, 25-fold higher than that of free insulin. We demonstrated that insulin, both in control animals and in those with hyperinsulinemia, travels bound to the receptor outer portion (ectodomain), which we called soluble insulin receptor, and that is released al higher amounts in response to plasma insulin increase; in rats with metabolic syndrome and hyperinsulinemia, plasma levels are much higher than in controls. Soluble insulin receptor increase in blood might be an early sign of metabolic syndrome.
Assuntos
Hiperinsulinismo/metabolismo , Resistência à Insulina/fisiologia , Insulina/metabolismo , Síndrome Metabólica/etiologia , Receptor de Insulina/metabolismo , Animais , Diabetes Mellitus Tipo 2/etiologia , Modelos Animais de Doenças , Intolerância à Glucose/etiologia , Humanos , Hipertensão/etiologia , Hipertrigliceridemia/etiologia , Insulina/sangue , Síndrome Metabólica/metabolismo , Obesidade Abdominal/etiologia , Ratos , Ratos WistarRESUMO
Interactions between extracellular matrix proteins and prostate carcinoma cells change dramatically during prostate tumor progression. We have concentrated on two key modifications that occur in the hemidesmosome in prostate carcinoma: loss of laminin-5 protein expression and altered basal cell polarity of the alpha6beta4 integrin. We previously demonstrated two cell line-specific isoforms (beta3A and beta3B) of the LAMB3 message. Cells expressing only the beta3B isoform did not translate the beta3 protein and were unable to assemble the laminin-5 trimer. One such cell line, LNCaP, was selected to determine whether restoration of the laminin-5 beta3A isoform would cause expression of a functional laminin-5 beta3 chain, assembly and secretion of the laminin-5 trimer, and reversion to a non-neoplastic phenotype. Laminin-5 beta3A cDNA was cloned and stably transfected into LNCaP cells. We observed the restoration of the beta3 protein, but a laminin-5 trimer was not secreted. Moreover, increased cell migration was demonstrated, and tumorigenicity was increased in SCID mice. A microarray analysis, performed between transfected and nontransfected LNCaP cells, showed most changing genes to be associated with signal transduction. The beta3 chain of laminin-5 may thus play an important role in signal transduction, which may enhance cell motility and tumorigenesis.
Assuntos
Carcinoma/metabolismo , Moléculas de Adesão Celular/biossíntese , Movimento Celular , Neoplasias da Próstata/metabolismo , Animais , Bioensaio , Carcinoma/patologia , Carcinoma/fisiopatologia , Adesão Celular/genética , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Membrana Celular/química , Perfilação da Expressão Gênica , Humanos , Integrina alfa6/análise , Integrina alfa6/metabolismo , Masculino , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/patologia , Neoplasias da Próstata/fisiopatologia , Isoformas de Proteínas/análise , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Transdução de Sinais/genética , Transfecção , CalininaRESUMO
In sequencing the beta3 chain of laminin 5 mRNA from LNCaP cells, we observed three different human cDNA clones (XM_001716, NM_000228 and L25541) in the GenBank that identified different sequences in the untranslated regions (UTR). XM_001716 and NM_000228 are almost identical cDNA clones with approximately 99% homology. However, they are quite different from L25541 in both the 5' UTR and the 3' UTR. Development of a PCR assay to specifically detect two of these different forms of the message led to the observation that they were differentially expressed in various cell lines. The message designated B3A (NM_000228, and XM_001716) was absent in LNCaP and MCF7 and greatly reduced in PC3-N, but was present in eight other epithelial cell lines. B3B (L25541) was present in all cell lines studied. The cell lines that failed to express the B3A form also failed to express the protein based on both immunoblotting and immunohistochemical analysis. It appears from this data that there are two isoforms of the beta3 mRNA, and that the 5' UTRs of the mRNAs play an important role in regulating translation of the beta3 protein. Since laminin 5 is lost in prostate carcinoma, the mechanism of control that results in the translation of the two forms of message may be important in tumorigenesis.
Assuntos
Laminina/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Imunofluorescência , Humanos , Immunoblotting , Laminina/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA/química , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The metabolic syndrome describes a group of signs that increase the likelihood for developing type 2 diabetes mellitus, cardiovascular diseases and some types of cancer. The action of insulin depends on its binding to membrane receptors on its target cells. We wonder if blood insulin could travel bound to proteins and if, in the presence of hyperinsulinemia, a soluble insulin receptor might be generated. We used young adult Wistar rats (which have no predisposition to obesity or diabetes), whose drinking water was added 20 % of sugar and that were fed a standard diet ad libitum for two and six months. They were compared with control rats under the same conditions, but that had running water for consumption. At two months, the rats developed central obesity, moderate hypertension, high triglyceride levels, hyperinsulinemia, glucose intolerance and insulin resistance, i.e., metabolic syndrome. Electrophoresis of the rats plasma proteins was performed, followed by Western Blot (WB) for insulin and for the outer portion of the insulin receptor. The bands corresponding to insulin and to the receptor external part were at the same molecular weight level, 25-fold higher than that of free insulin. We demonstrated that insulin, both in control animals and in those with hyperinsulinemia, travels bound to the receptor outer portion (ectodomain), which we called soluble insulin receptor, and that is released al higher amounts in response to plasma insulin increase; in rats with metabolic syndrome and hyperinsulinemia, plasma levels are much higher than in controls. Soluble insulin receptor increase in blood might be an early sign of metabolic syndrome.