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)     

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.     

A bioluminescent assay for measuring glucose uptake

Identifying activators and inhibitors of glucose uptake is critical for both diabetes management and anticancer therapy. To facilitate such studies, easy-to-use nonradioactive assays are desired. Here we describe a bioluminescent glucose uptake assay for measuring glucose transport in cells. The assay is based on the uptake of 2-deoxyglucose and the enzymatic detection of the 2-deoxyglucose-6-phosphate that accumulates. Uptake can be measured from a variety of cell types, it can be inhibited by known glucose transporter inhibitors, and the bioluminescent assay yields similar results when compared with the radioactive method. With HCT 116 cells, glucose uptake can be detected in as little as 5000 cells and remains linear up to 50,000 cells with signal-to-background values ranging from 5 to 45. The assay can be used to screen for glucose transporter inhibitors, or by multiplexing with viability readouts, changes in glucose uptake can be differentiated from overall effects on cell health. The assay also can provide a relevant end point for measuring insulin sensitivity. With adipocytes and myotubes, insulin-dependent increases in glucose uptake have been measured with 10- and 2-fold assay windows, respectively. Significant assay signals of 2-fold or more have also been measured with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and skeletal myoblasts.     

Inhibition of intestinal glucose uptake by aporphines and secoaporphines

The effects of aporphines and secoaporphines on glucose uptake by isolated intestinal brush-border membrane vesicles (BBMV) or basolateral membrane vesicles (BLMV) and glucose absorption during in situ intestinal perfusion were studied. Of the tested compounds, N-allylsecoboldine was the most potent glucose uptake inhibitor, with IC50 values of 159 microM and 121 microM, respectively, for uptake by BBMV and BLMV. While thaliporphine competitively inhibited glucose uptake by both membrane preparations, inhibition by N-allylsecoboldine was competitive using BBMV and noncompetitive using BLMV. In addition, N-allylsecoboldine significantly reduced both glucose absorption during in situ intestinal perfusion and blood glucose levels in the oral glucose tolerance test. The results demonstrate that levels of both aporphines and secoaporphines achievable by oral administration have an inhibitory effect on intestinal glucose uptake and suggest that the hypoglycemic effects of these compounds merit attention.     

Expression of glucose transporter GLUT3 by endotoxin in cultured rat astrocytes: the role of nitric oxide

The induction of nitric oxide (NO) synthase in astrocytes by endotoxin and/or cytokine treatment is associated with increased glucose consumption and glycolysis, but the mechanism whereby this phenomenon occurs remains obscure. In this work, we have addressed this issue and found that incubation of cultured rat astrocytes with lipopolysaccharide (LPS; 1 microg/mL) for 24 h increased the level of constitutively expressed GLUT1 glucose transporter mRNA, and triggered GLUT3 mRNA expression, which was absent in normal astrocytes. The occurrence of GLUT3 protein after LPS treatment was corroborated by western blotting and immunocytochemistry. A 4-h incubation of astrocytes in the absence of glucose, or under an oxygen-poor (3%) atmosphere also resulted in GLUT3 mRNA overexpression. Experiments performed with 2-deoxy-D-[U-14C]glucose (at 0.1 mM of D-glucose) confirmed that LPS (0.1-10 microg/mL) dose-dependently increased the rate of glucose uptake (by a factor of 1.6 at 1 microg/mL of LPS), which was paralleled with the increase in NO synthesis. Furthermore, blockade of NO synthase with 2-amino-5,6-dihydro-6-methyl-(4H)-1,3-thiazine (AMT; 50 microM) partially (by 45%) prevented the LPS-mediated increase in glucose uptake. Finally, incubation of astrocytes with the NO donor 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA; 100 microM) increased by a factor of 1.4 the rate of glucose uptake. We conclude that the increase in GLUT3-driven glucose uptake in astrocytes would have a neuroprotective role under conditions in which NO formation is combined with hypoglycaemia, such as in brain ischemia.     

Modulation of adipose tissue inflammation by bioactive food compounds

Adipose tissue has an important endocrine function in the regulation of whole-body metabolism. Obesity leads to a chronic low-grade inflammation of the adipose tissue, which disrupts this endocrine function and results in metabolic derangements, such as type-2 diabetes. Dietary bioactive compounds, such as polyphenols and certain fatty acids, are known to suppress both systemic and adipose tissue inflammation and have the potential to improve these obesity-associated metabolic disorders. Mechanistically, polyphenolic compounds including non-flavonoids, such as curcumin and resveratrol, and flavonoids, such as catechins (tea-polyphenols), quercetin and isoflavones, suppress nuclear factor-κB (NF-κB) and mitogen-activated protein (MAP) kinases (MAPK) pathways while activating the 5′ adenosine monophosphate-activated protein kinase (AMPK) pathway in adipose tissue. Dietary polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), conjugated linoleic acid (CLA) and monounsaturated fatty acids (MUFA), such as oleic acid, also impart anti-inflammatory effects through several mechanisms. These include activation of AMPK and peroxisome proliferator-activated receptor gamma (PPAR-γ), as well as suppression of toll-like receptors (TLRs) and NF-κB pathway. This review discusses the major molecular mechanisms of dietary polyphenols and fatty acids, alone or in combination, which are responsible for adipose tissue-associated anti-inflammatory effects.     

Isosteres of ester derived glucose uptake inhibitors

Glucose transporters (GLUTs) facilitate glucose uptake and are overexpressed in most cancer cells. Inhibition of glucose transport has been shown to be an effective method to slow the growth of cancer cells both in vitro and in vivo. We have previously reported on the anticancer activity of an ester derived glucose uptake inhibitor. Due to the hydrolytic instability of the ester linkage we have prepared a series of isosteres of the ester moiety. Of all of the isosteres prepared, the amine linkage showed the most promise. Several additional analogues of the amine-linked compounds were also prepared to improve the overall activity.     

Modulation of the leptin-induced white adipose tissue lipolysis by nitric oxide

The present study tested the hypothesis that nitric oxide (NO) is involved in the leptin-induced stimulation of lipolysis. The effect of intravenous (iv) administration of leptin (10, 100 and 1000 microg/kg body weight) or vehicle on serum NO concentrations and glycerol release from white adipocytes of Wistar rats was examined. One hour after injection, the three leptin doses tested increased serum NO concentrations 15.1%, 23.4% and 60.0%, respectively (P<.001 vs. baseline). The effect of leptin on NO concentrations was significantly dose dependent on linear trend testing (P=.0001). Simple linear regression analysis showed that the lipolytic rate measured was significantly correlated with serum NO concentrations (P=.0025; r=.52). In order to gain further insight into the potential underlying mechanisms, the effect of leptin on lipolysis was studied in the setting of nitric oxide synthase (NOS) inhibition or acute ganglionic blockade. The stimulatory effect of leptin on lipolysis was significantly decreased (P<.05) under NOS inhibition. On the contrary, the leptin-induced lipolysis was unaltered in pharmacologically induced ganglionic blockade. The lack of effect on isoproterenol-, forskolin- and dibutyryl-cyclic AMP-stimulated lipolysis suggests that leptin does not interfere with the signal transduction pathway at the beta-adrenergic receptor, the adenylate cyclase and the protein kinase A levels. These findings suggest that NO is a potential regulator of leptin-induced lipolysis.     

Interleukin-15 increases glucose uptake in skeletal muscle An antidiabetogenic effect of the cytokine

Previous studies have demonstrated that interleukin-15 (IL-15) has important anabolic effects on muscle protein metabolism. In the present investigation we have analysed the effects of IL-15 on glucose metabolism in skeletal muscle. Administration of a single dose of the cytokine (100 μg/kg body weight) resulted in a 32% increase on glucose uptake (as measured by the uptake of 2-deoxyglucose) in skeletal muscle. The effects observed on glucose uptake were direct since in vitro incubations of rat EDL muscles in the presence of the cytokine resulted in a 30% increase in glucose uptake. Similarly, IL-15 increased glucose uptake in C2C12 cell cultures, this being related with an increase in both glucose oxidation to CO₂ and the incorporation into muscle lipid. The effects of the cytokine were associated with an increase in GLUT-4 mRNA, suggesting a higher effect in insulin sensitivity. In conclusion, the data presented here indicate that IL-15 facilitates glucose metabolism in skeletal muscle and, therefore, a possible role of the cytokine as an antidiabetogenic drug merits future investigations.     

Effects of decavanadate and insulin enhancing vanadium compounds on glucose uptake in isolated rat adipocytes

The effects of different vanadium compounds namely pyridine-2,6-dicarboxylatedioxovanadium(V) (V5-dipic), bis(maltolato) oxovanadium(IV) (BMOV) and amavadine, and oligovanadates namely metavanadate and decavanadate were analysed on basal and insulin stimulated glucose uptake in rat adipocytes. Decavanadate (50 μM), manifest a higher increases (6-fold) on glucose uptake compared with basal, followed by BMOV (1 mM) and metavanadate (1 mM) solutions (3-fold) whereas V5 dipic and amavadine had no effect. Decavanadate (100 μM) also shows the highest insulin like activity when compared with the others compounds studied. In the presence of insulin (10 nM), only decavanadate increases (50%) the glucose uptake when compared with insulin stimulated glucose uptake whereas BMOV and metavanadate, had no effect and V5 dipic and amavadine prevent the stimulation to about half of the basal value. Decavanadate is also able to reduce or eradicate the suppressor effect caused by dexamethasone on glucose uptake at the level of the adipocytes. Altogether, vanadium compounds and oligovanadates with several structures and coordination spheres reveal different effects on glucose uptake in rat primary adipocytes.     

2-NBDG as a fluorescent indicator for direct glucose uptake measurement

Evaluation of glucose uptake ability in cells plays a fundamental role in diabetes mellitus research. In this study, we describe a sensitive and non-radioactive assay for direct and rapid measuring glucose uptake in single, living cells. The assay is based on direct incubation of mammalian cells with a fluorescent d-glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG) followed by flow cytometric detection of fluorescence produced by the cells. A series of experiments were conducted to define optimal conditions for this assay. By this technique, it was found that insulin lost its physiological effects on cells in vitro meanwhile some other anti-diabetic drugs facilitated the cell glucose uptake rates with mechanisms which likely to be different from those of insulin or those that were generally accepted of each drug. Our findings show that this technology has potential for applications in both medicine and research.     

A novel mechanism underlying the susceptibility of neuronal cells to nitric oxide: the occurrence and regulation of protein S-nitrosylation is the checkpoint

The susceptibility of neuronal cells to nitric oxide (NO) is a key issue in NO-mediated neurotoxicity. However, the underlying mechanism remains unclear. As a cyclic guanosine monophosphate (cGMP)-independent NO signaling pathway, S -nitrosylation (or S -nitrosation) has been suggested to occur as a post-translational modification in parallel with O-phosphorylation. The underlying mechanism of the involvement of protein S -nitrosylation in the susceptibility of neuronal cells to NO has been little investigated. In this study, we focused on the role of S -nitrosothiols (RSNO) in the susceptibility of a cerebellar cell line R2 to NO. Our results showed the following: (i) S -nitrosoglutathione (GSNO) induced a burst of RSNO in GSH-depleted R2 cells, the majority of which were primarily contributed by the S -nitrosylation of proteins (Pro-SNOs), and was followed by severe neuronal necrosis; (ii) the elevation in the level of Pro-SNOs resulted from a dysfunction of S -nitroglutathione reductase (GSNOR) as a result of its substrate, GSNO, being unavailable in GSH-depleted cells. In the meantime, the suppression of GSNOR increased NO-mediated neurotoxicity in R2 cells, as well as in cerebellar granule neurons; (iii) Our results also demonstrate that the burst of RSNO is the "checkpoint" of cell fate: if RSNO can be reduced to free thiol proteins, cells will survive; if they are further oxidized, cells will die; and (iv) GSH-ethyl ester and Vitamin C protected R2 cells against GSNO neurotoxicity through two distinct mechanisms: by inhibiting the elevation of Pro-SNOs and by reducing Pro-SNOs to free thiol proteins, respectively. A novel mechanism underlying the susceptibility of neuronal cells to NO is proposed and some potential strategies to prevent the NO-mediated neurotoxicity are discussed.     

The effect of creatine supplementation on glucose uptake in rat skeletal muscle

Glucose transport in muscle is a function of the muscle metabolic state, as evidenced by the increase in glucose transport which occurs with conditions of altered aerobic metabolism such as hypoxia or contractile activity. The energy state of the muscle can be determined by the muscle phosphocreatine concentration. Dietary supplementation of creatine has been shown to increase both phosphocreatine (PCr) and creatine (TCr) levels in muscle, although not in the same proportion, so that the PCr/TCr ratio falls suggesting an altered energy state in the cell. The purpose of this study was to determine the effect of increased creatine content on glucose uptake in muscle. PCr and TCr were determined in plantaris muscles from rats following five weeks of dietary supplementation of creatine monohydrate (300 mg/kg/day). (3)H-2-deoxyglucose uptake was measured in epitrochlearis muscles incubated in the presence or absence of a maximally stimulating dose of insulin. Despite a significant increase in creatine content in muscle, neither basal nor insulin-stimulated glucose uptake was altered in creatine supplemented rats. Since PCr levels were not increased with creatine supplementation, these results suggest that the actual concentration of PCr is a more important determinant of glucose uptake than the PCr/TCr ratio.     

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 role of nitric oxide in lung innate immunity: Modulation by surfactant protein-A

Surfactant protein A (SP-A) and alveolar macrophages are essential components of lung innate immunity. Alveolar macrophages phagocytose and kill pathogens by the production of reactive oxygen and nitrogen species. In particular, peroxynitrite, the reaction product of superoxide and nitric oxide, appears to have potent antimicrobial effects. SP-A stimulates alveolar macrophages to phagocytose and kill pathogens and is important in host defense. However, SP-A has diverse effects on both innate and adaptive immunity, and may stimulate or inhibit immune function. SP-A appears to mediate toxic or protective effects depending on the immune status of the lung. In contrast to mouse or rat cells, it has been difficult to demonstrate nitric oxide production by human macrophages. We have recently demonstrated that human macrophages produce nitric oxide and use it to kill Klebsiella pneumoniae. SP-A either stimulates or inhibits this process, depending on the activation state of the macrophage. Given its diverse effects on immune function, SP-A may prove to be an effective therapy for both infectious and inflammatory diseases of the lung.     

Design,synthesis and glucose uptake activity of some novel glitazones

Herein, we report a library consisting of some novel glitazones containing thiazolidinedione and its bioisosteres, rhodanine and oxadiazolidine ring structures as their basic scaffold for their antidiabetic activity. Twelve novel glitazones with diverse chemical structures were designed and synthesized by adopting appropriate synthetic schemes and analyzed. Later, subjected to in vitro glucose uptake assay in the absence and presence of insulin to confirm their antidiabetic activity using rat hemi-diaphragm. The titled compounds exhibited glucose uptake activity ranging weak to significant activity. Compounds 4, 5, 9, 11, 15, 16, 19 and 20 showed considerable glucose uptake activity apart from rosiglitazone, a standard drug. Compound 16 happens to be the candidate compound from this study to investigate further. The illustration about their design, synthesis, analysis and glucose uptake activity is reported here along with the in vitro and in silico study based structure–activity relationships.     

The role of S-nitrosoglutathione reductase (GSNOR) in human disease and therapy

S-nitrosoglutathione reductase (GSNOR), or ADH5, is an enzyme in the alcohol dehydrogenase (ADH) family. It is unique when compared to other ADH enzymes in that primary short-chain alcohols are not its principle substrate. GSNOR metabolizes S-nitrosoglutathione (GSNO), S-hydroxymethylglutathione (the spontaneous adduct of formaldehyde and glutathione), and some alcohols. GSNOR modulates reactive nitric oxide (?NO) availability in the cell by catalyzing the breakdown of GSNO, and indirectly regulates S-nitrosothiols (RSNOs) through GSNO-mediated protein S-nitrosation. The dysregulation of GSNOR can significantly alter cellular homeostasis, leading to disease. GSNOR plays an important regulatory role in smooth muscle relaxation, immune function, inflammation, neuronal development and cancer progression, among many other processes. In recent years, the therapeutic inhibition of GSNOR has been investigated to treat asthma, cystic fibrosis and interstitial lung disease (ILD). The direct action of ?NO on cellular pathways, as well as the important regulatory role of protein S-nitrosation, is closely tied to GSNOR regulation and defines this enzyme as an important therapeutic target.     

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.