Genoveva Durán-Reyes

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.

Hyperglycemia induces apoptosis and p53 mobilization to mitochondria in RINm5F cells.

The mechanisms related to hyperglycemia-induced pancreatic β-cell apoptosis are poorly defined. Rat insulin-producing cells (RINm5F) cultured in high glucose concentrations (30 mM) showed increased apoptosis and protein p53 translocation to mitochondria. In addition, hyperglycemia induced both the disruption of mitochondrial membrane potential (Δ < eqid1 > m), and an increase in reactive oxygen species (ROS), as shown by fluorescence changes of JC-1 and dichlorodihydrofluorescein-diacetate (DCDHF-DA), respectively. The increased intracellular ROS by high glucose exposure was blunted by mitochondrial-function and NADPH-oxidase inhibitors. We postulate that the concomitant mobilization of p53 protein to the mitochondria and the subsequent changes on the Δ < eqid2 > m, lead to an important pancreatic β-cell apoptosis mechanism induced by oxidative stress caused by hyperglycemia.

Pro-oxidating properties of melatonin in the in vitro interaction with the singlet oxygen.

In an aqueous system, the oxidation of the erythrocyte membrane by the singlet oxygen formed during the photoactivation of the rose bengal coloring was examined. The effects of the singlet oxygen on lipids and proteins were studied through the simultaneous quantification of peroxidation products, lipoperoxides and carbonyl groups, the oxidation of protein SH groups and the activity of the glyceraldehyde 3-phosphate dehydrogenase (G3PD) associated with the erythrocyte membrane. The antioxidant activity of melatonin was tested and compared to that of two antioxidants in extreme cases of hydrosolubility, ascorbate and beta-carotene, with the purpose of comparing the protective ability of melatonin against singlet oxygen. The results show the expected effect even at low (0.125-0.75 mM; 0.015-0.90 mM, respectively) for ascorbate and beta-carotene, antioxidants known to possess important antioxidant qualities against singlet oxygen. It is shown that melatonin, under the conditions described, and at the concentrations at which the other two compounds are efficacious, not only confers little antioxidant protection, but that a pro-oxidant tendency was proven both on lipids and proteins, as well as on G3PD enzymatic activity. The results show that the antioxidant protective effect that melatonin can exert on biological systems is probably not by a direct interaction with oxidant species, but probably, as has been previously proposed, through the regulation of antioxidant defense systems. The formation of secondary oxidation products, such as melatonin-derived endoperoxides, may explain the evidence found on pro-oxidant qualities of this molecule.

Nitric oxide synthesis inhibition suppresses implantation and decreases cGMP concentration and protein peroxidation

The effect of Nw-nitro-L-arginine on embryonic implantation and cGMP carbonyl group concentration was assessed at the rat implantation site (IS) and non-implantation site (NIS). The intraluminal administration of 25 microg (2.3 mM) of Nw-nitro-L-arginine inhibited implantation in 34.7% and embryo survival (100%), while in addition, decreasing cGMP concentration both at the site (1664.2 +/- 333.8 pmoles/mg of protein for the control and 1321 +/- 384.3 for those treated), as well as at the NIS (1203.7 +/- 200 to 780.2 +/- 168.5). Carbonyl group concentration was considerably less at the implantation site treated with Nw-nitro-L-arginine than in the control (0.062 +/- 0.012 nmoles/mg of protein and 0.45 +/- 0.1, respectively). Nonetheless, the NIS was not significantly different (0.12 +/- 0.04 and 0.15 +/- 0.05). Our results show that a nitric oxide (NO) dependent system parallel to the formation of cGMP and protein peroxidation products is important at the blastocyst implantation site in order for the endometrium to acquire the necessary properties for an adequate receptivity.

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.

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.

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 β-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 (1,134.4 ± 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 µIU/ml, respectively) and similar to those of the non-diabetic group (127.2 ± 34.1 mg/dl and 41.7 ± 13.9 µIU/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 β-cell damage and diabetes.

Effect of dexamethasone as an inhibitor of implantation and embryo development in rat; lysosomal role

In order to learn the mechanism of action of dexamethasone administration as an efficient inhibitor of estrogen activity in different tissues, the subcellular enzymatic distribution of two lysosomal enzymes: acid phosphatase (E.C.3.1.3.2.) and beta glucuronidase (E.C.3.2.1.3.1.) were measured. The rats were treated during preimplantation with dexamethasone (0.8 mg on days 3 and 4) or saline (controls). In the control group, the nuclear activity of lysosomal enzymes was significantly less in the implantation site tissue than in the treated group (p < 0.05). There were no modifications on the undecidualized endometrium under the steroid treatment. The lysosomal subfraction showed an opposite response. The steroid treatment produced an increase of activity in the decidualized tissue (1.9 +/- 0.4 to 4.9 +/- 0.4) while the nuclear enzymatic activity decreased under treatment; and simultaneously, the embryonic development was 100% abolished. From the results presented herein, it is proposed that the inhibitory effect of dexamethasone upon implantation is due to an inadequate biochemical differentiation at the implantation site, related to the inhibition of lysosomal movement toward the nucleus, and consequently to lysosomal enzymatic release and metabolic role.

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.

O-GlcNAc-Selective-N-Acetyl-β-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-β-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 µmol/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 β-cell function index (HOMA-β) 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 Km 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.