RESUMEN
The involvement of the clathrin-mediated endocytic internalization route in the uptake of cholera toxin (CT) was investigated using different cell lines, including the human intestinal Caco-2 and T84 cell lines, green monkey Vero cells, SH-SY5Y neuroblastoma cells and Madin-Darby canine kidney cells. Suppression of the clathrin-mediated endocytic pathway by classical biochemical procedures, like intracellular acidification and potassium depletion, inhibited cholera toxin uptake by up to about 50% as well as its ability to raise intracellular levels of cAMP. Also prior exposure of these cell types to the cationic amphiphilic drug chlorpromazine reduced the functional uptake of cholera toxin, even to a greater extent. These effects were dose- and cell type-dependent, suggesting an involvement of clathrin-mediated endocytosis in the functional uptake of cholera toxin. For a more straightforward approach to study the role of the clathrin-mediated uptake in the internalization of cholera toxin, a Caco-2(eps-) cell line was exploited. These Caco-2(eps-) cells constitutively suppress the expression of epsin, an essential accessory protein of clathrin-mediated endocytosis, thereby selectively blocking this internalization route. CT uptake was found to be reduced by over 60% in Caco-2(eps-) paralleled by a diminished ability of CT to raise the level of cAMP. The data presented suggest that the clathrin-mediated uptake route fulfils an important role in the functional internalization of cholera toxin in several cell types.
Asunto(s)
Toxina del Cólera/metabolismo , Clatrina/fisiología , Endocitosis/fisiología , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Clorpromazina/farmacología , Perros , Humanos , Potasio/metabolismoRESUMEN
Increased lipid peroxidation contributes to diabetic complications and redox-active iron is known to play an important role in catalyzing peroxidation reactions. We aimed to investigate if diabetes affects the capacity of plasma to protect against iron-driven lipid peroxidation and to identify underlying factors. Glycemic control, serum iron, proteins involved in iron homeostasis, plasma iron-binding antioxidant capacity in a liposomal model, and non-transferrin-bound iron were measured in 40 type 1 and 67 type 2 diabetic patients compared to 100 nondiabetic healthy control subjects. Iron-binding antioxidant capacity was significantly lower in the plasma of diabetic subjects (83 +/- 6 and 84 +/- 5% in type 1 and type 2 diabetes versus 88 +/- 6% in control subjects, p < 0.0005). The contribution of transferrin, ceruloplasmin, and albumin concentrations to the iron-binding antioxidant capacity was lost in diabetes (explaining only 4.2 and 6.3% of the variance in type 1 and type 2 diabetes versus 13.9% in control subjects). This observation could not be explained by differences in Tf glycation, lipid, or inflammatory status and was not associated with higher non-transferrin-bound iron levels. Iron-binding antioxidant capacity is decreased in diabetes mellitus.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Hierro/metabolismo , Peroxidación de Lípido/fisiología , Adulto , Anciano , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/fisiopatología , Femenino , Humanos , Hierro/sangre , Masculino , Persona de Mediana Edad , Transferrina/metabolismoRESUMEN
BACKGROUND: In vitro glycation of transferrin leads to increased oxidative stress by impairing iron-binding antioxidant capacity. The aim of this study is to develop a method to evaluate in vivo transferrin glycation in diabetes. METHODS: We adapted the nitroblue tetrazolium assay to measure in micro-well plates the fructosamine content of transferrin isolated from serum by immunocomplexation. RESULTS: Introduction of the immunocomplexation step did not affect the analytical performance of the fructosamine measurement and analytical variability was lower than 7%. The diabetic group (n=107) had significantly higher transferrin glycation (1.39+/-1.12 versus 0.79+/-1.09 micromol fructosamine/g transferrin in the non-diabetic group, n=91, p<0.0005) and this was most pronounced in type 1 diabetes (1.95+/-1.02 versus 1.06+/-1.04 micromol fructosamine/g transferrin in type 2, p<0.0005). Transferrin glycation was associated with parameters of glycaemic control but did not correlate with serum iron or total iron-binding capacity. Total iron-binding capacity was lower in type 1 diabetes (63+/-9 versus 69+/-12 micromol/l in type 2, p<0.05) and was mainly determined by transferrin concentration. CONCLUSIONS: These results indicate that the adapted nitroblue tetrazolium assay combined with immunocomplexation of serum transferrin is suitable to detect differences in in vivo transferrin glycation between non-diabetic, type 1 and type 2 diabetic subjects.
Asunto(s)
Diabetes Mellitus/sangre , Inmunoensayo/métodos , Transferrina/análisis , Transferrina/metabolismo , Diabetes Mellitus/metabolismo , Femenino , Glicosilación , Humanos , Hierro/sangre , Masculino , Persona de Mediana Edad , Transferrina/químicaRESUMEN
BACKGROUND: Diabetes is an inflammatory condition associated with iron abnormalities and increased oxidative damage. We aimed to investigate how diabetes affects the interrelationships between these pathogenic mechanisms. METHODS: Glycaemic control, serum iron, proteins involved in iron homeostasis, global antioxidant capacity and levels of antioxidants and peroxidation products were measured in 39 type 1 and 67 type 2 diabetic patients and 100 control subjects. RESULTS: Although serum iron was lower in diabetes, serum ferritin was elevated in type 2 diabetes (p = 0.02). This increase was not related to inflammation (C-reactive protein) but inversely correlated with soluble transferrin receptors (r = - 0.38, p = 0.002). Haptoglobin was higher in both type 1 and type 2 diabetes (p < 0.001) and haemopexin was higher in type 2 diabetes (p < 0.001). The relation between C-reactive protein and haemopexin was lost in type 2 diabetes (r = 0.15, p = 0.27 vs r = 0.63, p < 0.001 in type 1 diabetes and r = 0.36, p = 0.001 in controls). Haemopexin levels were independently determined by triacylglycerol (R(2) = 0.43) and the diabetic state (R(2) = 0.13). Regarding oxidative stress status, lower antioxidant concentrations were found for retinol and uric acid in type 1 diabetes, alpha-tocopherol and ascorbate in type 2 diabetes and protein thiols in both types. These decreases were partially explained by metabolic-, inflammatory- and iron alterations. An additional independent effect of the diabetic state on the oxidative stress status could be identified (R(2) = 0.5-0.14). CONCLUSIONS: Circulating proteins, body iron stores, inflammation, oxidative stress and their interrelationships are abnormal in patients with diabetes and differ between type 1 and type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Inflamación/fisiopatología , Hierro/metabolismo , Estrés Oxidativo/fisiología , Adulto , Anciano , Antígenos/análisis , Antioxidantes/análisis , Glucemia/metabolismo , Índice de Masa Corporal , Proteína C-Reactiva/análisis , Ceruloplasmina/análisis , Femenino , Ferritinas/análisis , Fibrinógeno/análisis , Haptoglobinas/análisis , Hemopexina/análisis , Humanos , Masculino , Persona de Mediana Edad , Receptores de Transferrina/sangre , Transferrina/análisis , Factor de von Willebrand/inmunologíaRESUMEN
Activated monocytes release oxygen radicals by respiratory burst and oxidative damage can be accelerated by transition metals. We investigated the cell-mediated and metal-catalysed in vitro oxidation of low-density lipoproteins (LDL), as well as the impact of the metal-binding protein transferrin (Tf). LDL oxidation was measured by monitoring the increase in fluorescence (350/440 nm excitation/emission). Maximal respiratory burst by U937 cells was achieved after 96 h differentiation with retinoic acid and dihydroxyvitamin D3 followed by stimulation with opsonised zymosan. Addition of activated cells resulted in the LDL oxidation, even in the absence of transition metals. Moreover, activated cells greatly enhanced metal-catalysed oxidative modifications, especially in the presence of copper. By binding metals, Tf was able to strongly impair this process. In conclusion, by generating oxygen radicals, activated U937 cells were able to oxidise LDL. The oxidising process was most pronounced in the presence of copper and could be blocked by Tf.
Asunto(s)
Cobre/farmacología , Hierro/farmacología , Lipoproteínas LDL/metabolismo , Transferrina/metabolismo , Catálisis , Diferenciación Celular , Línea Celular Tumoral , Humanos , Oxidación-Reducción/efectos de los fármacosRESUMEN
Free iron is capable of stimulating the production of free radicals which cause oxidative damage such as lipid peroxidation. One of the most important mechanisms of antioxidant defense is thus the sequestration of iron in a redox-inactive form by transferrin. In diabetes mellitus, increased oxidative stress and lipid peroxidation contribute to chronic complications but it is not known if this is related to abnormalities in transferrin function. In this study we investigated the role of transferrin concentration and glycation. The antioxidant capacity of apotransferrin to inhibit lipid peroxidation by iron-binding decreased in a concentration-dependent manner from 89% at > or = 2 mg/ml to 42% at 0.5 mg/ml. Pre-incubation of apotransferrin with glucose for 14 days resulted in a concentration-dependent increase of glycation: 1, 5 and 13 micromol fructosamine/g transferrin at 0, 5.6 and 33.3 mmol/l glucose respectively, p < 0.001. This was accompanied by a decrease in the iron-binding antioxidant capacity of apotransferrin. In contrast, transferrin glycation by up to 33.3 mmol/l glucose did not affect chemiluminescence-quenching antioxidant capacity, which is iron-independent. Colorimetric evaluation of total iron binding capacity in the presence of an excess of iron (iron/transferrin molar ratio = 2.4) also decreased from 0.726 to 0.696 and 0.585mg/g transferrin after 0, 5.6 and 33.3 mmol/l glucose, respectively, p < 0.01. In conclusion, these results suggest that lower transferrin concentration and its glycation can, by enhancing the pro-oxidant effects of iron, contribute to the increased lipid peroxidation observed in diabetes.
Asunto(s)
Diabetes Mellitus/metabolismo , Peroxidación de Lípido , Transferrina/química , Animales , Antioxidantes/farmacología , Apoproteínas/química , Encéfalo/metabolismo , Bovinos , Relación Dosis-Respuesta a Droga , Radicales Libres , Glucosa/metabolismo , Humanos , Hierro/metabolismo , Hierro/farmacología , Metabolismo de los Lípidos , Liposomas/metabolismo , Mediciones Luminiscentes , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Factores de Tiempo , Transferrina/biosíntesis , Transferrina/metabolismoRESUMEN
Prenylcysteine carboxymethyltransferase (pcCMT) is an enzyme that catalyzes the post-translational carboxymethylation of isoprenylated proteins ensuring a more efficient membrane attachment and proper guiding to a specific target membrane. In this paper, we report on modulation of pcCMT activity in retinoic acid (RA)-treated SH-SY5Y neuroblastoma cells using N-acetyl-S-farnesyl-L-cysteine (AFC) as artificial methyl acceptor. In addition, the methylation of endogenous proteins was followed by the vapor phase equilibrium assay and the storage phosphor screen (P-screen) technique with S-adenosyl-[3H-methyl] methionine (AdoMet) as methyl donor. Methylation of AFC was reduced to 75% of that of the control, the most prominent decrease being observed with the post-nuclear membrane fraction as enzyme source. With regard to protein methylation both screening methods yielded analogous results showing the [3H]-labeling of endogenous proteins in the 21-25kDa molecular mass (MM) range to be diminished by nearly 50%. This questions the role of protein carboxymethylation as an essential component of the differentiation process in SH-SY5Y neuroblastoma cells. The P-screen technique revealed that the methylation of other molecular mass proteins was also affected. Both S-adenosylhomocysteine (AdoHcy) and AFC (AdoHcy being the most effective) inhibited endogenous methylation. An interesting feature was that AFC inhibited the protein methylation proportionally more effective in RA-treated cells. Finally, the levels of three small guanosine-5'-triphosphate (GTP) binding proteins were screened upon differentiation showing rab3A to be increased while rhoA and H-ras were decreased.