The effect of nitric oxide on glucose metabolism

Nitric oxide (NO) is an important bioactive signaling molecule that mediates a variety of normal physiological functions, which, if altered, could contribute to the genesis of many pathological conditions, including diabetes. In this study, we examined the possible diabetogenicity of NO by noting differences in the cellular binding of insulin in dogs treated with the NO donor, S-nitrosoglutathione (GSNO) compared to captopril-treated controls. GSNO administration resulted in an abnormality in glucose metabolism which was attributed to decreased binding of insulin to its receptor on the cell membrane of mononuclear leucocytes, 11.60 ± 0.60% in GSNO-treated dogs compared with 18.10 ± 1.90% in captopril-treated control (p < 0.05). The decreased insulin binding was attributed to decreased insulin receptor sites per cell, 21.43 ± 2.51 × 10⁴ in GSNO-treated dogs compared with 26.60 ± 1.57 × 10⁴ in captopril-treated controls (p < 0.05). Average affinity analysis of the binding data demonstrated that this decrease in insulin binding was also due to a decrease in average affinity of the receptor on mononuclear leucocytes for insulin. This was evident by a decrease in empty and filled site affinities in GSNO-treated dogs compared with that of captopril-treated dogs (p < 0.05). It appears that GSNO is exerting its effect by decreasing the number of insulin receptor sites and/or decreasing the average receptor affinity. These results provide evidence for a novel role of NO as a modulator of insulin binding and the involvement of NO in the aetiology of diabetes mellitus. (Mol Cell Biochem 263: 29–34, 2004)     

Skeletal muscle nitric oxide signaling and exercise: a focus on glucose metabolism

Nitric oxide (NO) is an important vasodilator and regulator in the cardiovascular system, and this link was the subject of a Nobel prize in 1998. However, NO also plays many other regulatory roles, including thrombosis, immune function, neural activity, and gastrointestinal function. Low concentrations of NO are thought to have important signaling effects. In contrast, high concentrations of NO can interact with reactive oxygen species, causing damage to cells and cellular components. A less-recognized site of NO production is within skeletal muscle, where small increases are thought to have beneficial effects such as regulating glucose uptake and possibly blood flow, but higher levels of production are thought to lead to deleterious effects such as an association with insulin resistance. This review will discuss the role of NO in skeletal muscle during and following exercise, including in mitochondrial biogenesis, muscle efficiency, and blood flow with a particular focus on its potential role in regulating skeletal muscle glucose uptake during exercise.     

The arachidonic acid effect on platelet nitric oxide level

Arachidonic acid can act as a second messenger regulating many cellular processes among which is nitric oxide (NO) formation. The aim of the present study was to investigate the molecular mechanisms involved in the arachidonic acid effect on platelet NO level. Thus NO, cGMP and superoxide anion level, the phosphorylation status of nitric oxide synthase, the protein kinase C (PKC), and NADPH oxidase activation were measured. Arachidonic acid dose-dependently reduced NO and cGMP level. The thromboxane A₂ mimetic U46619 behaved in a similar way. The arachidonic acid or U46619 effect on NO concentration was abolished by the inhibitor of the thromboxane A₂ receptor SQ29548 and partially reversed by the PKC inhibitor GF109203X or by the phospholipase C pathway inhibitor U73122. Moreover, it was shown that arachidonic acid activated PKC and decreased nitric oxide synthase (eNOS) activities. The phosphorylation of the inhibiting eNOSthr495 residue mediated by PKC was increased by arachidonic acid, while no changes at the activating ser1177 residue were shown. Finally, arachidonic acid induced NADPH oxidase activation and superoxide anion formation. These effects were greatly reduced by GF109203X, U73122, and apocynin. Likely arachidonic acid reducing NO bioavailability through all these mechanisms could potentiate its platelet aggregating power.     

The effect of the nitric oxide donor sodium nitroprusside on glucose uptake in human primary skeletal muscle cells

Nitric oxide (NO) has been implicated as an important signaling molecule in the insulin-independent, contraction-mediated glucose uptake pathway and may represent a novel strategy for blood glucose control in patients with type 2 diabetes (T2DM). The current study sought to determine whether the NO donor, sodium nitroprusside (SNP) increases glucose uptake in primary human skeletal muscle cells (HSkMC) derived from both healthy individuals and patients with T2DM. Vastus lateralis muscle cell cultures were derived from seven males with T2DM (aged 54 ± 2 years, BMI 31.7 ± 1.2 kg/m², fasting plasma glucose 9.52 ± 0.80 mmol/L) and eight healthy individuals (aged 46 ± 2 years, BMI 27.1 ± 1.5 kg/m², fasting plasma glucose 4.69 ± 0.12 mmol/L). Cultures were treated with both therapeutic (0.2 and 2 μM) and supratherapeutic (3, 10 and 30 mM) concentrations of SNP. An additional NO donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) was also examined at a concentration of 50 μM. Glucose uptake was significantly increased following both 30 and 60 min incubations with the supratherapeutic SNP treatments (P = 0.03) but not the therapeutic SNP doses (P = 0.60) or SNAP (P = 0.54). There was no difference in the response between the healthy and T2DM cell lines with any treatment or dose. The current study demonstrates that glucose uptake is elevated by supratherapeutic, but not therapeutic doses of SNP in human primary skeletal muscle cells derived from both healthy volunteers and patients with T2D. These data confirm that nitric oxide donors have potential therapeutic utility to increase glucose uptake in humans, but that SNP only achieves this in supratherapeutic doses. Further study to delineate mechanisms and the therapeutic window is warranted.     

一氧化氮在炎性疼痛中的作用

一氧化氮（nitric oxide,NO）是细胞内重要的信使分子和神经递质,它参与多种生命活动,包括炎性疼痛.NO对炎性疼痛的发展和维持起到了重要的作用.研究NO在疼痛中所起到的作用及其机制有利于阐明痛觉生理和发现疼痛治疗的新手段.目前研究表明,脊髓水平NO参与炎性疼痛调制的可能机制主要有NO/cGMP途径、参与调控即刻早期基因、与其他神经递质的协同作用.另外研究表明,3种类型的一氧化氮合酶（nitric oxide synthases,NOS）在炎性疼痛过程中被激活或者有不同程度的增强表达.     

Role of metabolism pathway interaction of formaldehyde and nitric oxide in the mechanism of their toxic effect. 2. Toxic effect of nitric oxide

Toxic properties of NO in organism are realized under its hyperproduction and inhibition of the system of anti-oxidant protection as a result of complex chemical transformations, the transient metals, oxygen, superoxide and other radicals being their main participant. Here direct paths (through formation of nitrosil complexes with the gem and nongem iron) of the toxic action of NO and the path mediated by active forms of nitrogen are found, which disturb various biomolecules and subcellular component through the reactions of S- and N-nitrozation, nitration, oxidation, desamination and other reaction, cause metabolic disbalance and death of cells by the type of apoptosis or necrosis. A possible mechanism of the death of cells caused by NO was considered on the example of thymocytes. According to this mechanism one of early stages of this death is a decrease of the cell fund of AP, intensification of catabolism of adenine nucleotides and transformation of xanthine oxidoreductase from D-form (xanthine dehydrogenase) of O-forms (xantine oxidase) which catalizes formation of cytotoxic molecules of superoxide and hydroperoxide. This cytotoxic mechanism which includes transformation of xanthine oxidase system, is probably, universal and does not depend essentially on the starting factor.     

Role of interaction of metabolism pathways of formaldehyde and nitric oxide in the mechanism of their toxic effect. 3. Main metabolism sites of formaldehyde and nitric oxide mediating their effect

Data are presented concerning the basic metabolism sites, the reaction paths crossing in them and regulatory and toxical effect of formaldehyde and nitric oxide being mediated through them. In particular, they include: glutathione-formaldehyde-dependent dehydrogenase path of S-nitrosoglutathione reduction, semi-carbaside-sensitive amino-oxidase (SSAO) and NO-synthase systems; transformation of thioproline and metallothioneines, including nitrosation reactions. Possibilities of hexamethylenetetramine synthesis in the organism as well as its metabolism in conditions of formaldehyde hyperproduction and nitrosative stress are discussed. The role of metabolism sites, common for formaldehyde and nitrogen oxide, in the mechanisms of toxical effect of these compounds and development of pathologic states is considered.     

Dual effect of nitric oxide on phenoloxidase-mediated melanization

The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO₂ at the active site of PO followed by oxidation of DOPA by NO₂. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect’s haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.     

Rat liver-mediated metabolism of hydroxyurea to nitric oxide

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography-mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.     

Role of nitric oxide in cellular iron metabolism

Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) which are located in the 3 untranslated region (UTR) and the 5 UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO, a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO⁺ (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon- (IFN-) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN--treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO⁺-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.     

The long-term effect of mesh bioprosthesis in inguinal hernia repair on testicular nitric oxide metabolism and apoptosis in rat testis

Polypropylene mesh is the most widely used material in inguinal hernia repair. Although polypropylene mesh is known as an inert material, it is experimentally proven that mesh generates a chronic inflammatory tissue reaction. The aim of the present study was to investigate the long-term effects of polypropylene mesh material used in inguinal hernia operations on testicular function, testicular nitric oxide (NO) metabolism and germ cell-specific apoptosis in rats. The study comprised 40 male rats that were randomly allocated into two groups. In group 1, the left spermatic cord was elevated and a 0.5 x 1 cm polypropylene mesh was placed behind the left inguinal spermatic cord and group 2 consisted of the sham-operated controls. Blood samples were taken at 6 months preoperatively and postoperatively after to assess luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels for hormonal evaluation. Testicular NO was evaluated by the Griess method, apoptosis by a TUNEL method and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) expressions by immunohistochemical staining. Mild (+) eNOS expression was observed in all specimens. Mild (+) iNOS expression was only detected in ipsilateral testis of the mesh-implanted study group. Apoptotic cells were not detected in any samples. We are of the opinion that long-term polypropylene mesh implantation has no effect on testicular hormonal function and only a limited effect on nitric oxide levels and this effect is not sufficient to cause apoptosis in testis that could lead to infertility. It seems that mesh implantation is a reliable method in inguinal hernia repair; however, further work is required by more sensitive methods to fully elucidate the potential testicular damage.     

The effect of different antioxidants on nitric oxide production in hypertensive rats

The imbalance between nitric oxide (NO) and reactive oxygen species (ROS) production appears to be a common feature of experimental and human hypertension. Previously, different antioxidants and/or scavengers of oxygen free radicals were shown to activate nitric oxide synthase (NO synthase, NOS) and to increase the expression of both endothelial and neuronal NO synthase isoforms leading to blood pressure reduction. On the other hand, various antihypertensive drugs have been documented to possess antioxidant properties, which may contribute to their beneficial effect on blood pressure. This review is focused on the effects of antioxidant treatment in different models of experimental hypertension with a special attention to the prevention of oxidative damage and the augmentation of NO synthase activity and expression of NOS isoforms.     

The synthesis and effect of fluorinated chalcone derivatives on nitric oxide production

Dimethoxy- and trimethoxychalcone derivatives, with various patterns of fluorination, were synthesized and evaluated for their influence on nitric oxide production. Some of them, chalcones 1, 5, 7, 10, 11 and 17, inhibited NO production with an IC(50) in the submicromolar range; 17 is especially noteworthy because of its potency (IC(50) 30nM). These effects were not the consequence of a direct inhibitory action on enzyme activity but the inhibition of enzyme expression.     

Role of thiamine thiol form in nitric oxide metabolism

In alkaline media the thiamine cyclic form is converted into a thiol form (pK(a) 9.2) with an opened thiazole ring. The thiamine thiol form releases nitric oxide from S-nitrosoglutathione (GSNO). Thiamine disulfide, mixed thiamine disulfide with glutathione, and nitric oxide are produced in the reaction. Free glutathione was recorded in small amounts. The concentration of formed nitric oxide agreed well with the concentration of degraded GSNO. The concentration of released nitric oxide was determined under anaerobic conditions spectrophotometrically by production of nitrosohemoglobin. In air, the release of nitric oxide was recorded by the production of nitrite or the oxidation of oxyhemoglobin to methemoglobin. The concentration of the thiol form in the body under physiological pH values (7.2-7.4) did not exceed 1.5-2.0%. We believe that due to the exchange reactions between the thiamine thiol form and S-nitrosocysteine protein residues, nitric oxide can be released and mixed thiamine-protein disulfides are formed. The mixed thiamine disulfides (including thiamine ester disulfides) as well as the thiamine disulfide form are quite easily reduced by low molecular weight thiols to form the thiamine cyclic form with a closed thiazole ring. A possible role of the thiamine thiol form in releasing deposited nitric oxide from low-molecular-weight S-nitrosothiols and protein S-nitrosothiols and in regulation of blood flow in the vascular bed is discussed.     

The co-immobilization of P450-type nitric oxide reductase and glucose dehydrogenase for the continuous reduction of nitric oxide via cofactor recycling

The co-immobilization of enzymes on target surfaces facilitates the development of self-contained, multi-enzyme biocatalytic platforms. This generally entails the co-immobilization of an enzyme with catalytic value in combination with another enzyme that performs a complementary function, such as the recycling of a critical cofactor. In this study, we co-immobilized two enzymes from different biological sources for the continuous reduction of nitric oxide, using epoxide- and carboxyl-functionalized hyper-porous microspheres. Successful co-immobilization of a fungal nitric oxide reductase (a member of the cytochrome P450 enzyme family) and a bacterial glucose dehydrogenase was obtained with the carboxyl-functionalized microspheres, with enzyme activity maintenance of 158% for nitric oxide reductase and 104% for glucose dehydrogenase. The optimal stoichiometric ratio of these two enzymes was subsequently determined to enable the two independent chemical reactions to be catalyzed concomitantly, allowing for near-synchronous cofactor conversion rates. This dual-enzyme system provides a novel research tool with potential for in vitro investigations of nitric oxide, and further demonstrates the successful immobilization of a P450 enzyme with potential application towards the immobilization of other cytochrome P450 enzymes.     

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.     

Impairment of endothelial nitric oxide production by acute glucose overload

We examined the effects of acute glucose overload (pretreatment for 3 h with 23 mM D-glucose) on the cellular productivity of nitric oxide (NO) in bovine aortic endothelial cells (BAEC). We had previously reported (Kimura C, Oike M, and Ito Y. Circ Res, 82: 677-685, 1998) that glucose overload impairs Ca(2+) mobilization due to an accumulation of superoxide anions (O(2)(-)) in BAEC. In control cells, ATP induced an increase in NO production, assessed by diaminofluorescein 2 (DAF-2), an NO-sensitive fluorescent dye, mainly due to Ca(2+) entry. In contrast, ATP-induced increase in DAF-2 fluorescence was impaired by glucose overload, which was restored by superoxide dismutase, but not by catalase or deferoxamine. Furthermore, pyrogallol, an O(2)(-) donor, also attenuated ATP-induced increase in DAF-2 fluorescence. In contrast, a nonspecific intracellular Ca(2+) concentration increase induced by the Ca(2+) ionophore A-23187, which depletes the intracellular store sites, elevated DAF-2 fluorescence in both control and high D-glucose-treated cells in Ca(2+)-free solution. These results indicate that glucose overload impairs NO production by the O(2)(-)-mediated attenuation of Ca(2+) entry.     

The role of nitric oxide on bone metabolism in ovariectomized rats following chronic ethanol intake

This experimental study was designed to examine the effect of nitric oxide (NO) on bone metabolism in ovariectomized rats following chronic ethanol treatment. Chronic ethanol intake was produced by gradual substitution (within 3 weeks) of tap water in diet with 5,10,15 and finally 20% of ethanol. Thereafter, the rats were maintained under these conditions for a duration of 4 months. The rats were divided into two groups. The first group received sham operation (SHAM) and the rats in Group II were ovariectomized (OVX). Five weeks after the SHAM and ovariectomy, the rats were treated with ethanol for 4 months. After this period of ethanol administration, the NOS inhibitor N(W)-nitro-L-arginine methyl ester (L-NAME) was given for three weeks along with ethanol to the same rats. Serum interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, NO, calcium (Ca), phosphorous (P), parathyroid hormone (PTH), 25 HydroxyvitaminD3 [25(OH)D3], alkaline phosphatase (ALP), bone alkaline phosphatase (b-ALP), alanine amino transferase (ALT), aspartate amino transferase (AST), gamma-glutamyltransferase (GGT) levels were measured in different stages of the experiment. IL-1beta, IL-6, TNFalpha and NO levels increased after ethanol administration in SHAM and OVX rats.The decrease in serum Ca was significant while the changes in P, PTH and 25 (OH)D3 levels were not. ALP and b-ALP levels were significantly decreased; ALT, AST and GGT levels were significantly increased. In ovariectomized and SHAM rats, administration of L-NAME together with ethanol, produced a significant increase in IL-1beta, IL-6 and TNFalpha levels. In this group, Ca and P levels were significantly increased, PTH and 25 (OH)D3 levels were significantly decreased. Also, there was a significant decrease in ALT, AST, ALP, b-ALP, and GGT levels. NO increase due to alcohol intake may function as a protective mechanism preventing bone resorption in cases of estrogen insufficiency.