Moreover, a high-fat diet significantly inhibited EC insulin-stimulated Akt phosphorylation and FITC-insulin uptake that was partially reversed by SNP in rats. Finally, inhibition of S -nitrosylation by knockdown of thioredoxin-interacting protein completely eliminated SNP-enhanced FITC-insulin uptake.

We conclude that NO directly promotes EC insulin transport by enhancing protein S -nitrosylation. NO also inhibits PTP1B activity, thereby enhancing insulin signaling. Before insulin can act on myocytes, it must first traverse the continuous vascular endothelium in skeletal muscle.

Insulin delivery to muscle is affected by blood flow 1 , flow distribution 2 , and insulin transendothelial transport TET 3 , 4. Importantly, insulin delivery to muscle interstitial fluid is a rate-limiting step in the peripheral action of insulin 5 , 6 and is delayed in insulin-resistant, obese humans, suggesting a significant role for this transport process in peripheral insulin resistance 7 , 8.

Endothelial dysfunction, secondary to reduced nitric oxide NO bioavailability, is an early and prominent feature of insulin resistance.

Endothelial NO synthase NOS-3 or eNOS produces NO from l -arginine, and eNOS is activated by insulin at physiologic concentrations. Knockout of eNOS or inhibiting insulin signaling by endothelium-specific knockout of IRS2, leading to the reduction of eNOS activity in the vascular endothelial cell EC , produces metabolic insulin resistance 9 , In addition, endothelial-specific knockout of IRS2 inhibits insulin-induced microvascular recruitment and reduces insulin delivery to muscle interstitium.

However, it is not known whether the reduced insulin delivery is a result of reduced blood flow, altered flow distribution, impaired insulin TET, or a combination of these We and others have previously shown that insulin induces vasodilation by enhancing NO production to facilitate its own delivery to the peripheral tissues in vivo 1 , 2.

Whether NO directly affects insulin uptake and TET has not been examined. The insulin receptor and caveolae mediate EC insulin uptake 4 , 11 — 13 , and this process is blunted by either inhibiting intracellular insulin signaling or treating with tumor necrosis factor-α TNF-α.

Conversely, stimulating intracellular insulin signaling by inhibiting protein-tyrosine phosphatase 1B PTP1B enhances insulin uptake In the current study, we found that exogenously delivered NO stimulated both the uptake and the TET of insulin by aortic ECs.

We also found that NO partially or fully restored insulin uptake by the cells pretreated with inhibitors of insulin signaling pathways To explain these findings, we examined pathways downstream of NO production by which the NO might act on insulin uptake.

We found that exogenously delivered NO can directly promote insulin transport independent of eNOS activity through enhancing protein S -nitrosylation, including that of PTP1B, without affecting the soluble guanylyl cyclase sGC -cyclic guanosine monophosphate cGMP pathway and overcome the impaired insulin transport seen with experimental insulin resistance.

Bovine aorta ECs bAECs passage numbers 2—8; BioWhittaker, Inc. These experiments were performed as previously described 4 , Briefly, bAECs were seeded onto Transwell inserts 6. Louis, MO. The transendothelial electrical resistances were monitored daily with an Epithelial Voltohmmeter and EndOhm chamber WPI, Sarasota, FL.

At selected times, μL fluid was removed from the bottom chamber and replaced with μL EBM to ensure hydrostatic balance. The concentration of I-insulin was measured with a γ counter.

The percentage of insulin transported was calculated. Rats were killed by CO 2. The surrounding fascia was carefully removed, and the vessel was divided into small segments and cut open.

After stabilization in a cell culture incubator for 1 h, these aortic segments were treated with 0. After fixation with cold methanol, the endothelial face of the vessel was placed face down onto a coverslip that was precoated with poly- l -lysine Sigma-Aldrich , pressure was applied briefly, the vessel wall was removed, and the coverslip with adherent ECs was processed for immunocytochemical staining see immunocytochemistry.

The study procedure was approved by the animal care and use committee of the University of Virginia. Western blotting was performed as described previously 13 , This procedure was followed by incubation with a species-specific secondary antibody coupled to horseradish peroxidase Amersham [GE Healthcare Life Sciences], Piscataway, NJ , and the blots were developed with an enhanced chemiluminescence Western blotting kit Amersham [GE Healthcare Life Sciences].

The developed films were scanned with a densitometer Molecular Dynamics, Amersham, Piscataway, NJ and quantified with the use of ImageQuant 5. Real-time RT-PCR assay was performed as described previously 13 , Briefly, total RNAs were extracted from the cultured bAECs with an RNeasy kit Qiagen and were reverse transcribed with the iScript cDNA synthesis kit Bio-Rad.

The cDNA products were then amplified with iQ SYBR Green Supermix on an iCycler apparatus Bio-Rad. Standard curves for each mRNA were generated by serial dilution of cDNA synthesized from the extracted total RNA and was included in each iCycler real-time RT-PCR experiment.

The specificity of the desired product was verified by analysis of the melting curve. A specific small interfering RNA siRNA duplex against bovine Txnip mRNA and a scrambled siRNA control were purchased from Dharmacon, Inc. Lafayette, CO.

Forty-eight hours after transfection, cells were serum starved for 6 h followed by insulin treatment as described previously 13 , The double-staining protocols were the same as described previously 4 , Briefly, the methanol-fixed bAECs were washed three times in TBS, permeabilized in TBS containing 0.

The following primary antibodies were used: rabbit polyclonal anti-FITC Molecular Probes, Inc. The cells were washed three times in TBS and then incubated with species-specific secondary antibodies conjugated with a fluorochrome cyanine Cy2 or Cy3 Jackson ImmunoResearch, West Grove, PA at dilutions for 45 min at room temperature.

The cells were washed three times in TBS and then coverslipped with antifade mounting medium with DAPI. Intracellular cGMP concentration of the cultured bAECs was measured with a cGMP enzyme immunosorbent assay kit Cayman Chemical. Protein concentration was measured by the Bradford method.

The measured cGMP values were normalized against the corresponding protein concentrations. Arterial serum glucose concentrations were measured with a glucose colorimetric assay kit Cayman Chemical. Serum insulin Mercodia AB, Uppsala, Sweden and triglyceride Cayman Chemical concentrations were measured with ELISA.

The biotin derivatization was detected by the included fluorescein-conjugated avidin. In these experiments, assay of the protein S -nitrosylation was determined in combination with the immunocytochemical staining for PTP1B with the monoclonal primary antibody against PTP1B followed by a species-specific secondary antibody conjugated with Cy3 and visualized by confocal imaging.

The PTP1B activity of bAECs was measured after precipitation of PTP1B with a modified immunoprecipitation procedure described previously 15 , The lysates were gently rocked at 4°C for 15 min and then centrifuged at 14, g for 10 min at 4°C.

Equal amounts of protein samples μg of total protein were immunoprecipitated with the monoclonal anti-PTP1B antibody at 4°C overnight. Immunoprecipitates were washed five times with TBS, and the residue TBS buffer was removed.

The sample mixtures were incubated for 30 min at 30°C. After the reaction, μL aliquots were placed into half-area well plates, and 25 μL red reagent plus 40 μL assay buffer were added to each sample well and gently mixed. After incubation at room temperature for 30 min, the absorbance was measured at nm with a plate reader.

The immunocytochemical labeling was examined with a confocal microscope as described previously 4 , 12 — Confocal imaging was performed with a Leica SP5 X imaging system equipped with ultraviolet nm , tunable — nm white light and argon ion lasers , , , , nm ; ×40 and ×60 1. During image acquisition, the individual microscopic field was selected to include a similar number of cells but was otherwise random.

To quantify fluorescence intensity, the images from randomly selected microscopic fields containing a similar number of nuclei staining were outlined, and the integrated fluorescence intensities were measured with Image J software.

In the case Fig. Digital images were processed identically with Adobe Photoshop. Data are presented as mean ± SEM. Statistical comparisons among different groups were made with one-way ANOVA with Student-Newman-Keuls post hoc testing. We first examined the effect of l - N G -nitro- l -arginine methyl ester l -NAME inhibition of NOS on FITC-insulin uptake.

l -NAME added with l -arginine blocked the increased uptake seen with l -arginine alone Fig. We then examined whether the effect of l -arginine on FITC-insulin uptake could be mimicked by giving NO to bAECs.

Adding modest concentrations 0. However, SNP at higher concentrations did not stimulate insulin uptake Fig. Of note, 0. NO directly promotes EC FITC-insulin uptake. bAECs were serum starved for 6 h then pretreated with or without 0.

A : Effects of LNA on FITC-insulin uptake. B : Representative confocal images of bAECs stained for FITC from three independent experiments. C : The histograms indicate the dose response of FITC-insulin uptake to SNP treatment.

D : Quantification of the fluorescent intensity of FITC for each experimental condition indicated in the confocal images. E : Effects of LNA on SNP-stimulated increase of FITC-insulin uptake.

Next, we examined the effect of SNP on I-insulin TET with a Transwell device 4 , In aggregate, these data suggest that the NO donor SNP may directly promote insulin transport in an eNOS activity-independent fashion. SNP promotes insulin TET. Percent transport of total added I-insulin at 60 min was calculated.

We previously reported that insulin transport by bAECs depends on its intracellular insulin signaling as either general inhibition of tyrosine kinases genistein or more-specific inhibition of Src PP1 , phosphatidyl-inositol-3 kinase PI3K wortmannin , or mitogen-activated protein kinase MAPK PD 12 ; each inhibited FITC-insulin uptake.

Therefore, we tested whether adding SNP to bAECs relieved the inhibition of FITC-insulin uptake induced by blocking these intracellular insulin signaling pathways. Figure 3 and Supplementary Fig.

Adding SNP, however, completely rescued both wortmannin- and PP1-inhibited insulin uptake Fig. SNP did not significantly affect genistein-inhibited FITC-insulin uptake Supplementary Fig. These data indicate that NO is able to promote FITC-insulin uptake despite preinhibiting some intracellular insulin signaling pathways.

Effects of SNP on FITC-insulin uptake by ECs pretreated with inhibitors of insulin action. A : Representative confocal images of bAECs stained for FITC from three independent experiments.

B and C : Quantitative analysis of cellular insulin uptake for each experimental condition. E : Quantitative analysis of the cellular insulin uptake presented in D.

We previously used TNF-α treatment of ECs as an in vitro model of insulin resistance and showed that treatment of bAECs with TNF-α inhibited FITC-insulin uptake Figure 3D shows that adding 0. We next examined the pathways by which SNP promoted FITC-insulin transport.

The selective, irreversible, heme-site inhibitor of sGC 1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalinone ODQ 20 completely eliminated the enhanced FITC-insulin uptake induced by SNP compared with vehicle control Fig. However, the membrane-permeable cGMP analog 8-bromo-cGMP over a range of concentrations had no significant effects on FITC-insulin uptake Fig.

In addition, treatment of ECs with insulin with or without SNP did not significantly affect the intracellular cGMP levels Supplementary Fig. In aggregate, these data suggest that SNP-enhanced insulin transport may not be mediated by activation of sGC.

Effects of cGMP analog and ODQ on insulin uptake. bAECs were serum starved for 6 h then pretreated with either 8-bromo-cGMP 0.

B : The histograms indicate the dose response of FITC-insulin uptake to 8-bromo-cGMP treatment. C : Quantitative analysis of cellular insulin uptake for each experimental condition. cGMP10, cGMP 0. Because NO also regulates cellular function by S -nitrosylation, we next examined the effect of SNP treatment on protein S -nitrosylation.

PLoS ONE 10 4 : e Academic Editor: Aldrin V. Gomes, University of California, Davis, UNITED STATES. Received: May 5, ; Accepted: March 23, ; Published: April 20, Copyright: © Adela et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are available in the paper and its Supporting Information files. Additional information can be obtained from Dr. Sanjay K Banerjee and Dr.

Naveen Reddy. RA is a scholar in the Fogarty International Center of the National Institutes of Health training program under Award Number D43 TW SKN is thankful to DST, New Delhi for supporting with junior research fellowship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Metabolic syndrome is a serious health concern. The global burden of diabetes mellitus has been estimated at million and going to rise to million by the year [ 2 ].

Increasing incidence of morbidity and mortality due to cardiovascular complications including coronary artery diseases has been observed in Type 2 diabetic patients [ 3 ]. Diabetes is a metabolic disorder characterised by chronic hyperglycaemia.

The long-term effects of diabetes mellitus include cellular injury, inflammation and failure of various organs [ 4 ]. The complications of diabetes are divided into macro vascular complications i.

Among all complications, endothelial dysfunction is a common problem in all diabetic patients. Endothelial cells secrete different mediators such as vasodilators i. Hyperglycaemia and other metabolic changes may lead to impairment of nitric oxide NO production [ 6 ].

Impairment of endothelial function in T2DM patients ultimately leads to cardiovascular diseases. Thus, endothelial dysfunction is the early feature of cardiovascular complications in T2DM [ 7 ]. Nitric oxide is a gaseous molecule secreted by the endothelium and a major modulator of endothelial function [ 8 ].

NO is synthesized from L-arginine by the family of enzymes called nitric oxide synthases NOSs viz. neuronal NOS nNOS , endothelial NOS eNOS and inducible NOS iNOS [ 9 ].

NO is a key regulatory molecule with extensive metabolic, vascular, and cellular effects [ 10 ]. While low levels of NO is beneficial for several physiological and cellular functions, high levels of NO may cause detrimental effects in the cells.

High levels of NO may react with superoxide anion to generate peroxynitrite radical, which binds to proteins and thus affects their function [ 11 ].

Altered serum NO levels in T2DM were reported by different investigators previously [ 12 — 14 ]. The serum NO data in T2DM patients that reported by different scientific literature is controversial.

Some research articles reported increased NO levels in diabetes patients [ 13 ] whereas others reported the opposite [ 14 ]. In the present study, we have considered diabetic patients as a separate group compared to other diabetic patients with cardiovascular complications.

We were interested in knowing if the serum NO levels were altered due to the duration of diabetes and the presence of any cardiovascular complications along with diabetes. Till now, there was no study that reported the nitric oxide levels in diabetic patients having cardiovascular complications.

Therefore, the present study was designed to understand the alteration in nitric oxide levels with T2DM and T2DM with cardiovascular complications in South Indian patients and to find whether hyperglycaemia can induce NO production in endothelial cells.

Our study showed an increased level of nitric oxide in Indian T2DM patients, but not in patients with coronary artery disease CAD alone. Similarly, diabetic rats with hyperglycaemia also showed higher NO levels as compared to controls.

Our study indicated that hyperglycaemia is responsible in generation of high levels of NO from HUVEC cells through induction of iNOS and eNOS gene expression. Sample of patients, including men and women aged 35—65 years, was taken from the Mediciti Hospital, Hyderabad, a city from southern part of India.

The study conforms to the principles outlined in the Declaration of Helsinki and was approved by the Mediciti Ethics Committee Institutional Hyderabad. All patients were given detailed information of the study and they gave written consent before enrolling into the study.

Group 1 Control CT subjects had no prior history of T2DM, hypertension, coronary artery diseases or any other cardiovascular diseases, and were not taking medication for any chronic medical condition.

Fasting blood glucose, HbA1c and blood chemistry were normal. This group had no prior history of T2DM. Coronary artery disease patients were identified in the Cardiac Catheterization Unit of Mediciti hospital. After coronary angiogram, all patients were evaluated by cardiologists in the inpatient setting.

If patients were found to have any evidence of CAD, demographic, clinical, and angiographic data were collected from all such patients. Fasting sample were collected prior to the percutaneous coronary intervention or coronary artery bypass graft.

Clinical history and complete physical examination including measurements of blood pressure, was collected and conducted for all the participants. Blood samples were collected by venipuncture after an overnight fast, using Becton Dickison Vacutainer Red colour coded Tubes and blood was allowed to clot by leaving at room temperature for 15 min.

After centrifugation at g for 15 min at 4°C, serum was collected in 1. Height and weight were obtained using standardised techniques and instruments. Fasting blood glucose levels were measured by the FreeStyle optimum glucometer Abbott Diabetes Care, Australia. Creatinine Siemens DF33A and uric acid Siemens BA were measured by Siemens automated analyser Dimension Xpand plus , estimated Glomerular Filtration Rate eGFR calculated from creatinine by using Modification of Diet in Renal Disease MDRD formula.

Serum Insulin and C-peptide levels were measured by Bio-Rad Multiplex assay kit Catalog: AM. Assay was performed as per the manufacturer instructions. All animal experiments were undertaken with the approval of Institutional Animal Ethical Committee of Indian Institute of Chemical Technology IICT , Hyderabad, India.

Male Sprague-Dawley SD rats weighing between — gm were purchased from Teena Labs, Hyderabad, India. Animals were housed in BIOSAFE, an animal quarantine facility of IICT. p in freshly prepared citrate buffer.

After 72 hours of fasting, blood glucose level of rats was measured. Control group rats Con group were administered equal volume of citrate buffer i. p to nullify its effect. Rats were given free access to water and food throughout the study. After 8 weeks, blood was collected from rats to fractionise serum for further biochemical analysis.

The absorbance of the solution was read on a plate reader at nm. To quantify the NO production, a standard curve was generated using sodium nitrite. The cells of the passage three P3 were used for performing the experiments. Human umbilical vein endothelial cells HUVEC were seeded in a 96 well plate at a density of around 1X 10 4 cells per well and grown to confluence.

To simulate the in-vivo diabetic conditions, the cells were incubated with 10, 25, 50 and mM D-Glucose Sigma for two different time periods of 4 and 8 hours. We added different concentration of mannitol to each well during experimentation to maintain similar osmolarity.

Vascular endothelial growth factor VEGF was used as a positive control while the untreated cells served as control. After incubation, the supernatant media of each of the treatment groups were collected separately and their nitric oxide levels were quantified using nitric oxide measurement kit Arbor assay kit, MI, USA.

The exact concentrations of NO, produced in response to treatment with different concentration of glucose were quantified from the NO standard curve. The cells were exposed to 10, 25, 50 and mM D-Glucose treatments for two different time periods of 4 and 8 hours.

After the incubation, the cells were washed and incubated with 5μM DAF-2DA Sigma for 30 min. Briefly, 5 × 10 6 endothelial cells were incubated with 1 ml of TRIzol reagent for 5 minutes.

The resulting cell lysates were then mixed with 0. The upper aqueous layer was collected into RNase-free Eppendorf tubes and mixed with 0. Samples were then centrifuged at 11, rpm for 15 minutes at 4°C.

DNase treatment was further carried out to remove DNA contamination from isolated RNA. cDNA was prepared from isolated RNA 5 μg using 1μl of reverse transcriptase RT enzyme, 1 μl dNTP mix 10 mM , 4 μl 5X reaction buffer and 10 picomole oligo dT and RNase free water.

Total 10μl reaction mixture was denatured at 72°C for 3 min followed by sudden cooling for 10min and extension at 42°C for 60—90min.

After cDNA preparation, real-time PCR was performed for gene expression analysis. Detailed sequences of all primers used in this study have been summarized in Table 1. Ten nanogram of cDNA were analysed on StepOnePlus Applied Biosystem using Absolute SYBR Green ROX PCR Master Mix Takara as described before [ 18 ].

Fold-change analysis was based on normalizing with GAPDH. Patient clinical characteristics are represented as median Inter Quartile Range IQR for continuous and as percentages for categorical variables.

Animal and cell culture results are expressed as mean ± SEM. Statistical comparisons were done by One way Analysis of Variance ANOVA for cell culture related results and t—test was used to see the difference between the two groups in animal study. Non-normally distributed data is expressed as median IQR.

This was determined by using Kolmogorov-Smirnov test, followed by log transformation. Spearman rank correlation was used to compare NO levels in fasting blood glucose FBS and glycated hemoglobin HbA1c. ANOVA and Kruskal-Wallis and Man-Whitney, t-test analysis done by Graph Pad Prism version 5.

Human serum NO levels in Control CT and Type 2 diabetes T2DM. Data were represented as box median IQR and whisker plots. Human serum NO levels in Control CT , Coronary artery disease CAD and Type 2 diabetes with coronary artery disease DMCD.

Human serum NO levels in Control CT and Type 2 diabetes with hypertension DMHT. Human serum NO levels in two group of patients having diabetic duration below 5 years and above 5 years. For this study, we took fifty healthy subjects that represent Group 1 CT. While twenty six T2DM patients with no other complications were included in Group 2 T2DM , forty six T2DM with hypertensive patients were included in Group 3 DMHT.

Total twenty nine coronary artery disease patients twenty four acute myocardial infarction patients and five unstable angina pectoris patients were included in the Group 4 CAD.

Total thirty eight T2DM with coronary artery disease patients thirty one acute myocardial infarction and seven unstable angina pectoris patients were recruited in Group 5 DMCD. Single, double and triple vessel disease patients were included in both group 4 and group 5.

Table 2 shows the clinical characteristics of the study subjects. The mean age of T2DM, DMHT and CAD subjects were similar when compared to control subjects. However, the mean age of DMCD subjects was relatively higher when compared with controls.

There was no significant difference in height, weight and BMI among all the groups. Systolic BP was similar in control and T2DM patients, but significantly higher in DMHT, CAD, and DMCD.

Diastolic BP was similar in control, T2DM, CAD, DMCD patients, and higher in DMHT when compared to control and T2DM. Table 2 shows the biochemical characteristics of the study subjects. T2DM, DMHT, DMCD patients had higher fasting blood glucose and HbA1c levels when compared to the control and CAD subjects.

There was no significant difference in creatinine eGFR and uric acid levels among all the groups. Insulin and C-peptide levels were not significantly changed in the diseases groups as compared to control subjects, except diabetes with coronary artery diseases groups.

S1 Table. The duration median Inter Quartile Range of diabetes among the subjects of three groups i. Similarly, the duration of hypertension among DMHT, CAD and DMCD subjects were 4. While All CAD and DMCD subjects were receiving anti platelet therapy and other lipid lowering therapy for prophylaxis of the coronary artery disease.

Serum nitric oxide levels median Inter Quartile Range were significantly higher in T2DM There was no statistically significant difference in the NO levels between DMHT Serum NO levels in DMCD However there were no significant changes in CAD All the diabetic subjects from T2DM, DMHT, DMCD groups were further divided into two groups based on duration of diabetes i.

Nitric oxide As expected, blood glucose levels were significantly high in hyperglycaemic Dia rats induced by streptozocin STZ , when compared to Control Con rats Fig 2A. Serum nitric oxide levels Blood glucose levels in diabetic rats DIA after 8 weeks of STZ administration.

Reduced production of NO [i. This chapter focuses on the role of impaired NO metabolism in T2D. Asymmetric dimethylarginine ADMA , an endogenous competitive inhibitor of nitric oxide NO synthase NOS isoenzymes, can substantially inhibit vascular NO production at concentrations that are observed in pathophysiological conditions.

Such pathological elevated ADMA levels lead to a decreased NO bioavailability and the development of diabetes complications, including cardiovascular diseases, nephropathy, and retinopathy; elevated ADMA levels also increase the mortality risk in these patients. Here, we discuss current documents indicating how disrupted ADMA metabolism contributes to the development of T2D and its complications.

The role of other endogenous methylarginines, i. The nitrate NO3 -nitrite NO2 -nitric oxide NO pathway, as a storage reservoir for endogenous NO production, is dependent on the oral bacteria with NO3- reducing capacity.

Undesirable changes of oral microbiota towards a decreased load of health-related NO3-reducing bacteria and an overgrowth of pathogenic species, leading to subsequent decreased NO2 production in the oral cavity and decreased systemic NO availability, are now considered risk factors for the development of insulin resistance and type 2 diabetes T2D.

This chapter discusses available evidence focusing on oral microbiota dysbiosis in T2D, especially NO3-reducing bacteria and their metabolic activity including NO3-reductase and NO2-reductase activity , affecting net oral NO2 accumulation and the NO3-NO2-NO pathway.

Nitric oxide NO , a multifunctional gasotransmitter, is now considered an endocrine hormone that essentially contributes to the regulation of glucose and insulin homeostasis.

Here, we discuss current genetic data linking NO metabolism to metabolic disorders, especially insulin resistance and type 2 diabetes T2D. Although several gene variants of NO synthases [NOSs, i. Nitric oxide NO , a gaseous free radical, is a key signaling molecule in the different phases of the normal wound healing process.

The beneficial effects of NO in wound healing are related to its antibacterial properties, regulation of inflammatory response, stimulation of proliferation and differentiation of keratinocytes and fibroblasts, and promotion of angiogenesis and collagen deposition.

NO deficiency is an important mechanism responsible for poor healing in diabetic wounds. In this chapter, the function of NO in diabetic wound healing and the possible therapeutic significance of NO in the treatment of diabetic wounds are discussed.

Current knowledge supports this notion that NO-based intervention is a promising therapeutic approach for diabetic wound healing. Osteoporosis affects million people worldwide. Osteoporosis in subjects with diabetes is called diabetoporosis, and type 2 diabetes T2D contributes to and aggravates osteoporotic fractures.

Hyperglycemia, insulin resistance, bone vasculature impairment, increased inflammation, oxidative stress, and bone marrow adiposity contribute to a higher incidence of osteoporotic fractures in T2D. Decreased nitric oxide NO bioavailability due to lower endothelial NO synthase eNOS -derived NO and higher inducible NOS iNOS -derived NO is one of the main mechanisms of the diabetoporosis.

Available data indicates that T2D increases osteoclast-mediated bone resorption and decreases osteoblast-mediated bone formation, mediated in part by reducing eNOS-derived NO and increasing iNOS-derived NO.

NO donors delay osteoporosis and decrease osteoporotic fractures in subjects with T2D, suggesting the potential therapeutic implication of NO-based interventions for diabetoporosis. Uric acid UA is the end product of purine catabolism in humans.

Hyperuricemia can induce pancreatic β-cell death and impaired insulin secretion. It can also disrupt insulin-induced glucose disposal and insulin signaling in different insulin-sensitive tissues, including cardiomyocytes, skeletal muscle cells, adipocytes, hepatocytes, and endothelial cells.

These events lead to the development of systemic insulin resistance and impaired glucose metabolism. Induction of inflammation, oxidative stress, and impairment of nitric oxide NO metabolism mediate hyperuricemia-induced insulin resistance and dysglycemia.

This chapter is focused on the potential mediatory role of NO metabolism on hyperuricemia-induced dysglycemia and insulin resistance.

Page: 28 Author: Tara Ranjbar, Jennifer L. According to the World Health Organization WHO , the prevalence of obesity across the globe has nearly tripled since , with 39 million children under the age of 5 being overweight or obese in Obesity is the most common risk factor for developing type 2diabetes T2D , which may lead to elevated serum triglycerides, hypertension, and insulin resistance.

In the pathogenesis of T2D, there is a reduction in nitric oxide NO bioavailability. Restoration of NO levels has been associated with many favorable metabolic effects in T2D. Drugs that potentiate NO levels may have a role in improving T2D-associated adverse effects.

Current medications approved for use in the management of T2D include biguanides, thiazolidinediones, sulfonylureas, meglitinides, dipeptidyl peptidase-4 DPP-4 inhibitors, glucagon-like peptide-1 GLP- 1 receptor agonists, alpha-glucosidase inhibitors, and sodium-glucose co-transporter 2 SGLT2 inhibitors.

These drugs mitigate the many adverse effects associated with T2D.

Increased force of VSM contraction is mediated via Rho-Rho kinase ROK - and PKC-dependent MLCP inhibition. cGMP exerts its effects in VSMCs by cGMP-dependent protein kinase PKG, or cGK and by affecting cGMP-binding phosphodiesterase [ ].

In addition, cGMP-PKG inhibits PKC activation [ ]. However, the role of VASP in regulating vascular tone remains to be elucidated [ ]. Aortic rings of wild-type and VASP null mice showed similar relaxant responses to cGMP, ACh, and SNP, indicating that VASP is not essential for vasodilator-induced relaxation of VSM [ ].

For example, in rat aortic VSMCs, S-nitrosylation of RhoA by S-nitrosocystein inhibits the Rho-ROK-MLCP pathway, suggesting a cGMP-independent pathway for vasodilation [ ].

Mechanisms acting at the post-receptor level include a defective cGMP catabolism and b hyperglycemia-induced downregulation of protein kinase G PKG. Other ROS generators e. In addition, it was not due to different protein expressions of sGC subunits α1 and β1 [ 18 ].

Although not entirely determined, two mechanisms seem to result in sGC desensitization: 1 ROS-induced sGC oxidation, and 2 defective cGMP catabolism. Likewise, other vascular beds, e. Defective cGMP catabolism may also contribute to sGC desensitization in diabetic vessels.

PDEs control the abundance of cGMP [ ]. Among 11 families of PDE that have been identified [ ], PDE1, PDE3, and PDE5 degrade cGMP in VSM [ ], and PDE5 is the main one involved in cGMP catabolism in the smooth muscle [ ]. cGMP binds to PDE5 and increases its activity which means PDE5 activity increases following increased cGMP production [ ].

In addition, PKG-I phosphorylates PDE5 and prolongs its activation by increasing its affinity for cGMP [ ].

In VSMCs isolated from the aorta, baseline cGMP concentrations were about 3 times 1. This was not affected by the NOS inhibitor L -NMMA, which rules out sGC hyperactivation [ 18 ]. In addition, sGC inhibitors ODQ and Ambroxol decreased baseline cGMP in VSMCs from OZR but not controls. Still, baseline cGMP remained higher in VSMCs from OZR, suggesting defective cGMP catabolism [ 18 ].

Baseline PDE5 activity was lower in VSMCs from OZR, and the IBMX-induced increase in cGMP was smaller in VSMCs from OZR than in controls, suggesting defective PDE activity in VSMCs from OZR [ 18 ]. Hyperglycemia downregulates mRNA and protein levels of PKG-I in VSMCs through altered NOX signaling [ ].

In diabetic vessels, the ability of cGMP to activate PKG and PKG-dependent activation of PDE5 are also impaired [ 18 ]. No further evidence is available regarding T2D-induced changes in sGC downstream signaling. Beneficial effects of some pharmaceutical agents, including angiotensin-converting enzyme inhibitors e.

Furthermore, the use of nitroxyl donors, e. Quantifying the contribution of the impaired pathways i. Sun H, Saeedi P, Karuranga S, Pinkepank M, Ogurtsova K, Duncan BB, et al.

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Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents Subjects and Methods. Journal Article. Insulin Resistance Is Associated with Impaired Nitric Oxide Synthase Activity in Skeletal Muscle of Type 2 Diabetic Subjects. Kashyap , Sangeeta R. Oxford Academic.

Linda J. Jennifer Lamont. Bettie Sue S. Mandeep Bajaj. Swangjit Suraamornkul. Renata Belfort. Rachele Berria. Dean L. Kellogg, Jr. Yanjuan Liu. Ralph A. DeFronzo Ralph A. PDF Split View Views. Cite Cite Sangeeta R.

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First Published Online November 23, and L. contributed equally to this work. intracellular adhesion molecule-1;. insulin-stimulated glucose disposal;. vascular cellular adhesion molecule.

Insulin resistance: a multifaceted syndrome responsible for NIDDM, obesity, hypertension, dyslipidemia, and ASCVD. Google Scholar Crossref. Search ADS. Cardiovascular risk factors in confirmed prediabetic individuals: does the clock for coronary heart disease start ticking before the onset of clinical diabetes?

Google Scholar OpenURL Placeholder Text. Role of nitric oxide in cardiovascular disease: focus on the endothelium. Google Scholar PubMed.

OpenURL Placeholder Text. Insulin resistance, hyperlipemia, and hypertension in mice lacking endothelial nitric oxide synthase. Mice with gene disruption of both endothelial and neuronal nitric oxide synthase exhibit insulin resistance. Expression of nitric oxide synthase in skeletal muscle.

Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle. Abnormal endothelium-dependent vascular relaxation in patients with essential hypertension. Selective loss of microvascular endothelial function in human hypercholesterolemia.

Modulation of coronary vasomotor tone in humans. Insulin inhibits the expression of intercellular adhesion molecule-1 by human aortic endothelial cells through stimulation of nitric oxide. Rapid effects of glucose on the insulin signaling of endothelial NO generation and epithelial Na transport.

Decreased effect of insulin to stimulate skeletal muscle flow in obese men. Hyperinsulinemia produces both sympathetic neural activation and vasodilation in normal humans.

Insulin-mediated skeletal muscle vasodilation is nitric oxide dependent: a novel action of insulin to increase nitric oxide release. Pathogenesis of type 2 diabetes: metabolic and molecular implications for identifying diabetes genes.

Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro.

Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle. Evidence that nitric oxide increases glucose transport in skeletal muscle.

Nitric oxide stimulates glucose transport through insulin-independent GLUT4 translocation in 3T3—L1 adipocytes. N G -monomethyl- l -arginine alters insulin mediated calf blood flow but not glucose disposal in the elderly. Plasma nitric oxide concentrations are elevated in insulin-resistant healthy subjects.

Insulin resistance differentially affects the PI3-kinase and MAP kinase-mediated signaling in human muscle. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. ACE inhibitor improves insulin-resistance in diabetic mice mouse via bradykinin and nitric oxide.

Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. Obesity, atherosclerosis and the vascular endothelium: mechanisms of reduced nitric oxide bioavailability in obese humans. Free fatty acids elevation impairs insulin-mediated vasodilation and nitric oxide production.

Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Skeletal muscle insulin resistance in normoglycemic subjects with strong family history of type 2 diabetes is associated with decreased insulin stimulation of insulin receptor substrate-1 tyrosine phosphorylation.

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Endothelial nitric-oxide synthase type III is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases.

Activation of endothelium-derived nitric oxide production by the protein kinase Akt. The C termini of constitutive nitric-oxide synthases control electron flow through the flavin and heme domains and affect modulation by calmodulin.

Hyperglycemia inhibits endothelial nitric oxide synthase activity by post translational modification at the Akt site. Insulin-dependent activation of endothelial nitric oxide synthase is impaired by O-linked glycosylation modification of signaling proteins in human coronary endothelial cells.

Relationship between obesity, insulin sensitivity and coronary artery disease risk. The initial levels of GSH in the kidney in both groups was the lowest compared with all tissues assessed in the present study, consistent with a previous study Both treatments involving HBO led to a decrease, and the decrease was more pronounced in the combined treatment group.

Lung tissue is the first to be affected by sessions of HBO. The initial levels of 3NT were higher in the diabetic mice, and no significant change was observed in the final values in either the control or diabetic mice. Exercise increased the levels of 3NT, whereas HBO and the combined treatment did not have a significant effect.

The changes in GSH levels in lung tissues were similar to those observed in other tissues. The initial values of the control and diabetic mice were comparable, and the final values were significantly decreased in both groups compared to the respective initial value, but did not differ significantly between groups.

HBO reduced GSH levels, whereas exercise and the combined treatment did not have a notable effect. The development of diabetes did not significantly affect the initial, final or post-treatments values in the lamina propria. This confirmed the predominance of the effects of HBO over exercise in the exacerbation of nitrosative stress.

The initial levels of NO were not affected by diabetes. There were no significant changes in the final values of 3NT in both groups. The similar initials levels of GSH were followed by a sharp decrease in the final values of both groups in the mucosa.

The initial levels of NO were similar in the adipose tissues of both the control and diabetic mice. The initials level of NO in the spleen tissue did not differ significantly between the two groups. None of the treatments significantly altered the NO levels. The relatively low levels of 3NT in the control mice remained constant throughout the study.

The final value of 3NT was higher in the diabetic mice compared with the control group and the treatments did not significantly affect the 3NT levels.

The initial levels of GSH in the brain were not significantly affected by diabetes. Under conditions of relative stability of NO levels in the control group, this stability indicates a direct antioxidant affect against peroxynitrite: A greater level of GSH corresponded to a lower level of 3NT.

Table III shows which parameters were significantly altered in Table II. The development of diabetes did not affect the level of NO in the tissues assessed except the aorta Table IIIA. Conversely, induction of diabetes significantly increased the levels of 3NT in the majority of tissues with a reduction in the muscle and lamina propria , while decreasing the levels of GSH in adipose tissue and spleen, and increasing its levels in the aorta and lamina propria.

The control and diabetes groups showed a drastic and significant decrease in the initial levels of NO and GSH in all tissues between the two phases of the study.

The positive effects of treatments against oxidative stress are reflected by a decrease in 3NT levels or an increase in GSH levels 4. Some treatments resulted in a protective effect against nitrosative stress in the diabetic mice in certain tissues Table IIIB : Aorta, all treatments; and muscle, HBO.

However, a negative effect an increase in 3NT levels was observed more frequently: Liver, all treatments; kidney, all treatments; intestinal lamina propria, all treatments; muscle, exercise and combined treatment; lung, exercise; and spleen, combined treatment. An increase in GSH levels were observed following all treatments in the muscle, heart and kidney, after exercise in the mucosa and spleen; after HBO only in the brain; and after combined treatment in the spleen.

The results of the present study confirmed the successful establishment of a diet-induced diabetes mellitus type 2 model in mice. The age of mice is associated with the level of basal metabolism lower in adults , and increases the likelihood of developing obesity based on diet The possible effect of a change of basal metabolic levels in adult diabetic animals during the present study was eliminated by comparison of post-treatment levels of measured parameters to final levels of the diabetic group.

Oxidative stress served an important role in the pathogenesis of diabetes and complications linked to dysfunction of the endothelium. A possible initial mechanism of dysfunction of the endothelium may be associated with a reduction in the bioavailability of NO 6 , 49 , In the present study, the development of diabetes did not affect the levels of NO in the majority of tissues.

Indirectly, the stability of NO during diabetes emphasizes the importance of its homeostasis in tissues of mice in the experimental model used. The development of diabetes drastically increased the levels of 3NT in the majority of tissues, which is indicative of nitrosative stress 4 , 51 , as it is a product of a reaction of peroxynitrite derived from the reaction of NO with superoxide radicals with the tyrosine residues of proteins.

Conversely, the role of S-nitrosation in various signaling pathways associated with NO has also been discussed 25 and may be relevant for interpretation of the results of the present study considering the importance of S-nitrosation in the pathogenesis of diabetes.

Principally, the effect of NO in the short-term is the vasodilatation of large conduit vessels that can modulate the redox state in tissues. An increased production of superoxide in tissues is well-documented during the pathogenesis of diabetes 7 , 9 , 16 , suggesting increased elimination of NO by these superoxide radicals in the tissues of the diabetic mice.

Therefore, the lack of difference in the levels of NO and elevated levels of nitrosative stress observed in the majority of tissues in the diabetic mice suggests that any increased elimination of NO by superoxides was compensated for by an increase in its production in this animal model.

Excessive NO and superoxide production may disrupt the physiological balance between generation of peroxynitrite, antioxidant defense mechanisms and signaling pathways involving NO in the tissues of diabetic mice 4. GSH is a low-weight multifunctional molecule that also serves as the principal endogenous non-enzymatic antioxidant In the present study, no changes in the levels of GSH were observed in the majority of tissues in the diabetic mice.

In vivo studies have shown that the concentration of NO is closely associated with the synthesis of GSH in tissues. Increased production of NO inhibits the synthesis of GSH in rodent tissues, and a decreased production results in increased synthesis of GSH 24 , The stability observed in the levels of NO in the diabetic mice in the present study may therefore be associated with the stability in GSH levels in the majority of tissues in the present study.

In the initial values of the control group only, there was a significant negative correlation between the levels of GSH and 3NT, and a positive correlation between the initial levels of NO and final levels of 3NT in all tissues.

In the tissues of the control mice, the lower initial levels of nitrosative stress corresponded with an increase in antioxidant defense, which is manifested as higher levels of GSH.

A high level of nitrosative stress resulted in a low level of GSH as the compensatory mechanisms were overwhelmed by excessive production of peroxynitrite and consequently 3NT.

During phase 2 of the study, both the control and untreated diabetes groups displayed a similar decrease in the levels of NO and GSH in the majority of tissues, suggesting a possible age-related effect and thus may reflect a decrease in the basal levels of metabolism in adult mice.

The lower initial levels of 3NT were not significantly altered or significantly increased in the majority of the tissues in the control group between the two phases, whereas the elevated initial levels of 3NT in the diabetic mice were unchanged or decreased between the two phases.

The final levels of nitrosative stress as determined based on 3NT levels for the control group indicated either stability or an increase in levels, and in the diabetic mice, the levels were either stable or decreased. In the majority of these tissues the final 3NT levels were higher in the diabetic mice compared with the control group.

This difference coincided with a positive correlation between the final levels of GSH and NO in all tissues in the diabetic mice only, contradicting the known effect of NO on the synthesis of GSH in rodent tissues without diabetes 24 , 53 which was also observed in the control group in the present study.

The role of NO-related mechanisms were examined in processes of adaptation to exercise in tissues During a range of pathological processes and in all tissues and organs, NO was determined to be the primary molecule linked to the regulation of vascular endothelium tissue, and exercise may correct alterations in the bioavailability of this radical.

Enzymatic production of NO by endothelial nitric oxide synthase has a regulatory role in endothelial dysfunction 54 and NO participates in vascular adaptation to exercise 55 , Therefore, the association between NO and different nitric oxide synthases in diabetic tissues requires examination.

Studies have described the protective effect of HBO pre-treatment against oxidative stress produced by distinct factors in different animal models, including experimental damage to the rat spinal cord 27 , cerebral ischemia-reperfusion 28 , experimental myocardial infarct 53 , and the effect of ultraviolet light on mouse skin and liver HBO pretreatment is also reported to reduce the levels of indicators of oxidative stress in rat thoracic aorta, including NO 31 and to decrease oxidative stress in rat lungs In the present study, exercise and HBO did not affect the levels of NO in the majority of diabetic tissues, but the levels of nitrosative stress 3NT and GSH were increased in the majority of tissues, and contrary to the control group, where a negative correlation was observed between these parameters.

An increase in the levels of GSH was interpreted as a positive effect in relation to the redox equilibrium in tissues. All treatments did not alter or increase the levels of GSH in muscle, liver heart, kidney, lung and brain tissues except after HBO alone in the lung, or after combined treatment in the brain suggesting a positive effect on the redox equilibrium of tissues.

HBO alone increased the levels of GSH in the brain and decreased the levels in the lung. Conversely, exercise alone resulted in changes in GSH levels in the small intestine, where an increase was observed in the mucosa and a decrease was observed in the lamina propria, and this may be reflective of redistribution of GSH between different intestinal regions.

The combination treatment showed a similar effect on GSH levels as exercise in the majority of tissues.

Increased or unchanged levels of GSH by all treatments corresponded to an increase or unchanged levels of nitrosative stress. It is possible that the elevated levels of nitrosative stress facilitated the redistribution of GSH from liver to other tissues. In our previous study HBO-induced a notable decrease in the basal levels of GSH in the liver which coincided with an increase in the muscle, brain, small intestine and adipose tissue, without a significant change in the grade of GSH oxidation in the exercised mice That is, the change in GSH without a change in the oxidation of GSH following HBO, suggested that GSH was redistributed from the liver to the other, reflecting an improvement in the antioxidant capability of these tissues.

In conclusion, the development of diabetes in this animal model caused nitrosative stress in the majority of tissues without affecting GSH levels in the majority tissues, and all treatments resulted in an increase or stabilization of GSH levels in the muscle, liver, heart, kidney, lung and brain which coincided with an increase or stabilization in nitrosative stress in the same tissues.

We would like to thank our students Dr Pérez Castro CC, Franco-Valdillo A and Dr Toledo-Blas Mirelle for their assistance during the study.

All authors performed the experiments. AK, GGB and MDCCH performed the analysis and wrote the manuscript. LRGC and ELP performed the analysis and interpreted the data. All authors read and approved the final the manuscript. The study protocol was reviewed and approved by the Institutional Laboratory Use and Care Committee of the High Medical School, National Polytechnic Institute Mexico City, Mexico; approval no.

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