RESUMEN
AIMS/HYPOTHESIS: Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy. METHODS: Transgenic rats were developed by overexpression of full length GLO1. Diabetes was induced in wild-type (WT) and GLO1 rats and the animals were killed after 12 or 24 weeks of hyperglycaemia. N ε)-(Carboxyethyl)lysine (CEL), N(ε)-(carboxymethyl)lysine (CML) and MG-derived-hydroimidazalone-1 (MG-H1) were determined by immunohistochemistry and by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS). Müller glia dysfunction was determined by glial fibrillary acidic protein (GFAP) immunoreactivity and by spatial localisation of the potassium channel Kir4.1. Acellular capillaries were quantified in retinal flat mounts. RESULTS: GLO1 overexpression prevented CEL and MG-H1 accumulation in the diabetic retina when compared with WT diabetic counterparts (p < 0.01). Diabetes-related increases in Müller glial GFAP levels and loss of Kir4.1 at the vascular end-feet were significantly prevented by GLO1 overexpression (p < 0.05) at both 12- and 24-week time points. GLO1 diabetic animals showed fewer acellular capillaries than WT diabetic animals (p < 0.001) at 24 weeks' diabetes. CONCLUSIONS/INTERPRETATION: Detoxification of MG reduces AGE adduct accumulation, which, in turn, can prevent formation of key retinal neuroglial and vascular lesions as diabetes progresses. MG-derived AGEs play an important role in diabetic retinopathy.
Asunto(s)
Retinopatía Diabética/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Lactoilglutatión Liasa/biosíntesis , Neuroglía/metabolismo , Retina/metabolismo , Vasos Retinianos/metabolismo , Animales , Retinopatía Diabética/sangre , Retinopatía Diabética/patología , Retinopatía Diabética/prevención & control , Humanos , Hiperglucemia/metabolismo , Inmunohistoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Lactoilglutatión Liasa/genética , Microvasos/metabolismo , Microvasos/patología , Terapia Molecular Dirigida , Neuroglía/patología , Canales de Potasio de Rectificación Interna/metabolismo , Piruvaldehído , Ratas , Ratas Transgénicas , Proteínas Recombinantes/biosíntesis , Retina/enzimología , Retina/patología , Vasos Retinianos/patología , Factores de TiempoRESUMEN
Acrp30 is a circulating protein synthesized in adipose tissue. A single injection in mice of purified recombinant Acrp30 leads to a 2-3-fold elevation in circulating Acrp30 levels, which triggers a transient decrease in basal glucose levels. Similar treatment in ob/ob, NOD (non-obese diabetic) or streptozotocin-treated mice transiently abolishes hyperglycemia. This effect on glucose is not associated with an increase in insulin levels. Moreover, in isolated hepatocytes, Acrp30 increases the ability of sub-physiological levels of insulin to suppress glucose production. We thus propose that Acrp30 is a potent insulin enhancer linking adipose tissue and whole-body glucose metabolism.
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Adipocitos/metabolismo , Proteínas Sanguíneas/fisiología , Insulina/fisiología , Péptidos y Proteínas de Señalización Intercelular , Hígado/fisiología , Proteínas , Tiazolidinedionas , Adiponectina , Animales , Proteínas Sanguíneas/metabolismo , Línea Celular , Ingestión de Energía , Glucosa/biosíntesis , Humanos , Hipoglucemiantes/farmacología , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/metabolismo , Tiazoles/farmacologíaRESUMEN
Retinal neovascularization is a major cause of blindness in such disorders as retinopathy of prematurity, proliferative diabetic retinopathy and senile macular degeneration. Because ligation of vitronectin receptor-type integrins appears to be required for the survival and maturation of newly formed but not quiescent blood vessels in several vascular beds including the retina, blockade of this downstream adhesion receptor system was investigated. In a mouse model of hypoxia-induced retinal neovascularization twice daily administration of 1 to 20 mg cyclic alpha v-integrin antagonist peptide per kilogram of body weight reduced capillary proliferation in a dose-dependent fashion--maximum 76%--without obvious side effects. A cyclic control peptide displayed no inhibitory effect on neovascularization. These findings indicate that systemic application of vitronectin receptor antagonists appears to be clinically feasible and is efficient in preventing retinal neovascularization and superior to cytokine-blocking strategies.
Asunto(s)
Integrinas/antagonistas & inhibidores , Péptidos Cíclicos/uso terapéutico , Receptores de Vitronectina/antagonistas & inhibidores , Neovascularización Retiniana/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Péptidos Cíclicos/toxicidadRESUMEN
AIMS/HYPOTHESIS: Impaired nitric oxide (NO)-dependent vasorelaxation plays a key role in the development of diabetic vascular complications. We investigated the effect of hyperglycaemia on impaired vasoreactivity and a putative role therein of the AGE precursor methylglyoxal. METHODS: The effects of high glucose and methylglyoxal on NO-dependent vasorelaxation in isolated rat mesenteric arteries from wild-type and transgenic glyoxalase (GLO)-I (also known as GLO1) rats, i.e. the enzyme detoxifying methylglyoxal, were recorded in a wire myograph. AGE formation of the major methylglyoxal-adduct 5-hydro-5-methylimidazolone (MG-H1) was detected with an antibody against MG-H1 and quantified with ultra-performance liquid chromatography (tandem) mass spectrometry. Reactive oxygen species formation was measured with a 5-(and-6)-chloromethyl-2'7'-dichlorodihydrofluorescein diacetate acetyl ester probe and by immunohistochemistry with an antibody against nitrotyrosine. RESULTS: High glucose and methylglyoxal exposure of mesenteric arteries significantly reduced the efficacy of NO-dependent vasorelaxation (p < 0.05). This impairment was not observed in mesenteric arteries of GLO-I transgenic rats indicating a specific intracellular methylglyoxal effect. The diabetes-induced impaired potency (pD(2)) in mesenteric arteries of wild-type rats was significantly improved by GLO-I overexpression (p < 0.05). Methylglyoxal-modified albumin did not affect NO-dependent vasorelaxation, while under the same conditions the receptor for AGE ligand S100b did (p < 0.05). Methylglyoxal treatment of arteries increased intracellular staining of MG-H1 in endothelial cells and adventitia by fivefold accompanied by an eightfold increase in the oxidative stress marker nitrotyrosine. Antioxidant pre-incubation prevented methylglyoxal-induced impairment of vasoreactivity. CONCLUSIONS/INTERPRETATION: These data show that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by increased intracellular methylglyoxal levels in a pathway dependent on oxidative stress.
Asunto(s)
Endotelio Vascular/metabolismo , Hiperglucemia/metabolismo , Arterias Mesentéricas/metabolismo , Estrés Oxidativo/fisiología , Piruvaldehído/metabolismo , Vasodilatación/efectos de los fármacos , Análisis de Varianza , Animales , Recuento de Células , Línea Celular , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiopatología , Humanos , Hiperglucemia/fisiopatología , Inmunohistoquímica , Lactoilglutatión Liasa/genética , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiopatología , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacos , Piruvaldehído/farmacología , Ratas , Ratas Transgénicas , Especies Reactivas de Oxígeno/metabolismo , Vasodilatación/fisiologíaRESUMEN
A high-affinity macrophage receptor has been identified that recognizes proteins modified by a common in vivo process, long-term nonenzymatic reaction of glucose with proteins (AGE proteins). This receptor for glucose-modified proteins is now shown to be distinct from previously described scavenger receptors, using competition and crosscompetition experiments between AGE-modified protein and a variety of in vitro-modified scavenger receptor ligands, including unmodified BSA, unmodified low-density lipoproteins (LDL), acetyl-LDL, maleyl-BSA, and formaldehyde-treated BSA. Furthermore, the specific pattern of AGE-protein receptor inhibition by the polyanionic compounds polyinosinic acid, polyadenylic acid, polyglutamic acid, polycytidylic acid, fucoidin, and heparin was distinctly different from that of acetyl-LDL. By thus selectively recognizing a time-dependent in vivo protein modification, macrophages may preferentially degrade senescent macromolecules, thereby having an important role in the regulation of extracellular protein turnover.
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Moléculas de Adhesión Celular , Macrófagos/análisis , Complejo GPIb-IX de Glicoproteína Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Receptores Inmunológicos/análisis , Envejecimiento , Animales , Femenino , Glicosilación , Lipoproteínas LDL/metabolismo , Ratones , Proteínas/metabolismo , Receptores de LDL/análisis , Receptores Depuradores , Albúmina Sérica Bovina/metabolismoRESUMEN
We have previously shown that increased nonenzymatic glycosylation occurs in peripheral nervous tissue of diabetic humans and animals, primarily on the PO-protein of peripheral nerve myelin. The pathophysiologic mechanism by which this biochemical alteration leads to myelin breakdown and removal is not as yet understood. In the present study we show that advanced glycosylation end-product (AGE) adducts that form during long-term exposure of peripheral nerve myelin proteins to glucose in vitro and in vivo markedly alter the way in which myelin interacts with elicited macrophages. In this interaction, macrophages appear to specifically recognize AGEs on myelin, since AGE-BSA competes nearly as effectively as AGE-myelin, while neither unmodified BSA nor unmodified myelin compete. The failure of yeast mannan to interfere with macrophage recognition of AGE-myelin suggests that the mannose/fucose receptor does not mediate this process. Recognition of AGE-protein by macrophages is associated with endocytosis, as demonstrated by resistance of cell-associated radioactivity to removal by trypsin action, and by low temperature inhibition of ligand accumulation in the cellular fraction. 125I-labeled myelin that had been incubated in vitro with 50 mM glucose for 8 wk reached a steady state accumulation within thioglycolate-elicited macrophages that was five times greater than that of myelin incubated without glucose. Similarly, myelin isolated from rats having diabetes for 1.5-2.0 years duration had a steady state level that was 9 times greater than that of myelin from young rats, and 3.5 times greater than that of myelin from age-matched controls. In contrast, myelin isolated from rats having diabetes for 4-5 wk had the same degree of accumulation observed with myelin of age-matched normal rats. These data suggest that the amount of increased nonenzymatic glycosylation observed in the myelin of short-term diabetic rats had not yet resulted in the significant accumulation of AGE-myelin present both in vitro and in the long-term diabetic rats. The disappearance of acid-insoluble radioactivity from within the cells and the appearance of acid-soluble radioactivity released into the medium were very similar for the two groups, suggesting that the striking difference in accumulation seen between normal myelin and AGE-myelin is due primarily to increased uptake. Formation of irreversible AGE-adducts on myelin appears to promote the recognition and uptake of the modified myelin by macrophages. This interaction between AGE-myelin and macrophages may initiate or contribute to the segmental demyelination associated with diabetes and the normal aging of peripheral nerve.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Neuropatías Diabéticas/metabolismo , Macrófagos/metabolismo , Proteínas de la Mielina/metabolismo , Envejecimiento , Animales , Diabetes Mellitus Experimental/fisiopatología , Neuropatías Diabéticas/fisiopatología , Femenino , Glucosa/farmacología , Técnicas In Vitro , Cinética , Activación de Macrófagos , Ratones , Ratones Endogámicos , Ratas , Ratas EndogámicasRESUMEN
The chronic tissue damage associated with long-term diabetes mellitus may arise in part from in situ immune complex formation by accumulated immunoglobulins and/or antigens bound to long-lived structural proteins that have undergone excessive nonenzymatic glycosylation. In this report, we have tested this hypothesis using nonenzymatically glycosylated collagen. Binding of both albumin and IgG averaged four times the amount bound to unmodified collagen. Both albumin and IgG (anti-BSA) bound to nonenzymatically glycosylated collagen retained their ability to form immune complexes in situ with free antibody and antigen.
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Complejo Antígeno-Anticuerpo/metabolismo , Colágeno/análogos & derivados , Colágeno/metabolismo , Inmunoglobulina G/metabolismo , Lisina/metabolismo , Albúmina Sérica Bovina/metabolismoRESUMEN
Glucose can react nonenzymatically with amino groups of proteins to form covalent Amadori products. With time these adducts undergo further rearrangements to form irreversible advanced glycosylation endproducts (AGE), which accumulate with protein age. A specific AGE, 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI), has been identified on proteins in vivo. We have recently shown that a macrophage receptor specifically recognizes and internalizes proteins modified by AGE such as FFI, thus preferentially degrading senescent macromolecules. Reasoning that cellular turnover may be mediated by macrophage recognition of AGE-membrane proteins, we prepared human RBCs with FFI attached chemically. Human monocytes were incubated with either FFI-RBCs, IgG-opsonized RBCs, or PBS-treated RBCs. Erythrophagocytosis of FFI-RBCs was significantly higher than that of PBS-RBCs (55 vs. 4%; p less than 0.0025) and almost as high as that of IgG-RBCs (70%), and was competitively inhibited by AGE-BSA. AGE-RBCs were also prepared by incubating RBCs with various sugars. Human monocytes showed a 15% ingestion of glucose-RBCs, and a 26% ingestion of glucose-6-phosphate-RBCs, compared to 6% for PBS-RBCs. Similarly, diabetic mouse RBCs were phagocytosed by nearly three times more cells (21%) than normal mouse RBCs when exposed to syngeneic mouse macrophages. This phagocytosis was competitively inhibited (70%) by addition of excess AGE-BSA. The in vivo half-life of 51Cr-labeled mouse FFI-RBCs injected into syngeneic mice was reduced to 7 d, as compared to a half-life of 20 d for the controls. These data suggest that the macrophage receptor for the removal of glucose-modified proteins may also mediate the endocytosis of RBCs with AGE formed on their surface, and thus be responsible in part for the removal of some populations of aging cells.
Asunto(s)
Membrana Eritrocítica/metabolismo , Glicoproteínas/metabolismo , Macrófagos/inmunología , Fagocitosis , Animales , Supervivencia Celular , Diabetes Mellitus Experimental/inmunología , Glicosilación , Humanos , Imidazoles/metabolismo , Ratones , Ratones Endogámicos BALB C , Unión ProteicaRESUMEN
A stable, biologically active glycosylated insulin derivative that is complementary to the major combining site of concanavalin A has been synthesized. Hormone release is proportional to the quantity of glucose present. Glucose regulation of exogenous insulin delivery could have important applications in the therapy of diabetes mellitus.
Asunto(s)
Insulina/administración & dosificación , Glucemia/metabolismo , Concanavalina A/metabolismo , Insulina/análogos & derivados , Insulina/metabolismo , Maltosa , Receptores de Concanavalina A/metabolismoRESUMEN
Age-associated increases in collagen cross-linking and accumulation of advanced glycosylation products are both accelerated by diabetes, suggesting that glucose-derived cross-link formation may contribute to the development of chronic diabetic complications as well as certain physical changes of aging. Aminoguanidine, a nucleophilic hydrazine compound, prevented both the formation of fluorescent advanced nonenzymatic glycosylation products and the formation of glucose-derived collagen cross-links in vitro. Aminoguanidine administration to rats was equally effective in preventing diabetes-induced formation of fluorescent advanced nonenzymatic glycosylation products and cross-linking of arterial wall connective tissue protein in vivo. The identification of aminoguanidine as an inhibitor of advanced nonenzymatic glycosylation product formation now makes possible precise experimental definition of the pathogenetic significance of this process and suggests a potential clinical role for aminoguanidine in the future treatment of chronic diabetic complications.
Asunto(s)
Arterias/efectos de los fármacos , Diabetes Mellitus Experimental/tratamiento farmacológico , Guanidinas/farmacología , Animales , Arterias/metabolismo , Colágeno/metabolismo , Tejido Conectivo/efectos de los fármacos , Tejido Conectivo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Glucosa/metabolismo , Guanidinas/uso terapéutico , Masculino , Ratas , Ratas Endogámicas Lew , Albúmina Sérica Bovina/metabolismoRESUMEN
Proteins undergo a series of nonenzymatic reactions with glucose over time to form advanced glycosylation end products (AGEs). Macrophages have a receptor that recognizes the AGE moiety and mediates the uptake and degradation of AGE proteins. This removal process is associated with the production and secretion of cachectin (tumor necrosis factor) and interleukin-1, two cytokines with diverse and seemingly paradoxical biological activities. The localized release and action of these cytokines could account for the coordinated removal and replacement of senescent extracellular matrix components in normal tissue homeostasis.
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Interleucina-1/biosíntesis , Glicoproteínas de Membrana/fisiología , Monocitos/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Glicosilación , Humanos , Interleucina-1/genética , Cinética , Biosíntesis de Proteínas , ARN Mensajero/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Intracellular sugars are more reactive glycosylating agents than glucose. In vitro nonezymatic glycosylation of basic fibroblast growth factor (bFGF) by fructose, glucose-6-phosphate (G6P), or glyceraldehyde-3-phosphate (G3P) reduced high affinity heparin-binding activity of recombinant bFGF by 73, 77, and 89%, respectively. Mitogenic activity was reduced 40, 50, and 90%. To investigate the effects of bFGF glycosylation in GM7373 endothelial cells, we first demonstrated that GLUT-1 transporters were not downregulated by increased glucose concentration. In 30 mM glucose, the rate of glucose transport increased 11.6-fold, and the intracellular glucose concentration increased sixfold at 24 h and fivefold at 168 h. The level of total cytosolic protein modified by advanced glycosylation end-products (AGEs) was increased 13.8-fold at 168 h. Under these conditions, mitogenic activity of endothelial cell cytosol was reduced 70%. Anti-bFGF antibody completely neutralized the mitogenic activity at both 5 and 30 nM glucose, demonstrating that all the mitogenic activity was due to bFGF. Immunoblotting and ELISA showed that 30 mM glucose did not decrease detectable bFGF protein, suggesting that the marked decrease in bFGF mitogenic activity resulted from posttranslational modification of bFGF induced by elevated glucose concentration. Cytosolic AGE-bFGF was increased 6.1-fold at 168 h. These data are consistent with the hypothesis that nonenzymatic glycosylation of intracellular protein alters vascular cell function.
Asunto(s)
Diabetes Mellitus/metabolismo , Endotelio Vascular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Animales , Bovinos , División Celular , Células Cultivadas , Glucosa/farmacología , Transportador de Glucosa de Tipo 1 , Productos Finales de Glicación Avanzada/metabolismo , Glicosilación , Heparina/metabolismo , Humanos , Proteínas de Transporte de Monosacáridos/análisisRESUMEN
Hyperglycemia rapidly induces an increase in intracellular advanced glycation end products (AGEs) in bovine endothelial cells, causing an alteration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee. 1994. J. Clin. Invest. 94:110-117). Because sugar or sugar-adduct autoxidation is critical for AGE formation in vitro, we evaluated the role of reactive oxygen species (ROS) in intracellular AGE formation, using bovine aortic endothelial cells. 30 mM glucose increased intracellular ROS formation by 250% and lipid peroxidation by 330%, while not affecting ROS in the media. In cells depleted of glutathione, intracellular AGE accumulation increased linearly with ROS generation as measured by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 AU* mm/5x10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Deferoxamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hyperglycemia-induced formation of both ROS and AGE. To differentiate an effect of ROS generation on AGE formation from an effect of more distal oxidative processes, GM7373 endothelial cell lines were generated that stably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl-2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generation of intracellular AGEs, and that inhibition of oxidant pathways prevents intracellular AGE formation.
Asunto(s)
Antioxidantes/farmacología , Endotelio Vascular/metabolismo , Glucosa/farmacología , Productos Finales de Glicación Avanzada/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Animales , Bovinos , Células Cultivadas , Antagonismo de Drogas , Hiperglucemia/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2RESUMEN
Endothelial nitric oxide synthase (eNOS) is activated by phosphorylation of serine 1177 by the protein kinase Akt/PKB. Since hyperglycemia-induced mitochondrial superoxide overproduction increases O-linked N-acetylglucosamine modification and decreases O-linked phosphorylation of the transcription factor Sp1, the effect of hyperglycemia and the hexosamine pathway on eNOS was evaluated. In bovine aortic endothelial cells, hyperglycemia inhibited eNOS activity 67%, and treatment with glucosamine had a similar effect. Hyperglycemia-associated inhibition of eNOS was accompanied by a twofold increase in O-linked N-acetylglucosamine modification of eNOS and a reciprocal decrease in O-linked serine phosphorylation at residue 1177. Both the inhibition of eNOS and the changes in its post-translational modifications were reversed by antisense inhibition of glutamine:fructose-6-phosphate amidotransferase, the rate-limiting enzyme of the hexosamine pathway, or by blocking mitochondrial superoxide overproduction with uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD). Immunoblot analysis of cells expressing myc-tagged wild-type human eNOS confirmed the reciprocal increase in O-linked N-acetylglucosamine and decrease in O-linked serine 1177 phosphorylation in response to hyperglycemia. In contrast, when myc-tagged human eNOS carried a mutation at the Akt phosphorylation site (Ser1177), O-linked N-acetylglucosamine modification was unchanged by hyperglycemia and phospho-eNOS was undetectable. Similar changes in eNOS activity and covalent modification were found in aortae from diabetic animals. Chronic impairment of eNOS activity by this mechanism may partly explain the accelerated atherosclerosis of diabetes.
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Hiperglucemia/enzimología , Óxido Nítrico Sintasa/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Acetilglucosamina/metabolismo , Animales , Bovinos , Células Cultivadas , Diabetes Mellitus Experimental/enzimología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/citología , Humanos , Immunoblotting , Potenciales de la Membrana , Mutación , Óxido Nítrico Sintasa de Tipo III , Oligonucleótidos Antisentido/metabolismo , Oligonucleótidos Antisentido/farmacología , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-akt , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Previous studies showed that gammaIFN decreases metastatic hepatic tumor growth by stimulating Kupffer cells (KC). The present studies examine whether lymphocyte stimulation via cells engineered to secrete GM-CSF or IL-2 decreases hepatic tumor growth, and whether stimulation of both macrophages and lymphocytes is more effective than either individually. Rats were immunized with irradiated hepatoma cells transduced by herpes viral amplicon vectors containing the genes for GM-CSF, IL-2 or LacZ. On day 18, half of each group was treated with 5 x 10(4) U gammaIFN, or saline intraperitoneally for 3 d. On day 21, all rats received 5 x 10(5) hepatoma cells intrasplenically. On day 41, rats were killed and tumor nodules were counted. Separate rats underwent splenocyte and KC harvest for assessment of lymphocyte- and macrophage-mediated tumor cell kill in vitro. GM-CSF or IL-2 vaccines or gammaIFN decreased tumor nodules significantly (GM-CSF 13+/-4, IL-2 14+/-6 vs. control 75+/-24, P < 0.001). Combination therapy was more effective, and completely eliminated tumor in 4 of 12 IFN-GM-CSF and 8 of 11 IFN-IL-2 animals. Additional rats underwent partial hepatectomy, an immunosuppressive procedure known to accelerate the growth of hepatic tumor, following tumor challenge. Therapy was equally effective in this immunosuppressive setting. Vaccination is associated with enhancement of splenocyte-mediated tumoricidal activity, whereas the effect of gammaIFN is mediated by KC. GM-CSF and IL-2 vaccine therapy and pretreatment with gammaIFN represent effective strategies in reducing hepatic tumor. Combination therapy targets both lymphocytes and macrophages, and is more effective in reducing tumor than either therapy alone.
Asunto(s)
Interferón gamma/uso terapéutico , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/terapia , Simplexvirus/inmunología , Vacunas Virales/inmunología , Animales , División Celular/efectos de los fármacos , División Celular/inmunología , Citocinas/biosíntesis , Pruebas Inmunológicas de Citotoxicidad , Hepatectomía , Macrófagos del Hígado/citología , Neoplasias Hepáticas Experimentales/patología , Masculino , Metástasis de la Neoplasia , Ratas , Ratas Endogámicas BUF , Bazo/citologíaRESUMEN
Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.
Asunto(s)
Endocitosis , Endotelio Vascular/metabolismo , Endotelio Vascular/fisiología , Productos Finales de Glicación Avanzada/metabolismo , Lactoilglutatión Liasa/metabolismo , Acetona/análogos & derivados , Acetona/metabolismo , Aldehídos/metabolismo , Animales , Bovinos , Endotelio Vascular/enzimología , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Glutatión/análogos & derivados , Glutatión/farmacología , Glioxal/metabolismo , Hiperglucemia/metabolismo , Cetonas/metabolismo , Ácido Láctico/metabolismo , Lactoilglutatión Liasa/genética , Lisina/análogos & derivados , Lisina/metabolismo , Piruvaldehído/metabolismo , Transfección , Células Tumorales CultivadasRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-regulated nuclear receptor superfamily member. Liganded PPARgamma exerts diverse biological effects, promoting adipocyte differentiation, inhibiting tumor cellular proliferation, and regulating monocyte/macrophage and anti-inflammatory activities in vitro. In vivo studies with PPARgamma ligands showed enhancement of tumor growth, raising the possibility that reduced immune function and tumor surveillance may outweigh the direct inhibitory effects of PPARgamma ligands on cellular proliferation. Recent findings that PPARgamma ligands convey PPARgamma-independent activities through IkappaB kinase (IKK) raises important questions about the specific mechanisms through which PPARgamma ligands inhibit cellular proliferation. We investigated the mechanisms regulating the antiproliferative effect of PPARgamma. Herein PPARgamma, liganded by either natural (15d-PGJ(2) and PGD(2)) or synthetic ligands (BRL49653 and troglitazone), selectively inhibited expression of the cyclin D1 gene. The inhibition of S-phase entry and activity of the cyclin D1-dependent serine-threonine kinase (Cdk) by 15d-PGJ(2) was not observed in PPARgamma-deficient cells. Cyclin D1 overexpression reversed the S-phase inhibition by 15d-PGJ(2). Cyclin D1 repression was independent of IKK, as prostaglandins (PGs) which bound PPARgamma but lacked the IKK interactive cyclopentone ring carbonyl group repressed cyclin D1. Cyclin D1 repression by PPARgamma involved competition for limiting abundance of p300, directed through a c-Fos binding site of the cyclin D1 promoter. 15d-PGJ(2) enhanced recruitment of p300 to PPARgamma but reduced binding to c-Fos. The identification of distinct pathways through which eicosanoids regulate anti-inflammatory and antiproliferative effects may improve the utility of COX2 inhibitors.
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Ciclina D1/metabolismo , Inhibidores de Crecimiento/metabolismo , Proteínas Nucleares/metabolismo , Prostaglandina D2/análogos & derivados , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Sitios de Unión , División Celular , Ciclina D1/genética , Fase G1 , Regulación de la Expresión Génica/efectos de los fármacos , Inhibidores de Crecimiento/genética , Células HeLa , Humanos , Quinasa I-kappa B , Ligandos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Prostaglandina D2/metabolismo , Prostaglandina D2/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Elementos de Respuesta , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Pathogenesis of macrovascular complications of diabetes may involve an activation of the transcription factor nuclear factor-kappaB (NF-kappaB) by hyperglycemia and advanced glycosylation end products (AGEs). Activation of NF-kappaB is believed to be dependent on activation of the Rho family of GTPases. Although the precise mechanism of the Rho-mediated action is not completely understood, posttranslational modification of the Rho proteins by geranylgeranylation is required for their subsequent activation. We observed that in cultured vascular smooth muscle cells (VSMCs), insulin stimulated the activity of geranylgeranyltransferase (GGTase) I and increased the amounts of geranylgeranylated Rho-A from 47% to 60% (P:<0.05). GGTI-286, an inhibitor of GGTase I, blocked both effects of insulin. Increased availability of prenylated Rho-A significantly augmented the abilities of angiotensin II (Ang II), hyperglycemia, and AGEs to activate NF-kappaB, as measured by NF-kappaB response-element luciferase reporter activity. Preincubations of VSMCs with insulin for 24 hours doubled NF-kappaB transactivation by Ang II, hyperglycemia, and AGEs. This priming effect of insulin was completely inhibited by GGTI-286. We demonstrate for the first time, to our knowledge, that insulin potentiates NF-kappaB-dependent transcriptional activity induced by hyperglycemia, AGEs, and Ang II in VSMCs by increasing the activity of GGTase I and the availability of geranylgeranylated Rho-A.
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Angiotensina II/farmacología , Hiperglucemia/fisiopatología , Hiperinsulinismo/fisiopatología , FN-kappa B/genética , Transferasas Alquil y Aril/antagonistas & inhibidores , Transferasas Alquil y Aril/metabolismo , Animales , Bovinos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Genes Reporteros , Productos Finales de Glicación Avanzada/farmacología , Insulina/farmacología , Luciferasas/genética , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Factores de Tiempo , Transcripción Genética/efectos de los fármacos , TransfecciónRESUMEN
GLO1 (Glyoxalase1) is a ubiquitous cellular enzyme that detoxifies methylglyoxal (MG), which is a byproduct of glycolysis. Previously, we showed that ubiquitous overexpression of Glo1 reduced concentrations of MG and increased anxiety-like behavior, whereas systemic injection of MG reduced anxiety-like behavior. We further showed that MG is a competitive partial agonist at GABA-A receptors. Based on those data we hypothesized that modulation of GABAergic signaling by MG underlies Glo1 and MG's effects on anxiety-like behavior. As previous studies used ubiquitous overexpression, we sought to determine whether neuronal Glo1 overexpression was sufficient to increase anxiety-like behavior. We generated ROSA26 knock-in mice with a floxed-stop codon upstream from human Glo1 (FLOXGlo1KI) and bred them with mice expressing CRE recombinase under the direction of the Synapsin 1 promoter (Syn-CRE) to limit overexpression of Glo1 specifically to neurons. Furthermore, since previous administration of MG had been systemic, we sought to determine if direct microinjection of MG into the basolateral amygdala (BLA) was sufficient to reduce anxiety-like behavior. Thus, we performed bilateral microinjections of saline, MG (12µM or 24µM), or the positive control midazolam (4mM) directly into the BLA. FLOXGlo1KIxSyn-CRE mice showed significantly increased anxiety-like behavior compared to their FLOXGLO1xWT littermates. In addition, bilateral microinjection of MG and midazolam significantly decreased anxiety-like behavior compared to saline treated mice. These studies suggest that anatomically specific manipulations of Glo1 and MG are sufficient to induce changes in anxiety-like behavior.
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Ansiolíticos/administración & dosificación , Trastornos de Ansiedad/metabolismo , Complejo Nuclear Basolateral/metabolismo , Lactoilglutatión Liasa/metabolismo , Neuronas/metabolismo , Piruvaldehído/administración & dosificación , Animales , Trastornos de Ansiedad/tratamiento farmacológico , Complejo Nuclear Basolateral/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Agonistas de Receptores de GABA-A/administración & dosificación , Técnicas de Sustitución del Gen , Humanos , Lactoilglutatión Liasa/genética , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Microinyecciones , Midazolam/administración & dosificación , Neuronas/efectos de los fármacos , Regiones Promotoras Genéticas , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismoRESUMEN
Diabetes increases the risk of developing atherosclerotic arterial disease significantly. Although elevated glycohemoglobin was shown to be an independent risk factor in older women in the Framingham Heart Study, the relationship between hyperglycemia and macrovascular disease is complicated by the many other factors that influence atherogenesis in nondiabetic people. Studies in vitro suggest that chronic hyperglycemia may accelerate the atherogenic process through excessive glycation of various components of the arterial wall. These data are reviewed critically, and the biochemistry and pharmacological potential of the glycation-inhibitor aminoguanidine is discussed.