AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis.

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate-nitrite-NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (l-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate-nitrite-NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.

A Novel Method to Improve Perfusion of Ex Vivo Pumped Human Kidneys.

OBJECTIVE: To determine if addition of the S-nitrosylating agent ethyl nitrite (ENO) to the preservation solution can improve perfusion parameters in pumped human kidneys. BACKGROUND: A significant percentage of actively stored kidneys experience elevations in resistance and decreases in flow rate during the ex vivo storage period. Preclinical work indicates that renal status after brain death is negatively impacted by inflammation and reduced perfusion-processes regulated by protein S-nitrosylation. To translate these findings, we added ENO to the preservation solution in an attempt to reverse the perfusion deficits observed in nontransplanted pumped human kidneys. METHODS: After obtaining positive proof-of-concept results with swine kidneys, we studied donated human kidneys undergoing hypothermic pulsatile perfusion deemed unsuitable for transplantation. Control kidneys continued to be pumped a 4°C (ie, standard of care). In the experimental group, the preservation solution was aerated with 50 ppm ENO in nitrogen. Flow rate and perfusion were recorded for 10 hours followed by biochemical analysis of the kidney tissue. RESULTS: In controls, perfusion was constant during the monitoring period (ie, flow rate remained low and resistance stayed high). In contrast, the addition of ENO produced significant and sustained reductions in resistance and increases in flow rate. ENO-treated kidneys had higher levels of cyclic guanosine monophosphate, potentially explaining the perfusion benefits, and increased levels of interleukin-10, suggestive of an anti-inflammatory effect. CONCLUSIONS: S-Nitrosylation therapy restored the microcirculation and thus improved overall organ perfusion. Inclusion of ENO in the renal preservation solution holds promise to increase the number and quality of kidneys available for transplant.

Supplemental nitrite increases choroidal neovascularization in mice.

Low doses of nitrite, close to physiological levels, increase blood flow in normal and ischemic tissues through a nitric oxide (NO) dependent mechanism. Given that nitrite therapy and dietary supplementation with vegetables high in nitrate (e.g. beets) are gaining popularity we decided to determine if low doses of nitrite impact the development of choroidal neovascularization (CNV), a key feature of wet age related macular degeneration (AMD). Sodium nitrite (at 50 mg/L, 150 mg/L, and 300 mg/L), nitrate (1 g/L) or water alone were provided in the drinking water of C57BL/6 J mice aged 2 or 12 months. Mice were allowed to drink ad libitum for 1 week at which time laser-induced choroidal neovascularization (L-CNV) was induced. The mice continued to drink the supplemented water ad libitum for a further 14 days at which point optical coherence tomography (OCT) was performed to determine the volume of the CNV lesion. Blood was drawn to determine nitrite and nitrate levels and eyes taken for histology. CNV volume was 2.86 × 107 µm3 (±0.4 × 107) in young mice on water alone but CNV volume more than doubled to >6.9 × 107 µm3 (±0.8 × 107) in mice receiving 300 mg/L nitrite or 7.34 × 107 µm3 (±1.4 × 107) in 1 g/L nitrate (p < 0.01). A similar trend was observed in older mice. CNV volume was 5.3 × 107 µm3 (±0.5 × 107) in older mice on water alone but CNV volume almost doubled to approximately 9.3 × 107 µm3 (±1.1 × 107) in mice receiving 300 mg/L nitrite or 8.7 × 107 µm3 (±0.9 × 107) 1 g/L nitrate (p < 0.01). Plasma nitrite levels were highest in young mice receiving 150 mg/L in the drinking water with no changes in plasma nitrate observed. In older mice, drinking water nitrite did not significantly change plasma nitrite, but plasma nitrate was increased. Plasma nitrate was elevated in both young and old mice provided with nitrate supplemented drinking water. Our data demonstrate that the CNV lesion is larger in older mice compared to young and that therapeutic levels of oral nitrite increase the volume of CNV lesions in both young and older mice. Therapeutic nitrite or nitrate supplementation should be used with caution in the elderly population prone to CNV.

Direct comparison of inorganic nitrite and nitrate on vascular dysfunction and oxidative damage in experimental arterial hypertension.

Arterial hypertension is one of the major health risk factors leading to coronary artery disease, stroke or peripheral artery disease. Dietary uptake of inorganic nitrite (NO2-) and nitrate (NO3-) via vegetables leads to enhanced vascular NO bioavailability and provides antihypertensive effects. The present study aims to understand the underlying vasoprotective effects of nutritional NO2- and NO3- co-therapy in mice with angiotensin-II (AT-II)-induced arterial hypertension. High-dose AT-II (1 mg/kg/d, 1w, s. c.) was used to induce arterial hypertension in male C57BL/6 mice. Additional inorganic nitrite (7.5 mg/kg/d, p. o.) or nitrate (150 mg/kg/d, p. o.) were administered via the drinking water. Blood pressure (tail-cuff method) and endothelial function (isometric tension) were determined. Oxidative stress and inflammation markers were quantified in aorta, heart, kidney and blood. Co-treatment with inorganic nitrite, but not with nitrate, normalized vascular function, oxidative stress markers and inflammatory pathways in AT-II treated mice. Of note, the highly beneficial effects of nitrite on all parameters and the less pronounced protection by nitrate, as seen by improvement of some parameters, were observed despite no significant increase in plasma nitrite levels by both therapies. Methemoglobin levels tended to be higher upon nitrite/nitrate treatment. Nutritional nitric oxide precursors represent a non-pharmacological treatment option for hypertension that could be applied to the general population (e.g. by eating certain vegetables). The more beneficial effects of inorganic nitrite may rely on superior NO bioactivation and stronger blood pressure lowering effects. Future large-scale clinical studies should investigate whether hypertension and cardiovascular outcome in general can be influenced by dietary inorganic nitrite therapy.

Effects of age, sex and diet on salivary nitrate and nitrite in infants.

The inorganic anions nitrate and nitrite are oxidation products from endogenous nitric oxide (NO) generation and constituents in our diet. A nitrate-nitrite-NO pathway exists in which nitrate can be serially reduced to bioactive NO. The first step of this pathway occurs in the oral cavity where oral bacteria convert salivary nitrate to nitrite, whereafter nitrite is reduced to NO systemically by several enzymatic and non-enzymatic pathways. Data are scarce regarding salivary levels and oral conversion capacity of these anions in infants. We measured salivary nitrate and nitrate in infants at 4 and 12 months of age and related values to age, sex, dietary pattern and oral microbiome. Saliva was collected from a total of 188 infants at 4 and 12 months of age. Salivary nitrate, nitrite and nitrite/nitrate ratio as a measure of oral nitrate-reducing capacity were analyzed by HPLC and related to age, sex, type of diet (breast milk or formula) and oral microbiome. There was no difference in salivary nitrate, nitrite or nitrite/nitrate ratio between boys and girls at any age. At 4 months levels of these parameters were lower than what has been described in adults but they had all increased significantly at 12 months of age. At 4 months of age salivary nitrite/nitrate ratio was lower in breast-fed compared to formula-fed infants, but these differences disappeared at 12 months. Several bacterial species were associated with oral nitrate reducing capacity including Prevotella, Veillonella, Alloprevotella and Leptotrichia. We conclude that in infants there is an increase in salivary nitrate and nitrite as well as in oral nitrate-reductase capacity during the first year of life. Differences observed at 4 months of age between breast-fed and formula-fed infants disappear at one year of age.

Nitrite pharmacokinetics, safety and efficacy after experimental ventricular fibrillation cardiac arrest.

INTRODUCTION: Besides therapeutic hypothermia or targeted temperature management no novel therapies have been developed to improve outcomes of patients after cardiac arrest (CA). Recent studies suggest that nitrite reduces neurological damage after asphyxial CA. Nitrite is also implicated as a new mediator of remote post conditioning produced by tourniquet inflation-deflation, which is under active investigation in CA. However, little is known about brain penetration or pharmacokinetics (PK). Therefore, to define the optimal use of this agent, studies on the PK of nitrite in experimental ventricular fibrillation (VF) are needed. We tested the hypothesis that nitrite administered after resuscitation from VF is detectable in cerebrospinal fluid (CSF), brain and other organ tissues, produces no adverse hemodynamic effects, and improves neurologic outcome in rats. METHODS: After return of spontaneous circulation (ROSC) of 5 min untreated VF, adult male Sprague-Dawley rats were given intravenous nitrite (8 µM, 0.13 mg/kg) or placebo as a 5 min infusion beginning at 5 min after CA. Additionally, sham groups with and without nitrite treatment were also studied. Whole blood nitrite levels were serially measured. After 15 min, CSF, brain, heart and liver tissue were collected. In a second series, using a randomized and blinded treatment protocol, rats were treated with nitrite or placebo after arrest. Neurological deficit scoring (NDS) was performed daily and eight days after resuscitation, fear conditioning testing (FCT) and brain histology were assessed. RESULTS: In an initial series of experiments, rats (n = 21) were randomized to 4 groups: VF-CPR and nitrite therapy (n = 6), VF-CPR and placebo therapy (n = 5), sham (n = 5), or sham plus nitrite therapy (n = 5). Whole blood nitrite levels increased during drug infusion to 57.14 ±â€¯10.82 µM at 11 min post-resuscitation time (1 min after dose completion) in the VF nitrite group vs. 0.94 ±â€¯0.58 µM in the VF placebo group (p < 0.001). There was a significant difference between the treatment and placebo groups in nitrite levels in blood between 7.5 and 15 min after CPR start and between groups with respect to nitrite levels in CSF, brain, heart and liver. In a second series (n = 25 including 5 shams), 19 out of 20 animals survived until day 8. However, NDS, FCT and brain histology did not show any statistically significant difference between groups. CONCLUSIONS: Nitrite, administered early after ROSC from VF, was shown to cross the blood brain barrier after a 5 min VF cardiac arrest. We characterized the PK of intravenous nitrite administration after VF and were able to demonstrate nitrite safety in this feasibility study.

Effects of inorganic nitrate and nitrite consumption on cognitive function and cerebral blood flow: A systematic review and meta-analysis of randomized clinical trials.

We conducted a systematic review and meta-analysis of randomized clinical trials examining the effect of inorganic nitrate or nitrite supplementation on cognitive function (CF) and cerebral blood flow (CBF). Two databases (PubMed, Embase) were searched for articles from inception until May 2017. Inclusion criteria were: randomized clinical trials; participants >18 years old; trials comparing a nitrate/nitrite intervention with a control. Thirteen and nine trials were included in the meta-analysis to assess CF and CBF, respectively. Random-effects models were used and the effect size described as standardized mean differences (SMDs). A total of 297 participants (median of 23 per trial) were included for CF; 163 participants (median of 16 per trial) were included for CBF. Nitrate/nitrite supplementation did not influence CF (SMD +0.06, 95% CI: -0.06, 0.18, P = 0.32) or CBF under resting (SMD +0.14, 95% CI: -0.13, 0.41, P = 0.31), or stimulated conditions (SMD + 0.23, 95% CI: -0.11, 0.56, P = 0.19). The meta-regression showed an inverse association between duration of the intervention and CBF (P = 0.02) but no influence of age, BMI or dose (P < 0.05). Nitrate and nitrite supplementation did not modify CBF or CF. Further trials employing larger samples sizes and interventions with longer duration are warranted.

Contrasting effects of low versus high ascorbate doses on blood pressure responses to oral nitrite in L-NAME-induced hypertension.

Nitrite reduces blood pressure (BP) in both clinical and experimental hypertension. This effect is attributable to the formation of nitric oxide (NO) and other NO-related species, which may be improved by ascorbate or other antioxidants. However, the BP responses to oral nitrite result, at least in part, of increased gastric S-nitrosothiol formation. This study tested the hypothesis that ascorbate may destroy S-nitrosothiols and therefore not all doses of ascorbate enhance the BP responses to oral nitrite. We assessed the BP responses to oral sodim nitrite (0.2 mmol/kg) in L-NAME hypertensive rats pretreated with ascorbate (0, 0.02, 0.2, or 2 mmol/kg). Plasma and gastric wall concentrations of nitrite and nitroso compounds concentrations were determined using an ozone-based reductive chemiluminescence assay. Nitrate concentrations were determined using the Griess reaction. Free thiol concentrations were determined by a colorimetric assay. The BP responses to nitrite exhibited a bell-shape profile as they were not modified by ascorbate 0.02 mmol/l, whereas the 0.2 mmol/kg dose enhanced and the 2 mmol/kg dose attenuated BP responses. In parallel with BP responses, nitrite-induced increases in plasma nitrite and RSNO species were not modified by ascorbate 0.02 mmol/l, whereas the 0.2 mmol/kg dose enhanced and the 2 mmol/kg dose attenuated them. Similar experiments were carried out with an equimolar dose of S-nitrosogluthathione. Ascorbate dose-dependently impaired the BP responses to S-nitrosogluthathione, and the corresponding increases in plasma RSNO, but not in plasma nitrite concentrations. This is the first study to show that while ascorbate dose-dependently impairs the BP responses to oral S-nitrosogluthathione, there are contrasting effects when low versus high ascorbate doses are compared with respect to its effects on the blood pressure responses to oral nitrite administration. Our findings may have special implications to patients taking ascorbate, as high doses of this vitamin may impair protective mechanisms associated with nitrite or nitrate from dietary sources.

Reduced blood pressure responsiveness to skeletal muscle metaboreflex activation in older adults following inorganic nitrate supplementation.

The vasoactive molecule nitric oxide (NO) contributes to regulation of blood pressure (BP) at rest and during exercise. Age-related exaggerated increased BP responses during exercise have been proposed to be due in part to a decreased NO bioavailability and possibly an enhanced skeletal muscle metaboreflex. In the present study we sought to determine if age-related differences in BP responses to skeletal muscle metaboreflex activation exist. Additionally, since NO bioavailability can be improved with exogenous nitrate (NO3-) via the nitrate-nitrite-NO pathway, we tested the hypothesis that inorganic NO3- supplementation would reduce BP responses to muscle metaboreflex activation in healthy older adults. 13 older adults (67 ±â€¯1 years) participated in a randomized, double-blind, placebo controlled crossover study consisting of four weeks of NO3- supplementation [beetroot powder; 250 mg (∼4.03 mmol) of NO3- and 20 mg (∼0.29 mmol) of NO2-] and four weeks of placebo (beetroot powder devoid of NO3-/NO2-). Skeletal muscle metaboreflex testing consisted of isometric handgrip exercise (IHG) at 30% of maximal voluntary contraction immediately followed by post exercise forearm ischemia (PEI), which was achieved by inflation of a rapid pressure cuff (240 mmHg) around the upper arm. BP responses were analyzed as the change (Δ) from baseline to the end of IHG and PEI. An additional 10 young adults (25 ±â€¯1 years) were recruited to serve as a reference cohort and address if BP responses to skeletal muscle metaboreflex activation were greater with aging. BP responses to IHG were similar between the young and older adults. However, older adults demonstrated a greater increase in systolic BP during PEI (P < 0.05). Plasma NO3- and NO2-were increased following NO3- supplementation in older adults (P < 0.01). ΔSystolic BP (19 ±â€¯2 vs. 13±3 mmHg, P < 0.05), ΔDiastolic BP (7 ±â€¯1 vs. 5±1 mmHg, P < 0.05) and ΔMean arterial pressure (11 ±â€¯1 vs. 8±2 mmHg, P < 0.05) were reduced during PEI following four weeks of NO3-supplementation, whereas placebo had no effect on ΔSystolic BP (16 ±â€¯2 vs. 17±2 mmHg), ΔDiastolic BP (5 ±â€¯1 vs. 7±1 mmHg), and ΔMean arterial pressure (8 ±â€¯1 vs. 10±1 mmHg) during PEI (all P > 0.05). These data suggest that inorganic NO3- supplementation attenuates skeletal muscle metaboreflex mediated increases in BP during exercise in older adults.

Poppers and PrEP: Use of Pre-exposure Prophylaxis Among Men Who Have Sex with Men Who Use Inhaled Nitrites.

Men who have sex with men (MSM) commonly use inhaled nitrites, or poppers, though their use is a risk factor HIV seroconversion. Pre-exposure prophylaxis, or PrEP, is effective for HIV prevention, but is not widely used, and little is known regarding PrEP use and acceptability among MSM who use inhaled nitrites. We surveyed 580 MSM in Paris, France in 2016 about popper use, sexual behaviors including condomless anal intercourse (CAI), serosorting, and sexual positioning, PrEP use, PrEP candidacy, and interest in alternate PrEP delivery modalities. We included 444 HIV negative participants for the current study. 46.2% reported popper use in the prior 3 months. Using multivariate adjusted logistic regression, we found that popper users were more likely than non-users to consider themselves candidates for PrEP [adjusted relative risk ratio (aRRR) = 2.73; 95% CI 1.54-4.83], but they were not more likely to be current (aRRR = 1.54; 95% CI 0.71-3.33) or past (aRRR = 1.37; 95% CI 0.44-4.28) PrEP users. Mediation analyses indicated that increased CAI and serosorting partly explained the relationship between popper use and PrEP candidacy. There was considerable interest in alternate proposed PrEP delivery modalities, particularly long-acting injectable PrEP [adjusted risk ratio (aRR) = 1.43; 95% CI 1.15-1.79].

Dietary Nitrite Attenuates Elastase-Induced Pulmonary Emphysema in a Mouse Model.

Pulmonary emphysema (PE) is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is characterized by proteolytic destruction of the alveolar structure and subsequent inflammation of the respiratory tract. We hypothesized that nitrite attenuates the development of PE via anti-inflammatory actions. PE was induced by intratracheal instillation of porcine pancreas elastase (PPE) in mice. Dietary nitrite dose-dependently (50 and 150 mg/L in drinking water) attenuated emphysematous development and macrophage accumulation in the alveolar parenchyma 21 d after PPE treatment. The present study shows that dietary nitrite might be a possible nutritional strategy in preventing the development of PE in mice.

Oxidative stress and antioxidant responses in juvenile Brazilian flounder Paralichthys orbignyanus exposed to sublethal levels of nitrite.

This study evaluated the effects of short-term exposure to sublethal levels of nitrite on oxidative stress parameters and histology of juvenile Brazilian flounder Paralichthys orbignyanus. An assessment of fish recovery was also performed. Fish were exposed to 0.08 (control), 5.72, 10.43, and 15.27 NO2-N mg L-1 for 10 days followed by the same recovery time. Gill, liver, and muscle samples were collected after 1, 5, and 10 days of exposure and after recovery for the measurement of antioxidant capacity against peroxyl radicals (ACAP), glutathione-S-transferase (GST) activity, content of non-protein (NPSH) and protein thiols (PSH), and lipid peroxidation levels by thiobarbituric acid-reactive substances (TBARS) content. Nitrite exposure induced alterations which compromised the overall antioxidant system (reduced ACAP and GST activity) and enhanced oxidative damage in lipids and proteins. Increases in GST activity and NPSH and PSH contents were also demonstrated. The recovery period allowed for resumption of basal levels for all (treatment 5.72 NO2-N mg L-1) or some of the evaluated parameters (other treatments). In conclusion, exposure to nitrite concentrations from 5.72 to 15.27 NO2-N mg L-1 induced oxidative stress and antioxidant responses in juvenile Brazilian flounder. The 10-day recovery period was sufficient for a complete resumption of basal physiological condition of fish exposed to concentrations of up to 5.72 NO2-N mg L-1.

Methaemoglobinaemia: A Blue Light Emergency.

We present the case of a patient with severe methaemoglobinaemia secondary to "poppers". She presented with decreased level of consciousness and cyanosis. A methaemoglobin level was not available prior to treatment but the patient responded well and the diagnosis was confirmed afterwards.

Dietary nitrite reverses features of postmenopausal metabolic syndrome induced by high-fat diet and ovariectomy in mice.

Menopausal women are at greater risk of developing metabolic syndrome with reduced endothelial nitric oxide synthase (eNOS) activity. Hormone replacement therapy increases eNOS activity and normalizes some characteristics of metabolic syndrome. We hypothesized that nitric oxide (NO) supplementation should have a therapeutic effect on this syndrome. We examined the effect of dietary nitrite in a mouse model with postmenopausal metabolic syndrome induced by ovariectomy (OVX) and a high fat diet (HF). C57BL/6 female mice were divided into five groups, sham+normal fat diet (NF), sham+ HF, OVX+HF with or without sodium nitrite (50 mg and 150 mg/l) in the drinking water. Daily food intake and weekly body weight were monitored for 18 wk. OVX and HF significantly reduced plasma levels of nitrate/nitrite (NOx), and mice developed obesity with visceral hypertrophic adipocytes and increased transcriptional levels of monocyte chemoattractant protein-1, TNF-α, and IL-6 in visceral fat tissues. The proinflammatory state in the adipocytes provoked severe hepatosteatosis and insulin resistance in OVX+HF group compared with sham+NF group. However, dietary nitrite significantly suppressed adipocyte hypertrophy and transcriptions of proinflammatory cytokines in visceral fat in a dose-dependent manner. The improvement of visceral inflammatory state consequently reversed the hepatosteatosis and insulin resistance observed in OVX+HF mice. These results suggest that an endogenous NO defect might underlie postmenopausal metabolic syndrome and that dietary nitrite provides an alternative source of NO, subsequently compensating for metabolic impairments of this syndrome.

Both nitric oxide and nitrite prevent homocysteine-induced endoplasmic reticulum stress and subsequent apoptosis via cGMP-dependent pathway in neuronal cells.

Growing evidence indicates that endoplasmic reticulum (ER) stress and/or ER stress-mediated apoptosis may play a role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. The present study investigated the effects of non-cytotoxic concentrations of nitric oxide (NO) and nitrite, a metabolite of NO, on ER stress and ER stress-mediated apoptosis in Neuro-2a cells exposed to homocysteine (Hcy), an endogenous ER stress inducer. Hcy induced ER stress, as confirmed by inositol-requiring enzyme 1α (IRE1α) phosphorylation and X-box-binding protein-1 (Xbp1) mRNA splicing as well as C/EBP homologous protein (CHOP) expression, and apoptosis, as verified by Annexin V-positive cells. Surprisingly, non-cytotoxic NO (S-nitrosoglutathione) and nitrite markedly reduced Hcy-induced IRE1α phosphorylation, Xbp1 mRNA splicing, CHOP expression, and Annexin V-positive cells, indicating the cytoprotection of NO and nitrite against Hcy-induced ER stress and apoptosis. Moreover, inhibition of sGC/cGMP pathway abolished the cytoprotective effects of NO and nitrite, whereas cellular elevation of cGMP levels mimicked the cytoprotective actions of NO and nitrite. These findings provide the first evidence showing that both NO and nitrite can reduce ER stress and subsequent apoptosis via NO-sGC-cGMP pathway in neuronal cells and suggesting that NO and/or nitrite may have therapeutic value in the treatment of ER stress-associated neurodegenerative diseases.

Nitrite increases glucose-stimulated insulin secretion and islet insulin content in obese type 2 diabetic male rats.

PURPOSE: Reduced bioavailability of nitric oxide (NO) is associated with pathogenesis of type 2 diabetes. Nitrite can act as a substrate for generation of systemic NO. The aim of this study was to examine the effects of nitrite administration on glucose-stimulated insulin secretion (GSIS) and islet insulin content in obese type 2 diabetic rats. METHODS: Male rats were divided into 4 groups: Control, control + nitrite, diabetes, and diabetes + nitrite. Sodium nitrite (50 mg/L in drinking water) was administered for 8 weeks. Diabetes was induced using high-fat diet and low-dose of streptozotocine. Serum levels of fasting glucose, insulin, and lipid profile were measured and the insulin resistance/sensitivity indices were calculated every 2 weeks. Glycated hemoglobin (HbA1C) was measured every month. At the end of the study, tissue levels of glucose transporter 4 (GLUT4) protein and serum interleukin-1 beta (IL-1ß) were measured as well as glucose and insulin tolerance test were done. GSIS from isolated pancreatic islets and islet insulin content were also determined. RESULTS: Nitrite administration significantly increased insulin secretion in both control and diabetic rats in presence of 16.7 mM glucose. Nitrite also significantly increased islet insulin content by 27% and 39% in both control and diabetic rats, respectively. Nitrite decreased elevated serum IL-1ß in diabetic rats (4.0 ± 0.2 vs. 2.9 ± 0.2 pg/mL, P = 0.001). In diabetic rats, nitrite also significantly increased tissue levels of GLUT4 by 22% and 26% in soleus muscle and epididymal adipose tissue, respectively. In addition, nitrite significantly improved glucose and insulin tolerance, insulin sensitivity, lipid profile, and decreased fasting glucose and insulin, but had no effect on HbA1C. CONCLUSIONS: Long-term nitrite administration increased both insulin secretion and insulin content in obese type 2 diabetic rats. In addition, nitrite therapy had favorable effects on glucose tolerance, insulin resistance, inflammation, and dyslipidemia in type 2 diabetic rats.

S-nitrosation of calpains is associated with cardioprotection in myocardial I/R injury.

BACKGROUND: Myocardial infarction remains the single leading cause of death worldwide. Upon reperfusion of occluded arteries, deleterious cellular mediators particularly located at the mitochondria level can be activated, thus limiting the outcome in patients. This may lead to the so-called ischemia/reperfusion (I/R) injury. Calpains are cysteine proteases and mediators of caspase-independent cell death. Recently, they have emerged as central transmitters of cellular injury in several cardiac pathologies e.g. hypertrophy and acute I/R injury. METHODS: Here we investigated the role of cardiac calpains in acute I/R in relation to mitochondrial integrity and whether calpains can be effectively inhibited by posttranslational modification by S-nitrosation. Taking advantage of the a cardiomyocyte cell line (HL1), we determined S-nitrosation by the Biotin-switch approach, cell viability and intracellular calcium concentration after simulated ischemia and reoxygenation - all in dependence of supplementation with nitrite, which is known as an 'hypoxic nitric oxide (NO) donor'. Likewise, using an in vivo I/R model, calpain S-nitrosation, calpain activity and myocardial I/R injury were characterized in vivo. RESULTS: Nitrite administration resulted in an increased S-nitrosation of calpains, and this was associated with an improved cell-survival. No impact was detected on calcium levels. In line with these in vitro experiments, nitrite initiated calpain S-nitrosation in vivo and caused an infarct sparing effect in an in vivo myocardial I/R model. Using electron microscopy in combination with immuno-gold labeling we determined that calpain 10 increased, while calpain 2 decreased in the course of I/R. Nitrite, in turn, prevented an I/R induced increase of calpains 10 at mitochondria and reduced levels of calpain 1. CONCLUSION: Lethal myocardial injury remains a key aspect of myocardial I/R. We show that calpains, as key players in caspase-independent apoptosis, increasingly locate at mitochondria following I/R. Inhibitory post-translational modification by S-nitrosation of calpains reduces deleterious calpain activity in murine cardiomyocytes and in vivo.

Dietary nitrite supplementation attenuates cardiac remodeling in l-NAME-induced hypertensive rats.

Loss of nitric oxide (NO) bioavailability underlies the development of hypertensive heart disease. We investigated the effects of dietary nitrite on NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Sprague-Dawley rats were divided into five groups: an untreated control group, an l-NAME-treated group, and three other l-NAME-treated groups supplemented with 10 mg/L or 100 mg/L of nitrite or 100 mg/L of captopril in drinking water. After the 8-week experimental period, mean arterial blood pressure was measured, followed by sampling of blood and heart tissue for assessment of nitrite/nitrate levels in the plasma and heart, the plasma level of angiotensin II (AT II), and the heart transcriptional levels of AT II type 1 receptor (AT1R), transforming growth factor-ß1 (TGF-ß1), and connective tissue proteins such as type 1 collagen and fibronectin. Heart tissue was analyzed by histopathological morphometry, including assessments of ventricular and coronary vascular hypertrophy and fibrosis, as well as immunohistochemistry analyses of myocardial expression of AT1R. l-NAME treatment reduced the plasma nitrate level and led to the development of hypertension, with increased plasma levels of AT II and increased heart transcriptional levels of AT1R and TGF-ß1-mediated connective tissue proteins, showing myocardial and coronary arteriolar hypertrophy and fibrosis. However, dietary nitrite supplementation inhibited TGF-ß1-mediated cardiac remodeling by suppressing AT II and AT1R. These results suggest that dietary nitrite levels achievable via a daily high-vegetable diet could improve hypertensive heart disease by inhibiting AT II-AT1R-mediated cardiac remodeling.

Nitrite Prevents Right Ventricular Failure and Remodeling Induced by Pulmonary Artery Banding.

BACKGROUND: Nitrite has been shown to reduce right ventricle (RV) remodeling in experimental pulmonary hypertension. However, whether this effect is due to a reduction in RV afterload (ie, reduction in pulmonary artery pressure) or a direct effect on the RV itself remains unanswered. We hypothesize that nitrite has direct effects on RV remodeling and studied its effects in mice with pulmonary artery banding (PAB). METHODS AND RESULTS: PAB decreased exercise tolerance and reduced RV systolic and diastolic function. Nitrite treatment attenuated the decrease in RV systolic function and improved the RV diastolic function. Nitrite-treated mice with PAB had similar exercise tolerance compared with a control group. PAB induced RV hypertrophy and fibrosis which were associated with increased expression of phospho-Akt. Interestingly, nitrite treatment attenuated PAB-induced RV hypertrophy and reduced the expression of phospho-Akt in RV tissue from mice with PAB. In neonatal rat cardiac fibroblast, nitrite also attenuated hypoxia-induced increase in expression of phospho-Akt. CONCLUSION: Our study indicates that nitrite treatment has direct beneficial effects on RV and improves function and attenuates remodeling in RV exposed to chronic pressure overload. These beneficial effects, at least in part, could be due to the inhibition of the phospho-Akt (p-Akt) pathway activation.

The role of inorganic nitrate and nitrite in CVD.

CVD is the leading cause of death worldwide, a consequence of mostly poor lifestyle and dietary behaviours. Although whole fruit and vegetable consumption has been consistently shown to reduce CVD risk, the exact protective constituents of these foods are yet to be clearly identified. A recent and biologically plausible hypothesis supporting the cardioprotective effects of vegetables has been linked to their inorganic nitrate content. Approximately 60-80 % inorganic nitrate exposure in the human diet is contributed by vegetable consumption. Although inorganic nitrate is a relatively stable molecule, under specific conditions it can be metabolised in the body to produce NO via the newly discovered nitrate-nitrite-NO pathway. NO is a major signalling molecule in the human body, and has a key role in maintaining vascular tone, smooth muscle cell proliferation, platelet activity and inflammation. Currently, there is accumulating evidence demonstrating that inorganic nitrate can lead to lower blood pressure and improved vascular compliance in humans. The aim of this review is to present an informative, balanced and critical review of the current evidence investigating the role of inorganic nitrate and nitrite in the development, prevention and/or treatment of CVD. Although there is evidence supporting short-term inorganic nitrate intakes for reduced blood pressure, there is a severe lack of research examining the role of long-term nitrate intakes in the treatment and/or prevention of hard CVD outcomes, such as myocardial infarction and cardiovascular mortality. Epidemiological evidence is needed in this field to justify continued research efforts.