Increased nitric oxide production in platelets from severe chronic renal failure patients.

Nitric oxide (NO) production occurs through oxidation of the amino acid L-arginine by NO synthase (NOS). NO inhibits platelet activation by increasing the levels of cyclic guanosine monophosphate (cGMP), thus maintaining vascular homeostasis. Our group previously demonstrated (da Silva et al. 2005) an enhancement of the L-arginine-NO-cGMP pathway in platelets taken from chronic renal failure (CRF) patients on haemodialysis associated with reduced platelet aggregation. We investigate the platelet L-arginine-NO-cGMP pathway, platelet function, and inflammation from patients in CRF on conservative treatment. A total of 42 CRF patients and 42 controls (creatinine clearance = 27 ± 3 vs. 93 ± 1 mL per min per 1.73 m2, respectively) participated in this study. NOS activity and expression and cGMP concentration were measured in platelets. Platelet aggregation induced by collagen or ADP was evaluated and plasma levels of fibrinogen were determined by the Clauss method. A marked increase in basal NOS activity was seen in undialysed CRF patients compared with controls, accompanied by an elevation of fibrinogen plasma levels. There were no differences in expression of NOS and in cGMP levels. In this context, platelet aggregation was not affected. We provide the first evidence of increased intraplatelet NO biosynthesis in undialysed CRF patients, which can be an early marker of future haemostatic abnormalities during dialysis treatment.

Aerobic exercise training attenuates doxorubicin-induced ultrastructural changes in rat ventricular myocytes.

AIMS: To investigate the effects of aerobic exercise training on cardiomyocyte ultrastructure, oxidative stress, and activation of protein synthesis pathways in a model of cardiomyopathy induced by doxorubicin (Dox). MAIN METHODS: Male Sprague Dawley rats were randomly assigned to Control (saline, sedentary), Dox/sedentary (DoxSed), or Dox/exercise (DoxEx) groups. Saline or Dox were injected i.p. for 10 days (1 mg/kg/d). Aerobic exercise training was performed for 9 wks (starting with drug administration) on a treadmill, 5 d/wk, 30 min/d at 60% of maximum velocity. After euthanasia, the left ventricle (LV) was dissected, and processed for microscopy or frozen for Western blot and kinetic measurement of antioxidant enzymes activity. KEY FINDINGS: Dox resulted in a mortality of 31.2% of sedentary animals, whilst all animals from both Control and DoxEx groups survived. DoxSed animals presented increased LV connective tissue deposition alongside with massive sarcomeric disorganization with dissolution of myofibrils and wavy Z-lines. There was an increase in oxidative damage and a reduction in the activation of both Akt and ERK pathways in LV from DoxSed compared to Control group. Aerobic training caused notable changes in myocardial structure with reduced fibrosis and preservation of myofibrils integrity and sarcomere organization. This was associated with reduced LV oxidative damage and increased activity of antioxidant enzymes, and an increase in the activation of PI3K-Akt pathway. SIGNIFICANCE: Aerobic exercise training was effective in preventing mortality caused by Dox and in preserving LV ultrastructure, partially via activation of the physiological protein synthesis pathway, PI3K-Akt, and reducing oxidative stress.

Weight loss enhances hepatic antioxidant status in a NAFLD model induced by high-fat diet.

Nonalcoholic fatty liver disease (NAFLD) is a benign condition that can progress to more severe liver damage in a process mediated, in part, by disturbances in redox balance. Additionally, some argue that it is set to become the main cause of end-stage liver disease in the near future. Here, we investigated whether diet-induced weight loss is able to reverse hepatic lipid accumulation and reduce oxidative stress in liver from C57BL/6 mice fed a high-fat (HF) diet. Male C57BL/6 mice were divided into 4 groups: standard chow (SC; 10% energy from fat, 16 weeks); HF (50% energy from fat, 16 weeks); SC-HF (SC for 8 weeks followed by HF for 8 weeks); and HF-SC (HF for 8 weeks followed by SC for 8 weeks). The HF diet during 8 (SC-HF) and 16 weeks (HF) downregulated messenger RNA levels and protein expression of Nrf2 and endogenous antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) in the liver; caused liver steatosis; affected liver function markers; increased intra-abdominal and subcutaneous adipose tissue; and induced glucose intolerance and hypercholesterolemia compared with controls (SC). Diet-induced weight loss significantly reduced the intrahepatic lipid accumulation, improved glucose tolerance, and restored both gene and protein expression of the antioxidant enzymes. Our findings suggest that a dietary intervention aimed to induce weight loss may exert protective effects in NAFLD as it can reduce hepatic oxidative stress and intrahepatic lipid accumulation, which can hinder the progression of this condition to more severe states.

Modulation of the cationic amino acid transport system y+L by surface potential, ouabain and thrombin in human platelets: effects of uremia.

BACKGROUND: Nitric oxide (NO), a key endogenous mediator involved in the maintenance of platelet function, is synthesized from the amino acid L-arginine. We have shown that L-arginine transport in platelets is rate-limiting for NO synthesis. A disturbance in the L-arginine-NO pathway in platelets was previously described in chronic renal failure (CRF) patients. METHODS: Detailed kinetic studies were performed in platelets from controls (n = 60) and hemodialysis patients (n = 26). RESULTS: The transport of L-arginine in platelets is mediated via system y+L, which is competitively inhibited by L-leucine in the presence of Na+ and by the irreversible inhibitor pCMB. In platelets, system y+L is markedly stimulated by an Na+/K+-ATPase inhibitor, ouabain, and by changes in surface potential, while it is downregulated by intraplatelet amino acid depletion (zero-trans) and by thrombin. In CRF patients, activation of L-arginine transport was limited to well-nourished patients compared to malnourished patients and controls, where it was reduced and did not differ significantly among the groups under zero-trans conditions. CONCLUSION: Our results provide the first evidence that system y+L in platelets is modulated by zero-trans conditions, surface potential, thrombin and intraplatelet Na+ concentration. Our findings suggest that enhanced transport in CRF involves increased L-arginine exchange with intraplatelet neutral amino acids.

Erythrocyte nitric oxide availability and oxidative stress following exercise.

Growing evidence has shown that acute exercise impairs erythrocyte membrane structure and function as a consequence of increased physical and chemical stress. Erythrocyte-synthesized nitric oxide (NO) is known to modulate membrane fluidity, and its bioavailability depends on the balance between its production and scavenging by reactive oxygen species. Here, we investigated whether a maximal exercise test could affect erythrocyte NO bioavailability and oxidative stress. Twelve men (26±4 years old, V̇O2peak 44.1±4.3 mL·kg-1·min-1) performed a treadmill maximal cardiopulmonary exercise test. Blood was collected at rest and immediately after exercise for erythrocytes isolation. Maximal exercise caused an increase in erythrocytes count, haemoglobin and haematocrit levels. There was no change in L-arginine influx into erythrocytes after exercise. Yet, nitric oxide synthase activity, and thus, NO production, was increased after maximal test, as well cyclic GMP levels. In relation to biomarkers of oxidative stress, maximal test resulted in increased levels of lipid peroxidation, and diminished superoxide dismutase activity. Neither glutathione peroxidase nor catalase activity was affected by maximal test. Our findings demonstrate that the increased erythrocyte membrane rigidity caused by an acute bout of exercise may be caused, in part, by an increased lipid oxidative damage caused by ROS produced exogenously.

Early hepatic insult in the offspring of obese maternal mice.

We hypothesized that the maternal obesity initiates metabolic disorders associated with oxidative stress in the liver of offspring since early life. Mouse's mothers were assigned into 2 groups according to the diet offered (n = 10 per group): standard chow (SC) or high-fat diet (HF). The results revealed that HF offspring had an increase in body mass at day 10 (+25%, P < .05) and in glucose levels (+25%, P < .0001). Hepatic triacylglycerol was increased in HF offspring at day 1 and day 10 compared with SC offspring (+30%, P < .01 and +40%, P < .01) as was hepatic steatosis (+110%, P < .001; +145%, P < .0001). Fatty acid synthase was increased in HF offspring at day 1 (+450%, P < .01) and peroxisome proliferator activator receptor-γ was elevated at day 1 and day 10 (+140%, P < .01; +2741%, P < .01). Peroxisome proliferator activator receptor-α was diminished in HF offspring at day 10 compared with SC offspring (-100%, P < .01). Moreover, carnitine palmitoyl-CoA transferase-1 was decreased in HF offspring at day 1 and day 10 (-80%, P < .01; -60%, P < .05). In the HF offspring (compared with the SC offspring), the catalase and the superoxide dismutase were significantly lower in both days 1 and 10 (P < .05). In 10-day-old offspring, glutathione peroxidase 1 and glutathione reductase were lower in HF offspring than in SC offspring (P < .0001). Our findings suggest that the maternal obesity in mice induces an early oxidative dysfunction coupled with hepatic steatosis and might contribute to progressive liver injury later in life.

Serum level changes of long chain-polyunsaturated fatty acids in patients undergoing periodontal therapy combined with one year of omega-3 supplementation: a pilot randomized clinical trial.

PURPOSE: We aimed to investigate the impact of nonsurgical periodontal treatment combined with one-year dietary supplementation with omega (ω)-3 on the serum levels of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and arachidonic acid (AA). METHODS: Fifteen patients with chronic generalized periodontitis were treated with scaling and root planing. The test group consisted of seven patients (43.1±6.0 years) supplemented with ω-3, consisting of EPA plus DHA, three capsules, each of 300 mg of ω-3 (180-mg EPA/120-mg DHA), for 12 months. The control group was composed of eight patients (46.1±11.6 years) that took a placebo capsule for 12 months. The periodontal examination and the serum levels of DPA, EPA, DHA, and AA were performed at baseline (T0), and 4 (T1), and 12 (T2) months after therapy. RESULTS: In the test group, AA and DPA levels had been reduced significantly at T1 (P<0.05). AA and EPA levels had been increased significantly at T2 (P<0.05). The ΔEPA was significantly higher in the test compared to the placebo group at T2-T0 (P=0.02). The AA/EPA had decreased significantly at T1 and T2 relative to baseline (P<0.05). CONCLUSIONS: Nonsurgical periodontal treatment combined with ω-3 supplementation significantly increased the EPA levels and decreased the AA/EPA ratio in serum after one year follow-up. However, no effect on the clinical outcome of periodontal therapy was observed.

Effects of non-surgical periodontal treatment on the L-arginine-nitric oxide pathway and oxidative status in platelets.

Several studies have suggested an increase of cardiovascular disease (CVD) risk on periodontitis patients. An enhancement has been demonstrated on both platelet activation and oxidative stress on periodontitis patients, which may contribute for this association. Therefore, the aim of this study was to evaluate the effects of non-surgical periodontal treatment on the l-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway and oxidative status in platelets. A total of eight periodontitis patients and eight controls were included in this study. Clinical, laboratory and experimental evaluations were performed on baseline and 90 days after periodontal treatment (except for western blot analysis). The clinical periodontal evaluation included measurements of probing pocket depth (PPD), clinical attachment loss (CAL), % of sites with plaque and % of sites with bleeding on probing. We evaluated: l-[(3)H]arginine influx; nitric oxide synthase (NOS) and arginase enzymes activity and expression; expression of guanylate cyclase and phosphodiesterase-5 enzymes; cGMP levels; platelet aggregation; oxidative status through superoxide dismutase (SOD) and catalase activities, and measurement of reactive oxygen species (ROS) levels and C-reactive protein (CRP) levels. The initial results showed an activation of both l-arginine influx and via system y (+ )L associated with reduced intraplatelet cGMP levels in periodontitis patients and increased systemic levels of CRP. After periodontal treatment, there was a significant reduction of the % of sites with PPD 4-5mm, % of sites with CAL 4-5 mm, and an enhancement in cGMP levels and SOD activity. Moreover, CRP levels were reduced after treatment. Therefore, alterations in the intraplatelet l-arginine-NO-cGMP pathway and oxidant-antioxidant balance associated with a systemic inflammatory response may lead to platelet dysfunction, which may contribute to a higher risk of CVD in periodontitis.

Modulation of cytokines, resistin, and distribution of adipose tissue in C57BL/6 mice by different high-fat diets.

OBJECTIVE: To investigate whether changing the lipid source induces metabolic changes and/or modulates the adipose tissue distribution in mice fed with a high-fat (HF) diet. METHODS: C57BL/6 mice were subjected to a 10-wk control diet (10% fat) or an HF diet (60% fat) containing lard (HF-L), olive oil (HF-O), sunflower oil, or canola oil. Food intake and body weight were measured. At euthanasia, blood was collected and adipose tissue was dissected. Serum hormones and cytokines were determined. RESULTS: The plasma insulin levels were higher in the HF-L and HF-O groups than in the other three groups (P < 0.0001). The levels of resistin were highest in the HF-L and HF-O groups (P < 0.0001). Leptin expression was also highest in these two groups (P < 0.0001). Of the four groups, interleukin-6 was expressed at the highest level in the HF-L group (P < 0.0005), whereas adiponectin was expressed at the lowest level (P < 0.0001). The accumulation of subcutaneous and visceral adipose tissues was higher in the HF-L group compared with the other groups. This group was hypertrophic because of excess subcutaneous fat and epididymal fat in the adipocytes. However, the ratio of subcutaneous to visceral fat was significantly lower in the HF-L and HF-O groups compared with the other groups. CONCLUSION: In mice fed fat-rich diets, the level of adipokines, the distribution of adipose tissue, and the metabolism of carbohydrates are more significantly influenced by the lipid content rather than the absolute amount of lipid.