Albumin antioxidant response to stress in diabetic nephropathy progression.

BACKGROUND: A new component of the protein antioxidant capacity, designated Response Surplus (RS), was recently described. A major feature of this component is the close relationship between protein antioxidant capacity and molecular structure. Oxidative stress is associated with renal dysfunction in patients with renal failure, and plasma albumin is the target of massive oxidation in nephrotic syndrome and diabetic nephropathy. The aim of the present study was to explore the albumin redox state and the RS component of human albumin isolated from diabetic patients with progressive renal damage. METHODS/PRINCIPAL FINDINGS: Serum aliquots were collected and albumin isolated from 125 diabetic patients divided into 5 groups according to their estimated glomerular filtration rate (GFR). In addition to clinical and biochemical variables, the albumin redox state, including antioxidant capacity, thiol group content, and RS component, were evaluated. The albumin antioxidant capacity and thiol group content were reciprocally related to the RS component in association with GFR reduction. The GFR decline and RS component were significantly negatively correlated (R = -0.83, p<0.0001). Age, creatinine, thiol groups, and antioxidant capacity were also significantly related to the GFR decline (R = -0.47, p < 0.001; R = -0.68, p<0.0001; R = 0.44, p < 0.001; and R = 0.72, p < 0.0001). CONCLUSION/SIGNIFICANCE: The response of human albumin to stress in relation to the progression of diabetic renal disease was evaluated. The findings confirm that the albumin molecular structure is closely related to its redox state, and is a key factor in the progression of diabetes nephropathy.

Oral supplementation with glycine reduces oxidative stress in patients with metabolic syndrome, improving their systolic blood pressure.

Reactive oxygen species derived from abdominal fat and uncontrolled glucose metabolism are contributing factors to both oxidative stress and the development of metabolic syndrome (MetS). This study was designed to evaluate the effects of daily administration of an oral glycine supplement on antioxidant enzymes and lipid peroxidation in MetS patients. The study included 60 volunteers: 30 individuals that were supplemented with glycine (15 g/day) and 30 that were given a placebo for 3 months. We analysed thiobarbituric acid reactive substances (TBARS) and S-nitrosohemoglobin (SNO-Hb) in plasma; the enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) in erythrocytes; and the expression of CAT, GPX, and SOD2 in leukocytes. Individuals treated with glycine showed a 25% decrease in TBARS compared with the placebo-treated group. Furthermore, there was a 20% reduction in SOD-specific activity in the glycine-treated group, which correlated with SOD2 expression. G6PD activity and SNO-Hb levels increased in the glycine-treated male group. Systolic blood pressure (SBP) also showed a significant decrease in the glycine-treated men (p = 0.043). Glycine plays an important role in balancing the redox reactions in the human body, thus protecting against oxidative damage in MetS patients.

Protein antioxidant response to the stress and the relationship between molecular structure and antioxidant function.

BACKGROUND: Proteins have long been considered a principal target for oxidants as a result of their abundance in biological systems. However, there is increasing evidence about the significant antioxidant activity in proteins such as albumin. It is leading to new concepts that even consider albumin not only as an antioxidant but as the major antioxidant in plasma known to be exposed to continuous oxidative stress. Evidence presented here establishes a previously unrecognized relationship between proteins' antioxidant capacity and structural stress. METHODOLOGY/PRINCIPAL FINDINGS: A chemiluminiscence based antioxidant assay was achieved to quantify the antioxidant capacity of albumin and other proteins. The capabilities of proteins as antioxidants were presented, but in addition a new and powerful component of the protein antioxidant capacity was discovered. The intrinsic component, designated as Response Surplus (RS), represents a silent reserve of antioxidant power that awakens when proteins face a structural perturbation (stressor) such as temperature, short wave UV light, the same reactive oxygen species, and more extreme changes like glucose or aldehyde-mediated structural modifications. The work also highlights the importance of structural changes in protein antioxidant properties and the participation of sulfhydryl groups (SHs) in the RS antioxidant component. Based on recent evidence about the SH group chemistry, a possible model for explaining RS is proposed. CONCLUSIONS/SIGNIFICANCE: The data presented show the significant antioxidant behavior of proteins and demonstrate the existence of a previously unrecognized antioxidant response to the stress. Several implications, including changes in elementary concepts about antioxidants and protein function, should emerge from here.

O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase activity and mRNA expression in muscle is related to glucosamine-induced insulin resistance.

Glucosamine (GlcN)-induced insulin resistance is associated with an increase in O-linked-N-acetylglucosaminylated modified proteins (O-GlcNAcylated proteins). The role played by O-GlcNAc-selective-N-acetyl-beta-D-glucosaminidase (O-GlcNAcase), which removes O-N-acetyl-glucosamine residues from O-GlcNAcylated proteins, has not yet been demonstrated. We investigated whether GlcN-induced whole-body insulin resistance is related to tissue O-GlcNAcase activity and mRNA expression. GlcN (30 mumol/kg/min) or physiological saline (control) was intravenously infused into Sprague-Dawley rats for 2 h. After GlcN treatment, rats were subjected to the following: intravenous glucose tolerance test, insulin tolerance test or removal of the liver, muscle and pancreas. GlcN was found to provoke hyperglycemia compared to control (8.6 +/- 0.41 vs. 4.82 +/- 0.17 mM, p < 0.001). The insulin resistance index (HOMA-IR) increased (15.76 +/- 1.47 vs. 10.14 +/- 1.41, p < 0.001) and the beta-cell function index (HOMA-beta) diminished (182.69 +/- 22.37 vs. 592.01 +/- 103, p < 0.001). Liver glucose concentration was higher in the GlcN group than in the control group (0.37 +/- 0.04 vs. 0.24 +/- 0.038 mmol/g dry weight, p < 0.001). Insulin release index (insulin/glucose) was less in the GlcN group than in the control (2.2 +/- 0.1 vs. 8 +/- 0.8 at 120 min, p < 0.001). In the GlcN group, muscle O-GlcNAcase activity diminished (0.28 +/- 0.019 vs. 0.36 +/- 0.018 nmol of p-nitrophenyl/mg protein/min, p < 0.001), and K(m) increased (1.51 +/- 0.11 vs. 1.12 +/- 0.1 mM, p < 0.001) compared to the control. In the GlcN group, O-GlcNAcase activity/mRNA expression was altered (0.6 +/- 0.07 vs. 1 +/- 0.09 of control, p < 0.05). In conclusion, O-GlcNAcase activity is posttranslationally inhibited during GlcN-induced insulin resistance.

DD genotype of angiotensin-converting enzyme in type 2 diabetes mellitus with renal disease in Mexican Mestizos.

AIM: The DD genotype of angiotensin-converting enzyme (ACE) has been suggested as a major contributor of diabetic nephropathy in several populations. The purpose of the present study was to determine whether micro/macroalbuminuria is associated with ACE insertion/deletion (I/D) polymorphism in Mexican Mestizos with type 2 diabetes mellitus. METHODS: A total of 435 patients with type 2 diabetes mellitus, of whom 233 had albuminuria, were characterized for the ACE I/D polymorphism by the polymerase chain reaction method. RESULTS: Clinical and biochemical characteristics and frequencies according to DD, ID and II genotypes in patients with and without albuminuria showed no significant differences. However, only females with micro/macroalbuminuria showed higher frequency of a DD genotype than those without albuminuria (27.9%, 21.2% and 10.5%, respectively; P

Glycine increases mRNA adiponectin and diminishes pro-inflammatory adipokines expression in 3T3-L1 cells.

Obesity and type 2 diabetes course with chronic low-grade inflammation, where adiponectin is down-regulated and pro-inflammatory markers, like interleukin (IL)-6, tumor necrosis factor alpha (TNF-alpha), and C-reactive protein (CRP), are up-regulated. A treatment option to improve the micro- and macro-complications in type 2 diabetes is the use of glycine, which has been demonstrated previously to increase the expression of anti-inflammatory cytokine IL-10 in monocytes and down-regulate the expression of TNF-alpha in monocytes and Kupffer cells. Recently, our group demonstrated that glycine decreases the pro-inflammatory plasmatic cytokines in type 2 diabetes. The aim of this study was to test the effect of glycine on adipokines expression in 3T3-L1 cells. Cells were grown and differentiated in the presence of 10 mM glycine. After 2 days of confluence, cells were differentiated to adipocytes in the same medium supplemented with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine. The RNA was extracted at days 0 and 8 of differentiation (fibroblasts and mature adipocyte phenotypes, respectively). The expression of PPAR-gamma (peroxisome proliferator-activated receptor-gamma), adiponectin, resistin, IL-6 and TNF-alpha were analyzed by real-time PCR. We demonstrated that when 3T3-L1 cells were treated with glycine, IL-6, resistin and TNF-alpha mRNA expression was decreased, but surprisingly adiponectin and PPAR-gamma were up-regulated. In all cases the values were statistically significant (P<0.05) between glycine treatment and controls. These results show that glycine improves the pro-inflammatory profile and up-regulates adiponectin gene expression. Therefore, glycine could be useful as a modulator of the pro-inflammatory state observed in obesity and type 2 diabetes.

Formation of an adduct between insulin and the toxic lipoperoxidation product acrolein decreases both the hypoglycemic effect of the hormone in rat and glucose uptake in 3T3 adipocytes.

Lipid peroxidation induced by reactive oxygen species might modify circulating biomolecules because of the formation of alpha,beta-unsaturated or dicarbonylic aldehydes. In order to investigate the interaction between a lipoperoxidation product, acrolein, and a circulating protein, insulin, the acrolein-insulin adduct was obtained. To characterize the adduct, gel filtration chromatography, sodium dodecylsulfate-polyacrylamide gel electrophoresis and carbonyl determination were performed. Induction of hypoglycemia in the rat and stimulation of glucose uptake by 3T3 adipocytes were used to evaluate the biological efficiency of the adduct compared with that of native insulin (Mackness, B., Quarck, R., Verte, W., Mackness, M., and Holvoet, P. (2006) Arterioscler., Thromb. Vasc. Biol. 26, 1545-1550). Formation of the acrolein-insulin complex in vitro increased the carbonyl group concentration from 2.5 to 22.5 nmol/mg of protein, and it formed without intermolecular aggregates (Halliwell, B., and Whiteman, M. (2004) Br. J. Pharmacol. 142, 231-255. The hypoglycaemic effect 18 min after administration to the rat is decreased by 25% (Robertson, R. P. (2004) J. Biol. Chem. 279, 42351-42354. An adduct concentration of 94 nM, compared to 10 nM for native insulin, was required to obtain the A 50% (concentration needed to obtain 50% of maximum transport of glucose uptake by 3T3 adipocytes). In conclusion, formation of the acrolein-insulin adduct modifies the structure of insulin and decreases its hypoglycemic effect in rat and glucose uptake by 3T3 adipocytes. These results help explain how a toxic aldehyde prone to be produced in vivo can structurally modify insulin and change its biological action.

Glucose-6-phosphate dehydrogenase activity and NADPH/NADP+ ratio in liver and pancreas are dependent on the severity of hyperglycemia in rat.

Hyperglycemia is associated with metabolic disturbances affecting cell redox potential, particularly the NADPH/NADP+ ratio and reduced glutathione levels. Under oxidative stress, the NADPH supply for reduced glutathione regeneration is dependent on glucose-6-phosphate dehydrogenase. We assessed the effect of different hyperglycemic conditions on enzymatic activities involved in glutathione regeneration (glucose-6-phosphate dehydrogenase and glutathione reductase), NADP(H) and reduced glutathione concentrations in order to analyze the relative role of these enzymes in the control of glutathione restoration. Male Sprague-Dawley rats with mild, moderate and severe hyperglycemia were obtained using different regimens of streptozotocin and nicotinamide. Fifteen days after treatment, rats were killed and enzymatic activities, NADP(H) and reduced glutathione were measured in liver and pancreas. Severe hyperglycemia was associated with decreased body weight, plasma insulin, glucose-6-phosphate dehydrogenase activity, NADPH/NADP+ ratio and glutathione levels in the liver and pancreas, and enhanced NADP+ and glutathione reductase activity in the liver. Moderate hyperglycemia caused similar changes, although body weight and liver NADP+ concentration were not affected and pancreatic glutathione reductase activity decreased. Mild hyperglycemia was associated with a reduction in pancreatic glucose-6-phosphate dehydrogenase activity. Glucose-6-phosphate dehydrogenase, NADPH/NADP+ ratio and glutathione level, vary inversely in relation to blood glucose concentrations, whereas liver glutathione reductase was enhanced during severe hyperglycemia. We conclude that glucose-6-phosphate dehydrogenase and NADPH/NADP+ were highly sensitive to low levels of hyperglycemia. NADPH/NADP+ is regulated by glucose-6-phosphate dehydrogenase in the liver and pancreas, whereas levels of reduced glutathione are mainly dependent on the NADPH supply.

Diabetogenic effect of STZ diminishes with the loss of nitric oxide: role of ultraviolet light and carboxy-PTIO.

Nitric oxide has been demonstrated to participate in beta-cell damage during streptozotocin (STZ)-induced diabetes. STZ consists of 2-deoxy-D-glucose substituted by N-methyl-N-nitrosourea at C-2 and therefore can liberate (.) NO. However, it has not been proven whether (.) NO generation from STZ is responsible for the disease. We found that STZ treated in vitro with ultraviolet (UV) light liberated significantly more (.) NO than non-irradiated STZ (1134.4 +/- 104 vs. 256.9 +/- 240 nmol). Moreover, the diabetogenic effect of STZ was abolished by UV irradiation before its administration to experimental animals. In these animals the glucose and insulin values were significantly different from those of the diabetic group (151.3 +/- 16.6 vs. 364.6 +/- 63.4 mg/dl and 36.3 +/- 17.9 vs. 0.08 +/- 5.5 microIU/ml, respectively) and similar to those of the non-diabetic group (127.2 +/- 34.1 mg/dl and 41.7 +/- 13.9 microIU/ml, respectively). Carboxy-PTIO treatment returned glycemia to nearly normal levels in 60% of STZ-induced diabetic rats (157.5 +/- 11.8 vs. 364.6 +/- 63.6 mg/dl of the diabetic group). L-NAME and dexamethasone cannot return either glucose or insulin to normal levels. In conclusion, UV light increased (.) NO liberation from STZ and suppressed its diabetogenic activity. It is possible that the diabetogenic activity of STZ is related to the liberation of nitric oxide from STZ, since carboxy-PTIO scavenger had a protective effect, while L-NAME and dexamethasone did not. It is possible that an increase in (.) NO concentration into cell, independently of its endogenous or exogenous origin, can induce beta-cell damage and diabetes.

Importancia de los radicales libres durante el ciclo reproductor / Importance of free radicals during the reproductive cycle

Los productos de la peroxidación, los radicales libres y los compuestos antioxidantes aumentan notablemente durante la ovulación, la implantación y a medida que transcurre la gestación. La concentración del radical anión superóxido (O2-) aumenta aproximadamente seis veces en el proestro, mientras que su enzima reguladora, la superóxido dismutasa, disminuye. La presencia del radical superóxido está relacionada con el edema y la proliferación celular del estro. Este radical también está relacionada con el aumento de la fluidez y polaridad de las membranas durante la implantación. A medida que evoluciona la preñez aumentan los productos de lipoperoxidación y los compuestos antioxidantes, sugiriendo que los lipoperóxidos durante esta etapa evocan a los mecanismos de defensa, de tal manera que al final de la gestación los antioxidantes superan a los fenómenos peroxidativos. Por otro lado, el radical óxido nítrico ha cobrado una gran importancia durante la gestación, ya que es considerado como uno de los relajantes mas potentes del músculo liso. La inhición de su síntesis provoca signos semejantes a los de pre-eclampsia y su administración logra revertir muchas alteraciones vasculares. El papel de los radicales libres no se limita a efectos nocivos, sino que oportunamente y en concentraciones adecuadas, éstos participan como inductores de factores de crecimientos o en la fluidez de las membranas

Participación del líquido folicular en el desarrollo del folículo, la maduración del ovocito y la función espermática / Participation of follicular fluid in follicle development, oocyte maturity and spermatic function

El estudio de la composición del líquido folicular (LF) es de gran importancia en reproducción asistida, por la función que éste desempeña en la maduración del ovocito y en la capacitación espermática. La composición bioquímica del LF está influenciada por el estado de maduración del folículo y recíprocamente dicha composición predice el éxito de la subsecuente maduración folicular y el embarazo. En la mayoría de las especies las concentraciones elevadas de estradiol son comunes de los folículos de madurez intermedia la progesterona de los maduros y los andrógenos de los atrésicos. La concentración del estradiol está asociada con la capacidad fertilizante de los ovocitos y la de sus metabolitos con la estimulación de la producción de progesterona. Además, el IF regula la acción de las gonadotropinas, al favorecer la unión de éstas a su receptor. El LF contribuye regulando la meiosis, ya que contiene factores que estimulan la maduración del ovocito o que bloquean la habilidad de los gránulos corticales para modificar los componentes de la zona pelúcida. Asimismo, la exposición de los espermatozoides a este fluido favorece la reacción acrosomal, su movilidad y la capacidad para penetrar al óvulo

Participación del citoesqueleto en la fisiología del endometrio / Cytoskeleton participation in endometrium physiology

En el endometrio el citoesqueleto participa en todas las funciones mecánicas de la célula, en el movimiento y reacomodo de organelos y proteínas solubles y en el metabolismo en general. El epitelio endometrial por su morfología y aparente homogeneidad celular, se ha estudiado más que el estroma. Se sabe que los filamentos intermedios muestran un patrón característico típico de la clase celular. Durante la preñez y la pseudopreñez, en la región apical de las células epiteliales tanto luminales como glandulares predomina la queratina sobre la región basolateral, en tanto que la vimetina solo se encuentra en las células epiteliales luminales, y se incrementa el día de la implantación. En humanos y roedores, la desmina solo se expresa en la decidua. Se piensa que los filamentos intermedios, participan en la difusión de proteínas de membrana cambiando la polaridad. Los microfilamentos intervienen en la regulación de la forma y movilidad celular. En el epitelio luminal participan en las transformaciones de la superficie uterina, como son las microvellosidades. Al sistema de microtúbulos (MT) en el endometrio y otros órganos se le ha relacionado con la psisción y movimiento de orgánulos como son los lisosomas, mitocondrias o aparato de Golgio; además se ha demostrado que los MT también intervienen en la síntesis de DNA, ya que drogas como la colchicina impiden estos fenómenos

El sistema lisosomal en los mecanismos hormonales: revisión / Lysosomal system in hormonal mechanisms: review

Los lisosomas no deben considerarse sólo orgánulos que participan en la lisis de diferentes moléculas en proceso de digestión o defensa celular, ya que participan en diversos procesos de metabolismo celular, incluyendo los regulados hormonales. La actividad lisosomal se ve alterada minutos después de la administración de hormonas tanto esteroides como peptídicas incluyendo efectos como: labilización de membranas, cambio en la estructura química de las hidrolasas (latencia estructural) y migración perinuclear de lisosomas. Estas alteraciones demuestran que la actividad lisosomal es hormino-dependiente, y que hidrolasas como fosfatasa ácida pueden esta actuando de alguna manera a nivel de des-represión génica. Por otro lado, los lisosomas pueden propagar efectos hormonales desde la superficie de las células blanco hasta el núcleo. Es muy probable que ésta propagación se efectúe por medio de poblaciones especializadas de lisosomas que bajo el efecto hormonal migran a la región perinuclear. Se ha demostrado que ciertas substancias naturales y sintéticas que estabilizan membranas pueden bloquear esos eventos de distinto grado, de tal manera, que estos restablecen la labilización lisosomal y por consiguiente impiden la liberación enzimática. De una u otra forma, por lo anteriormente descrito, el sistema lisosomal participa en los mecanismos de comunicación autocrina, paracrina y endocrina