RESUMEN
OBJECTIVE: To determine the in vivo effects of intraarticular MMP-13. METHODS: Human recombinant MMP-13 was injected intraarticularly (i.a. ) into the hamster knee joint. MMP-13 activity, collagen and proteoglycan fragments, and hyaluronan were measured in synovial fluid. Antibody 9A4 was used to localize collagen damage. Western blotting was used to determine the size of type II collagen fragments. RESULTS: MMP-13 activity measurements showed greater than 98% of MMP-13 to be cleared instantly from the joint cavity. The remainder was cleared with a t(1/2)of 2 h. Immunohistochemical staining demonstrated collagen cleavage was limited to a thin superficial band on the surface of the articular cartilage whereas collagen damage extended more deeply into the synovial capsule and the menisci. The elevation of proteoglycan and hyaluronan in synovial fluid after MMP-13 was modest. Collagen fragments appeared in synovial fluid within 15 min following MMP-13. They were cleared with a half-life of circa 1.8 h and the predominant fragment was 32 kDa. CONCLUSIONS: Activated MMP-13 leads to tissue collagen damage with the release of collagen fragments. These fragments are measurable and could provide a method for assessment of cartilage collagen damage.
Asunto(s)
Cartílago Articular/efectos de los fármacos , Colagenasas/farmacología , Animales , Cartílago Articular/enzimología , Cartílago Articular/metabolismo , Colágeno/metabolismo , Colagenasas/farmacocinética , Cricetinae , Técnicas de Cultivo , Femenino , Humanos , Inyecciones Intraarticulares , Metaloproteinasa 13 de la Matriz , Mesocricetus , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Líquido Sinovial/enzimología , Líquido Sinovial/metabolismoRESUMEN
Experimental allergic encephalomyelitis is a murine model of preclinical autoimmune disease that has pathologic similarities to multiple sclerosis (MS). Although CD4+ T cells have been shown to play a crucial role in the development of disease, we recently demonstrated a link between the development of paralysis and eosinophil infiltration into the spinal cord. As such, CD4+ cells may initiate disease, but eosinophils may be the actual effector cells responsible for causing damage to myelin and causing paralysis. Because MS patients sometimes experience early visual problems, ie, optic neuritis, we explored whether an early eosinophil infiltrate was also observed in the optic nerves of SJL mice after the passive transfer of encephalitogenic T cells. Seven days after the passive transfer of myelin basic protein (MBP)-reactive T cell blasts, we observed a significant infiltration of eosinophils into the optic nerves of the mice. This infiltration persisted during the early phases of paralysis, then declined to baseline values by the peak of limb paralysis on Day 10, and remained at baseline during the remission phase of the disease. Remyelination of optic nerves was observed at this time. These results suggest that eosinophil infiltration into the optic nerve is one of the earliest events occurring after the passive transfer of encephalitogenic T cells in murine experimental allergic encephalomyelitis.
Asunto(s)
Encefalomielitis Autoinmune Experimental/patología , Neutrófilos/fisiología , Nervio Óptico/patología , Animales , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Inmunización Pasiva , Transfusión de Linfocitos , Ratones , Ratones Endogámicos , Proteína Básica de Mielina/inmunología , Neutrófilos/patología , Nervio Óptico/fisiopatología , Nervio Óptico/ultraestructura , Linfocitos T , Factores de TiempoRESUMEN
Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.
Asunto(s)
Benzopiranos/farmacología , Ácidos Carboxílicos/farmacología , Movimiento Celular/efectos de los fármacos , Encefalomielitis Autoinmune Experimental/etiología , Eosinófilos/efectos de los fármacos , Receptores de Leucotrieno B4/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Benzopiranos/uso terapéutico , Ácidos Carboxílicos/uso terapéutico , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/prevención & control , Femenino , Inmunización Pasiva , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Oligopéptidos/inmunología , Parálisis/prevención & control , Médula Espinal/patología , Linfocitos T/inmunologíaRESUMEN
The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter) expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and 360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice (approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in the basal state, which markedly increased after an insulin/glucose injection. In contrast, nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein in the basal state with a relatively small increase after an insulin/glucose challenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the glucose transport defect in db/db mice is at the level of glucose transporter translocation. Together, these data demonstrate that GLUT4 upregulation overcomes the glucose transporter translocation defect and alleviates insulin resistance in genetically diabetic mice, thus resulting in markedly improved glycemic control.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hiperglucemia/metabolismo , Proteínas de Transporte de Monosacáridos/biosíntesis , Proteínas Musculares , Tejido Adiposo/química , Tejido Adiposo/citología , Factores de Edad , Animales , Transporte Biológico , Glucemia/análisis , Peso Corporal , Compartimento Celular , Membrana Celular/química , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Tipo 2/genética , Carbohidratos de la Dieta/metabolismo , Femenino , Expresión Génica , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Hiperglucemia/genética , Insulina/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/aislamiento & purificación , Miocardio/química , Miocardio/citología , Distribución TisularRESUMEN
Human GLUT4 protein expression in muscle and adipose tissues of transgenic mice decreases plasma insulin and glucose levels and improves glucose tolerance compared with nontransgenic controls (Liu, M.-L., Gibbs, E. M., McCoid, S. C., Milici, A. J., Stukenbrok, H. A., McPherson, R. K., Treadway, J. L., and Pessin, J. E. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11346-11350). We examined the basis of improved glycemic control in hGLUT4 transgenic mice by determining glucose homeostasis and metabolic profiles in vivo. Glucose turnover experiments indicated a 1.4-fold greater systemic glucose clearance in hGLUT4 mice relative to controls (p < 0.05), whereas hepatic glucose production was similar despite 26% lower (p < 0.05) glucose levels. Glucose infusion rate during an euglycemic-hyperinsulinemic clamp was 2-fold greater (p < 0.05) in hGLUT4 mice versus controls, and skeletal muscle and heart glycogen content were increased 3-5-fold (p < 0.05). The increased peripheral glucose clearance in hGLUT4 mice was associated with increased (25-32%) basal and insulin-stimulated glucose transport rate in soleus muscle (p < 0.01), and increased muscle plasma membrane-associated GLUT4 protein. Fed hGLUT4 mice displayed 20-30% lower plasma glucose and insulin levels (p < 0.05) and 43% elevated glucagon levels (p < 0.001) compared with controls. Triglycerides, free fatty acids, and beta-hydroxy-butyrate were elevated 43-63% (p < 0.05) in hGLUT4 mice due to hypoinsulinemia-induced lipolysis. Free fatty acids and beta-hydroxybutyrate levels in hGLUT4 mice increased further upon fasting, and skeletal muscle glycogen levels decreased markedly compared with controls. The data demonstrate that high level expression of hGLUT4 increases systemic glucose clearance and muscle glucose utilization in vivo and also results in marked compensatory lipolysis and muscle glycogenolysis during a fast.
Asunto(s)
Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Proteínas Musculares , Animales , Ingestión de Alimentos , Ayuno , Femenino , Glucagón/sangre , Transportador de Glucosa de Tipo 4 , Homeostasis , Humanos , Insulina/sangre , Cinética , Masculino , Ratones , Ratones Transgénicos , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/metabolismoRESUMEN
To examine the physiological role of the GLUT4/muscle-fat specific facilitative glucose transporter in regulating glucose homeostasis, we have generated transgenic mice expressing high levels of this protein in an appropriate tissue-specific manner. Examination of two independent founder lines demonstrated that high-level expression of GLUT4 protein resulted in a marked reduction of fasting glucose levels (approximately 70 mg/dl) compared to wild-type mice (approximately 130 mg/dl). Surprisingly, 30 min following an oral glucose challenge the GLUT4 transgenic mice had only a slight elevation in plasma glucose levels (approximately 90 mg/dl), whereas wild-type mice displayed a typical 2- to 3-fold increase (approximately 250-300 mg/dl). In parallel to the changes in plasma glucose, insulin levels were approximately 2-fold lower in the transgenic mice compared to the wild-type mice. Furthermore, isolated adipocytes from the GLUT4 transgenic mice had increased basal glucose uptake and subcellular fractionation indicated elevated levels of cell surface-associated GLUT4 protein. Consistent with these results, in situ immunocytochemical localization of GLUT4 protein in adipocytes and cardiac myocytes indicated a marked increase in plasma membrane-associated GLUT4 protein in the basal state. Taken together these data demonstrate that increased expression of the human GLUT4 gene in vivo results in a constitutively high level of cell surface GLUT4 protein expression and more efficient metabolic control over fluctuations in plasma glucose concentrations.