Dietary nitrite improves insulin signaling through GLUT4 translocation

Free Radical Biology and Medicine 67 (2014) 51–57

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Original Contributions

Dietary nitrite improves insulin signaling through GLUT4 translocation Hong Jiang a, Ashley C. Torregrossa a, Amy Potts a, Dan Pierini b, Mayank Aranke c, Harsha K. Garg a, Nathan S. Bryan a,d,n a

Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, Health Science Center, Houston, TX 77030, USA California State University at Fullerton, Fullerton, CA 92831, USA c The University of Texas at Austin, Austin, TX 78712, USA d Graduate School of Biomedical Sciences, The University of Texas at Houston, Houston, TX 77030, USA b

art ic l e i nf o

a b s t r a c t

Article history: Received 19 June 2013 Received in revised form 14 October 2013 Accepted 15 October 2013 Available online 21 October 2013

Diabetes mellitus type 2 is a syndrome of disordered metabolism with inappropriate hyperglycemia owing to a reduction in the biological effectiveness of insulin. Type 2 diabetes is associated with an impaired nitric oxide (NO) pathway that probably serves as the key link between metabolic disorders and cardiovascular disease. Insulin-mediated translocation of GLUT4 involves the PI3K/Akt kinase signal cascade that results in activation of endothelial NO synthase (eNOS). eNOS is dysfunctional during diabetes. We hypothesize that loss of eNOS-derived NO terminates the signaling cascade and therefore cannot activate GLUT4 translocation and that dietary nitrite may repair this pathway. In this study, we administered 50 mg/L sodium nitrite to db/db diabetic mice for 4 weeks. After 4 weeks treatment, the db/db mice experienced less weight gain, improved fasting glucose levels, and reduced insulin levels. Cell culture experiments using CHO-HIRc-myc-GLUT4eGFP cell lines stably expressing insulin receptor and myc-GLUT4eGFP protein, as well as L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc), showed that NO, nitrite, and GSNO stimulate GLUT4 translocation independent of insulin, which is inhibited by NEM. Collectively our data suggest that nitrite improves insulin signaling through restoration of NO-dependent nitrosation of GLUT4 signaling translocation. These data suggest that NO-mediated nitrosation of GLUT4 by nitrite or other nitrosating agents is necessary and sufficient for GLUT4 translocation in target tissue. Description of this pathway may justify a high-nitrate/nitrite diet along with the glycemic index to provide a safe and nutritional regimen for the management and treatment of diabetes. & 2013 Elsevier Inc. All rights reserved.

Keywords: Nitric oxide Insulin Cell signaling GLUT4 Diabetes Nutrition Free radicals

The normal endothelium synthesizes and secretes a number of factors that are vital for the maintenance of cardiovascular homeostasis. The generation of nitric oxide (NO) by the constitutive enzyme endothelial nitric oxide synthase (eNOS) is essential for normal physiological regulation of blood flow and nutrient delivery to tissues [1]. In this regard, NO is a potent vasodilator as well as a powerful anti-platelet and anti-leukocyte factor [1]. NO is one of the most important signaling molecules in the body and loss of NO function is one of the earliest indicators or markers of disease. A number of previous experimental studies have demonstrated impaired endothelial function in animal models of diabetes mellitus [2–5]. Studies of isolated rat aortic tissue have revealed that endothelial cell NO release is impaired in the diabetic state and this defect can be only partially restored by the addition of the

n Corresponding author at: The University of Texas School of Medicine at Houston, Texas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, 1825 Pressler St. 530C, SRB 530B, Houston, TX 77030, United States. Fax: þ1 713 500 2447. E-mail address: [email protected] (N.S. Bryan).

0891-5849/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2013.10.809

NO precursor, L-arginine [2–4,6]. In addition, numerous clinical studies [7,8] have clearly documented severe endothelial dysfunction in humans that suffer from diabetes mellitus. Polymorphisms in the eNOS gene have predictive value for the development of diabetic complications [9]. The dysfunctional NO pathway in diabetics is thought to be the cause of the increased incidence of cardiovascular complications [1]. The potential mechanisms that may account for attenuated eNOS function and a reduction in endothelial NO synthesis in diabetics are numerous. The increases in circulating glucose, insulin, and cytokines that occur in type 2 diabetes have all been independently shown to impair eNOS enzyme activity in experimental studies [4,5]. All of these conditions acting independently or in unison could render the eNOS enzyme dysfunctional. The physiological significance of impaired eNOS function and reductions in vascular NO bioavailability may serve to reduce blood flow to various organs in patients with diabetes mellitus as well as disrupting insulin-dependent glucose uptake. Insulin has vasodilator actions that depend on endotheliumderived NO [10]. Part of this response and the key cardiovascular

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protective effects occur via a phosphatidylinositol 3′-kinase–protein kinase B–endothelial nitric oxide synthase (PI3K-Akt-eNOS)dependent signaling mechanism in addition to its metabolic modulation, which renders insulin a potent organ protector in multiple clinical applications. The PI3K pathway, which activates serine/threonine protein kinase Akt, enhances eNOS phosphorylation and NO production [11]. On a molecular level, metabolic insulin resistance results from impaired PI3K-dependent signaling in metabolic targets of insulin [12]. The similarities between insulin signaling related to glucose uptake and insulin signaling related to vasodilation explain the parallel impairment of glucose transporter 4 (GLUT4) translocation in fat and muscles and endothelial NO production in the vessels, respectively, under insulin-resistant conditions [13]. The PI3K-Akt pathway has been shown to promote NO production in healthy vascular endothelial cells, which provides support that the vascular endothelium is a physiological target of insulin that couples regulation of glucose metabolism with NO production and hemodynamics [14]. However, there is substantial evidence of impairment of this pathway in diabetes, through disruption of insulin secretion via Akt phosphorylation and expression [15,16] and/or eNOS phosphorylation and activation [11]. Furthermore, mice lacking endothelial nitric oxide synthase develop insulin resistance, hyperlipidemia, and hypertension mimicking the metabolic syndrome [17]. There is a clear association between diabetes and disruption of endothelium-derived NO through the insulin signaling pathway. To date, there has been no molecular mechanism proposed to fully explain how NO is required for insulin signaling in endothelial cells and subsequent glucose uptake in target cells such as adipocytes and skeletal muscle. It is not understood how insulin is transported across the endothelial barrier to reach interstitial compartments to bind to and activate insulin receptors on adipocytes, skeletal muscle, and hepatocytes, triggering glucose uptake and clearance from the circulation. A major effect of insulin regulation of blood glucose concentration is the enhancement of glucose uptake by the peripheral tissues. To stimulate insulin-induced glucose uptake by the skeletal muscles, adipocytes, and hepatocytes, insulin is thought to be delivered into the capillaries and cross the endothelial barrier to enter the interstitial space. It has been previously shown that insulin transport across capillaries is the rate-limiting step for insulin signaling [18]. Evidence suggests that there is an impairment of insulin delivery through the endothelial barrier in type 2 diabetes and obesity [19,20]. However, it is unknown how insulin receptor activation on endothelial cells affects insulin transport and binding to target cells that subsequently activate GLUT4 translocation. It is, however, evident that NO is part of this process, but the exact mechanism is not known. Several studies now demonstrate that exogenous nitrite contributes to whole body NO production and homeostasis and is an alternate source of NO in vivo. In fact, it has been shown that nitrite may be the endocrine mediator of NO-based signaling [21]. Nitrite can form nitrosothiols and regulate protein structure and function through nitrosation of critical cysteine residues on proteins [22–24]. Enhancing nitrite availability through therapeutic intervention by administering bolus nitrite before cardiovascular insult has shown remarkable effects in reducing the injury from myocardial infarction, ischemic liver and kidney injury, stroke, and cerebral vasospasm [25–30] in animal models. It was recently revealed that nitrite when given orally is rapidly absorbed with greater than 95% bioavailability [31]. Nitrite seems to have residual effects long after it has been metabolized and cleared from the body by preconditioning the myocardium when administered 24 h before ischemic insult [32]. Nitrite has also been shown to augment ischemia-induced angiogenesis and arteriogenesis [33]. Most recently inorganic nitrate was shown to reverse features of metabolic syndrome in eNOS-knockout mice through

repletion of nitrite and NO homeostasis [34]. In humans, dietary nitrate and nitrite sources have been demonstrated to lower blood pressure [35–37] and decrease oxygen consumption during submaximal and maximal aerobic exercise [38,39]. Collectively, these studies clearly reveal the benefits of nitrite as a means to restore or enhance NO bioavailability and/or homeostasis in an endotheliumindependent manner, offering a chance to correct NO homeostasis when endothelium-derived NO is insufficient or dysfunctional, such as in diabetes. Our hypothesis is that dietary nitrite can provide an endothelium-independent source of NO and recapitulate the insulin signaling cascade when eNOS may be dysfunctional. Restoration of NO-based signaling by nitrite will restore glucose uptake and ameliorate the diabetes phenotype.

Materials and methods Animal studies Eight-week-old mice homozygous for the diabetes spontaneous mutation (Leprdb) B6.BKS(D)-Leprdb/J were purchased from The Jackson Laboratory (Stock 000697). Mice homozygous for the diabetes spontaneous mutation (Leprdb) become obese at approximately 3 to 4 weeks of age. Elevations of plasma insulin begin at 10 to 14 days and elevations of blood sugar at 4 to 8 weeks. Homozygous mutant mice are polyphagic, polydipsic, and polyuric. The severity of disease on this genetic background leads to an uncontrolled rise in blood sugar, severe depletion of the insulinproducing β cells of the pancreatic islets, and death by 10 months of age. Exogenous insulin fails to control blood glucose levels and gluconeogenic enzyme activity increases. Therefore, this mouse model accurately reflects human type 2 diabetes mellitus. Animals were maintained on a standard chow diet (Purina 5001) and kept on a normal 12/12-h light cycle with a minimum of 10 days allowed for local vivarium acclimation before experimental use. A subset of mice was administered 50 mg/L sodium nitrite in their drinking water for 4 weeks. The drinking water was changed twice weekly to ensure accurate dosing. All experiments complied with federal and state regulations in accordance with the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council). Animals were fasted overnight before blood and tissue harvest to determine true steadystate levels. Vascular reactivity Male mice age 12 weeks were anesthetized with diethyl ether. A thoracotomy was performed to expose the thoracic and abdominal aorta. A 25-gauge syringe was inserted into the apex of the left ventricle and perfused free of blood with oxygenated Krebs Henseleit buffer. The right atrium was cut to provide an exit for blood. The aorta was removed and cleaned of fat and adventitia. The aorta was cut into 2-mm-long segments and mounted on a four-channel wire myograph (AD Instruments). Vessel rings were maintained in 10-ml organ baths with oxygenated PSS (95% O2 and 5% CO2) at 37 1C. Rings were allowed to equilibrate for 80 min with the buffer in each organ bath changed every 20 min. One and a half gram pretension was placed on each aortic ring (appropriate starting tension for optimal vasomotor function as determined in previous experiments). An eight-channel octal bridge (Powerlab) and data-acquisition software (Chart version 5.2.2) were used to record all force measurements. After equilibration for 80 min, 1 mM phenylephrine was added to each ring for submaximal contraction as determined by first contraction with KCl. After stabilization, a dose response to acetylcholine was added to the rings and

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Blood analysis Venous blood was obtained from the tail vein. Glucose determinations were made with a glucometer (True Track blood glucose monitoring system; CVS). Insulin was determined by ultra-sensitive mouse insulin ELISA kit (Crystal Chem, Inc., Cat. No. 90080). CHO-HIRc-myc-GLUT4eGFP cell culture CHO-HIRc-myc-GLUT4eGFP cells were maintained in Ham’s F12 medium plus 10% fetal bovine serum (FBS) with 1% P/S (100  penicillin and streptomycin stock). Cells were plated in 60-mm dishes and then allowed to attach and grow for 1 day. Cells were then serum starved in Ham’s F12 medium (2 ml) containing 1 mg/ ml bovine serum albumin for 3 h before experimentation. We then removed 1 ml of medium and placed the dish on a fluorescence microscope under a 20  objective, adding 1 ml of prewarmed reagents at a 2  concentration. We captured images at room temperature at the rate of one frame per minute for 25–45 min using a camera attached to a Nikon Eclipse TE2000E inverted fluorescence microscope. Analysis of images and conversion of videos to individual frames were done using Amira software (VSG Co., Burlington, MA, USA). L6-GLUT4myc cell culture L6-GLUT4myc cells were maintained in α-MEM supplemented with 10% FBS, blasticidin S (2 μg/ml), and 1% antibiotic/antimycotic solution (10,000 U/ml penicillin G, 10 mg/ml streptomycin, 25 μg/ ml amphotericin B) in a 5% CO2 incubator at 37 1C. Differentiation was induced by switching cells to medium supplemented with 2% FBS. Experiments were performed in differentiated myotubes 8 days after seeding. Imaging of GLUT4 The GLUT4 level at the cell surface of nonpermeabilized L6GLUT4myc myotubes was measured by an antibody-coupled FITC fluorescence assay. Four thousand myoblasts per well were seeded into 24-well tissue culture plates with differentiation medium. We allowed them to grow and differentiate into myotubes for 8 days. Cells were serum starved with a depletion medium (α-MEM) for 3 h at 37 1C before experimentation. Then the cells were treated with the different reagents for 25–30 min. After the indicated treatments, cells were washed in ice-cold phosphate-buffered saline (PBS) twice. Cells were then fixed in 3% paraformaldehyde for 10 min at 4 1C and then 20 min at room temperature and quenched with 50 mM NH4Cl in PBS for 10 min at 4 1C. The cells were blocked in 5% Blotto (Santa Cruz Biotechnology) for 15 min

250

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150 100

Weight (g)

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After L6-GLUT4myc cells were incubated with anti-c-Myc–FITC antibody, the cells were washed six times with cold PBS and then stained with DRAQ5 (Cell Signaling) 1:1000 in PBS for 45 min at room temperature. The cells on slides were mounted with Prolong Gold antifade reagent (Invitrogen) and visualized by confocal microscopy.

Results A report by Carlstrom et al. [34] demonstrating that dietary nitrate could reverse metabolic syndrome in eNOS-knockout mice prompted our investigations of using nitrite in a mouse model of diabetes. Preliminary experiments focused on providing proof of concept that nitrite could improve the diabetic condition in a relevant mouse model of diabetes. We chose the db/db mouse as a representative model. Our published data showing that nitrite can restore NO homeostasis in eNOS-knockout mice provide justification for using nitrite in a model of diabetes in which NO production and regulation is compromised [40]. We first began a treatment regimen of the db/db diabetic mice using a single dose of 50 mg/L sodium nitrite administered via their drinking water to determine if providing nitrite to the NO-insufficient diabetic mice could affect glucose management and symptoms related to diabetes. Diabetic mice were enrolled at 8 weeks and then administered 50 mg/L nitrite in the drinking water ad libitum for 4 weeks. We measured their fasting glucose levels and weight gain every week for 4 weeks. There was no significant difference in the starting weight or starting glucose levels in the two groups. As shown in Fig. 1, there was a 35% reduction in fasting glucose in the db/db mice given nitrite compared to the diabetic mice given nitrite-free water. Furthermore, the mice supplemented with nitrite gained less weight over the 4 weeks. To help understand these results, we analyzed fasting insulin levels after 4 weeks and found reduced insulin levels in the nitrite-fed diabetic mice. These data suggest an improvement in insulin sensitivity and insulin signaling whereby less insulin is needed to control glucose concentrations. There was no difference in the amount of food or water consumed. Biochemical analysis of skeletal muscle, visceral fat, and liver tissue revealed that nitrite-treated mice had significantly more nitrite and nitrosothiols in fat and liver but there were no significant changes in the skeletal muscle of treated vs untreated mice (data not shown). We also performed measures of

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and then incubated with mouse anti-c-Myc–FITC antibody (Invitrogen) solution (2.5 μg/ml in PBS with 5% Blotto) for 8 h at 4 1C. After labeling, excess antibodies were removed by extensive washing in ice-cold PBS. The fraction of GLUT4myc at the cell surface was measured by a fluorescence plate reader with excitation at 490 nm and emission at 525 nm.

Fasting Insulin (ng/ml)

tension was recorded. Increasing doses of acetylcholine were added every 5 min.

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20 15 10 5 0

Day 0

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Fig. 1. Sodium nitrite (50 mg/L) added to drinking water improves diabetes in db/db mice. (A) Four weeks of nitrite treatment leads to significantly improved fasting blood glucose levels. (B) Mice receiving nitrite gain less weight over 4 weeks treatment. (C) Fasting insulin levels improve with nitrite treatment. Data are the average 7 SEM of n ¼ 12 mice per group. * denotes p o 0.05.

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endothelial function by isolated aortic relaxation in an organ bath in both control and nitrite-treated mice. Although we did not observe a statistically significant change in endothelial function, there was a trend toward improvement with the nitrite treatment that seemed to improve over time. Perhaps a longer treatment period is needed for changes in endothelial function. These data suggest that nitrite provided in the drinking water can be taken up by select tissues to restore NO homeostasis affecting insulin signaling, glucose management, and weight gain after 4 weeks. These data are consistent with recently published reports of nitrate reversing the phenotype of metabolic syndrome [34] in which these investigators saw an increase in steady-state levels of nitrite after nitrate supplementation. Although there are clear positive effects of nitrite treatment on diabetic mice, it is not clear mechanistically what is happening at the cellular level. To gain more insight into how nitrite may be affecting insulin signaling, we acquired a cell culture system that allowed us to investigate the effects of nitrite, NO, and S-nitrosoglutathione (GSNO) on insulin signaling and GLUT4 translocation. We elected to use CHO-HIRc-myc-GLUT4eGFP cells, which stably express Myc and enhanced green fluorescent protein (eGFP)-tagged GLUT4 in addition to human insulin receptor (HIRc). These cells have previously been used in experiments showing insulin-mediated GLUT4 translocation with easy visualization by fluorescence

Control

microscopy due to GFP [41]. We treated these cells with insulin, NO donor DEA-NO (1 mM), sodium nitrite (1–100 mM), and GSNO (1 mM) and found that NO, nitrite, and GSNO were just as effective as insulin at GLUT4 translocation in this cell system at select concentrations. Using 1 mM nitrite, there was a slight but nonsignificant increase in GLUT4 translocation, whereas 10 and 100 mM nitrite show significant enhancement of GLUT4 translocation. As shown in Fig. 2, one can see the increase in fluorescence at the periphery of the cells as GLUT4 translocates over time. The intensity of the signal over time can therefore be quantified and graphically represented as illustrated in Fig. 3. Interestingly, the effects of NO, nitrite, and GSNO were all abolished by pretreatment of the cells with 10 mM N-ethylmaleimide (NEM), which alkylates free thiols, and there was no effect when pretreated with L-NGnitroarginine methyl ester (L-NAME). When pretreated with insulin and followed by administration of NO or nitrite, no additional changes to the effects of insulin were observed. The entire insulin signaling pathway remained intact in these cells. This is known because the insulin effects are abolished by cytochalasin B, genistein, and wortmannin [41]. To use a more biological cell culture system, we obtained L6 skeletal muscle cells stably expressing rat GLUT4 with a Myc epitope (L6-GLUT4myc). Similar to the above, cells were treated with nitrite, DEA-NO, and S-nitrosoglutathione to determine the

0

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Fig. 2. Representative photos of GLUT4:GFP translocation after treatment with saline, insulin (0.5 mM), or sodium nitrite (100 mM).

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dose dependency and relative efficacy of response. Fig. 4 shows representative photos of the fluorescence of GLUT4 translocation after treatment with saline, sodium nitrite, and insulin. Data were expressed as fold induction relative to unstimulated control cells as shown in Fig, 5. We were able to corroborate the findings in the CHO-HIRc-myc-GLUT4eGFP cells demonstrating a dose-dependent effect of nitrite and positive effects of 1 mM GSNO and NO on GLUT4 translocation. Consistent with the data from above, pretreatment of these cells with NEM prevented GLUT4 translocation by stimulus.

Discussion Our data show that authentic NO, nitrite, and GSNO, which are all potential products of eNOS production, are necessary and sufficient for GLUT4 translocation in target cells. These effects are independent of insulin and are completely blocked by pretreatment with NEM, suggesting a key role for cysteine thiols on GLUT4. The GLUT4 protein contains only two cysteine residues vicinal at positions 451 and 453 [42]. Our data suggest that one or both of these cysteine residues are nitrosated by nitrite or GSNO formed from NO production and that this posttranslational modification signals translocation of GLUT4 to the membrane. It is known that posttranslational modification of GLUT4 affects its subcellular localization and translocation [43]. However, only phosphorylation, N-glycosylation and ubiquitination,

2700 Control DEANO Nitrite NEM Insulin

palmitoylation, and SUMOylation have been considered. Whereas S-nitrosylation is a specific and fundamental posttranslational modification [44], no study has considered such a modification on GLUT4 until now. We also demonstrate that nitrite administered through the drinking water can improve the diabetic phenotype including lower fasting glucose levels, less weight gain, and improved insulin sensitivity. Because endothelial function or endothelium-derived NO is either compromised or dysfunctional during diabetes, this presents a substantial road-block in the signaling cascade. Restoring NO production through this pathway will probably provide benefits to diabetic patients and may even increase efficacy of existing drugs and prevent the unwanted cardiovascular side effects as recently unmasked by rosiglitazone. Although most antidiabetic drugs are effective at managing blood glucose levels, intensive treatment with these drugs in patients with established diabetes does not improve risk of cardiovascular disease (e.g., the ACCORD studies). Our data reveal that NO signaling is not only the key link between metabolic disorders and cardiovascular disease but may be the underlying cause of insulin resistance and metabolic syndrome owing to a breakdown in GLUT4 signaling. Human studies indicate that green leafy vegetables, which are known rich sources of nitrate and nitrite, are associated with an approximately 14% lower risk of developing type 2 diabetes [45]. Nitrite-mediated nitrosation of GLUT4 by dietary sources of nitrite and nitrate may be responsible for this observation. Our data suggest that insulin binding to endothelial cells promotes NO production that can then activate GLUT4

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Fig. 3. Quantitative analysis of the effects of insulin, NO, GSNO, and nitrite on GLUT4 translocation in CHO-HIRc-myc-GLUT4eGFP cells. Mean GFP fluorescence intensity at a defined area on the plasma membrane of the cells in images during the time course was measured by Amira software. Data represent the mean 7 SEM of three independent experiments.

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Fig. 5. Fold increase in GLUT4 translocation in L6 skeletal muscle cells after treatment with insulin, nitrite, NO, GSNO, and NEM. Data are the average 7 SEM of n ¼ 3 experiments done in triplicate. * denotes p o 0.05.

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Fig. 4. Representative photos of GLUT4 translocation in L6 myocytes after 30 min treatment with saline, 100 mM nitrite, and insulin.

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insuli

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Fig. 6. Proposed model of GLUT4 signaling to translocate by nitrite or GSNO.

translocation in target cells independent of insulin. This may limit the role and need for direct insulin binding to target tissue. As long as there is sufficient NO produced by the endothelial cells to reach underlying target tissues, glucose clearance can occur. Our proposed model is illustrated in Fig. 6. Insulin signaling in endothelial cells with subsequent production of NO or nitrite activates GLUT4 translocation on target cells in adipocytes, skeletal muscle, or hepatocytes without the need for insulin to reach and bind these target cells. Therefore, insulin-mediated NO production in endothelial cells may then lead to nitrite and/or GSNO that activates the underlying target tissues without the need for insulin transport across the endothelial barrier, especially in occluded junctions of skeletal muscle. This would provide a back-up system in vascular beds that feed skeletal muscle and adipose tissue where insulin transport may be compromised, but be dependent upon posttranslational modification of one or both of these cysteine residues by a nitrosating agent such as nitrite or GSNO. Therefore, strategies designed to restore NO-based cell signaling pathways would have a positive effect on insulin signaling and glucose clearance. We propose that nitrite or GSNO formed from endothelial NO production may act as the nitrosating agent to modify the GLUT4 transporter causing translocation to membrane for glucose uptake. Because NO itself is a poor nitrosating agent, we suspect that formation of nitrite and/or nitrosothiols is necessary for this modification. Lifestyle strategies including diets rich in nitrite and nitrate along with moderate physical exercise, which promotes NO production, may be the best approach for management of diabetes through restoration of NO homeostasis and GLUT4 function.

Acknowledgments We thank the IMM Microscopy Service Center for their support, expertise, and services. The CHO-HIRc-myc-GLUT4eGFP cells were a kind gift from Dr. Manoj Kumar Bhat in the National Center for Cell Science, Ganeshkhind, Pune 411 007, India. The L6-GLUT4myc cells were a kind gift from Dr. Amira Klip, Program in Cell Biology at The Hospital for Sick Children, Toronto, ON, Canada. N.S. Bryan and UTHealth have financial interests in Neogenis, Inc., a company that develops, produces, and sells nitric oxide-related products intended to improve health; develops diagnostics for nitric oxiderelated metabolites; and performs commercial measurement of nitric oxide metabolites in biological samples.

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