Nitric Oxide 44 (2015) 31–38

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Nitric Oxide j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n i o x

Dietary nitrite supplementation improves insulin resistance in type 2 diabetic KKAy mice Kazuo Ohtake a, Genya Nakano a, Nobuyuki Ehara a, Kunihiro Sonoda b, Junta Ito c, Hiroyuki Uchida a, Jun Kobayashi a,* a

Division of Pathophysiology, Department of Clinical Dietetics and Human Nutrition, Faculty of Pharmaceutical Science, Josai University, Saitama, Japan Department of Food and Nutritional Environment, College of Human Life and Environment, Kinjo Gakuin University, Nagoya, Japan c Division of Oral Anatomy, Department of Human Development and Fostering, Meikai University School of Dentistry, Saitama, Japan b

A R T I C L E

I N F O

Article history: Received 12 September 2014 Revised 3 November 2014 Available online 13 November 2014 Keywords: Dietary nitrite/nitrate Insulin resistance Type 2 diabetes mellitus Glucose transporter 4

A B S T R A C T

Background: Because insulin signaling is essential for endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, the loss of bioavailable NO might be a common molecular mechanism underlying the development of insulin resistance and endothelial dysfunction. Although dietary nitrite acts as a substrate for systemic NO generation, thereby serving as a physiological alternative source of NO for signaling, it is not precisely known how dietary nitrite affects type 2 diabetes mellitus. Here we report the therapeutic effects of dietary nitrite on the metabolic and histological features of KKAy diabetic mice. Methods: KKAy mice were divided into three groups (without nitrite, and with 50 mg/L and 150 mg/L nitrite in drinking water), and two groups of C57BL/6J mice served as controls (without nitrite and with 150 mg/L nitrite in drinking water). After 10 weeks, blood samples, visceral adipose tissues, and gastrocnemius muscles were collected after a 16-hour fast to assess the homeostasis model assessment of insulin resistance (HOMA-IR) levels, the histology of the adipose tissue, insulin-stimulated sequential signaling to glucose transporter 4 (GLUT4), and nitrite and nitrate contents in the muscle using an HPLC system. Results: KKAy mice developed obesity with enhanced fasting plasma levels of glucose and insulin and exhibited increased HOMA-IR scores compared with the C57BL/6J control mice. Dietary nitrite dosedependently reduced the size of the hypertrophic adipocytes and TNF-α transcription in the adipose tissue of KKAy diabetic mice, which also restored the insulin-mediated signal transduction, including p85 and Akt phosphorylation, and subsequently restored the GLUT4 expression in the skeletal muscles. Conclusions: These results suggest that dietary nitrite provides an alternative source of NO, and subsequently improves the insulin-mediated signaling and the metabolic and histological features in KKAy diabetic mice. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Insulin affects cells through binding to its receptor on the surface of insulin-responsive cells, thus regulating glucose homeostasis. In addition, a signaling pathway linking the insulin receptor to the activation of endothelial nitric oxide synthase (eNOS) in the vascular endothelium has also been recognized [1–4]. Therefore, a reciprocal relationship is considered to exist between insulin resistance and endothelial dysfunction in the pathophysiology underlying diabetes and metabolic syndrome [5]. Accumulating evidence has suggested that polymorphisms in the eNOS gene are associated with

* Corresponding author. Division of Pathophysiology, Department of Clinical Dietetics and Human Nutrition, Faculty of Pharmaceutical Science, Josai University, Saitama, 350-0295, Japan. Fax: +81 49 271 7209. E-mail address: [email protected] (J. Kobayashi). http://dx.doi.org/10.1016/j.niox.2014.11.009 1089-8603/© 2014 Elsevier Inc. All rights reserved.

the development of metabolic syndrome in humans (Fernandez ML 2004 [34]; Monti LD 2003 [35]), and eNOS-deficient mice display a number of features of metabolic syndrome characterized by hypertension, dyslipidemia, and insulin resistance, Duplain et al. 2001 [6]). These findings indicate that a loss of bioavailable nitric oxide (NO) might be a common molecular mechanism underlying the development of metabolic syndrome and insulin resistance [7]. It would therefore be reasonable to consider an exogenous NO donor as a pharmacological agent for the prevention and treatment of type 2 diabetes mellitus. It is well accepted that dietary nitrate and nitrite act as a substrate for systemic NO generation, serving as physiological alternative sources of NO for NO-based signaling, especially when endogenous NOS-derived NO is lacking (Bryan 2005 [36]). However, there have been few reports showing the effects of dietary nitrite/ nitrate on type 2 diabetes mellitus [8,9]. In the present study, we first characterized KKAy mice as a type 2 diabetic animal model in

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comparison with C57BL/6J control mice in terms of their insulin resistance, visceral adipose tissue, and insulin-stimulated signaling process with regard to glucose transporter 4 (GLUT4) expression. We then sought to investigate whether dietary nitrite supplementation would reverse the metabolic and histological features of KKAy diabetic mice and consequently improve their insulin resistance. 2. Materials and methods 2.1. Animals Specific pathogen-free male KKAy and C57BL/6J mice from CLEA Japan, Inc. (Tokyo, Japan) were allowed food (CE-2, CLEA Japan) and reverse osmosis (RO) water ad libitum beginning when they were 3 weeks old, and were kept on a 12/12 h light/dark cycle with at least 7 days of local vivarium acclimatization before experimental use. All of the experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Josai Life Science Center (H22040) and were consistent with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

performed with 1 μg of total RNA using Gene RED PCR Mix (NIPPON GENE) according to the manufacturer’s instructions: one cycle at 42 °C for 40 min, 95 °C for 5 min, and 4 °C for 5 min for reverse transcription; and 22–34 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min for PCR. PCR was performed with a RoboCycler 96 gradient temperature cycler (Stratagene). The oligonucleotides used as primers were synthesized by FASMAC (Kanagawa, Japan). The primer pairs were as follows: GAPDH: forward primer, 5′-agaacatcatccctgcatcc3′, reverse primer, 5′-tccaccaccctgttgctgta-3′ (367 bp, 22 cycles); TNF-α: forward primer, 5′-ggcaggtctactttggagtcattgc-3′, reverse primer, 5′-acattcgaggctccagtgaattcgg-3′ (300 bp, 34 cycles); MCP-1: forward primer, 5′-actgaagccagctctctcttcctc-3′, reverse primer, 5′ttccttcttggggtcagcacagac-3′ (274 bp, 28 cycles). The target mRNA expression levels were quantified relative to the expression of GAPDH. A portion of each PCR mixture was electrophoresed in 2% agarose gels in TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3), and the bands were visualized by ethidium bromide staining. The intensity of the PCR products was measured using a Gene Genius Bioimaging System (Syngene).

2.2. Experimental procedures

2.3. RT-PCR The adipose tissue samples stored at −80 °C were then extracted using an RNeasy Lipid Tissue Mini kit (QIAGEN) according to the manufacturer’s instructions. The total RNA concentrations were quantified by the NanoDrop system (Thermo Fisher ScientificTM). RT-PCR was

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Starting when they were 4 weeks old, the KKAy mice were divided into three groups: without nitrite (KKAy-0 mg/L, n = 10), and with 50 mg/L (KKAy-50 mg/L, n = 10) and 150 mg/L nitrite (KKAy-150 mg/ L, n = 10) in drinking water. Two groups of C57BL/6J mice served as controls: without nitrite (C57BL/6J-0 mg/L, n = 8) and with 150 mg/L nitrite (C57BL/6J-150 mg/L, n = 8) in drinking water. The mice were housed in individual cages in a temperature- and humidity-controlled room. Obesity was confirmed by checking the body weight (once a week), food intake (once a week), and casual blood glucose levels (once every 2 weeks) during the 10-week experimental period. The casual blood glucose levels were determined with a GlucocardTM device (GT-1661, Arkray, Inc., Kyoto, Japan). Blood samples were obtained from the cut tip of the tail. At 13 weeks of age, intraperitoneal glucose tolerance tests (IPGTT) were performed. Following an overnight fast, the animals were administered an intraperitoneal dose of 20% glucose solution (1.5 g/ kg). Blood samples were collected from a tail vein before and 15, 30, 60, 90 and 120 min after the injection. At 14 weeks of age, the animals were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) following a 16hour fasting period. To determine the homeostasis model assessment as an index of insulin resistance (HOMA-IR) levels, blood samples (about 200 μL) were collected from the tail vein before necropsy. The blood glucose levels were determined with the GlucocardTM (GT1661, Arkray, Inc., Kyoto, Japan). The plasma insulin concentrations were determined with a Mouse Insulin ELISA kit (Shibayagi, Gunma, Japan). The HOMA-IR was calculated as the fasting plasma glucose [mmol/L] × fasting plasma insulin [mU/L]/22.5 [10]. Thereafter, animals were intraperitoneally administered insulin (10 U/kg, diluted in saline). This produced a maximal sustained increase in the IR, IRS-1, and p85 phosphorylation within 5 minutes [11]. At 5 minutes after the injection, the gastrocnemius muscles were excised and frozen immediately at −80 °C until use. Visceral adipose tissue specimens were also excised, weighed, and stored at −80 °C until use, or were fixed in 10% (w/v) neutral buffered formalin solution.

Weeks Fig. 1. The time course of the changes in body weight (A), food intake (B), and casual blood glucose levels (C) with or without dietary nitrite supplementation. The KKAy groups show remarkable increases in body weight, food intake and casual blood glucose levels compared with the C57BL/6J groups over the course of this study. The dietary nitrite has no impact on the changes in body weight, food intake, or casual blood glucose levels in either the KKAy or C57BL/6J groups. ◆: KKAy-0 mg/L, Ë: KKAy50 mg/L, ▲: KKAy-150 mg/L, ◇: C57BL/6J-0 mg/L, △: C57BL/6J-150 mg/L. The results are shown as the means ± SE (n = 8–10).

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The adipose tissue specimens were fixed in 10% neutral buffered formalin solution and embedded in paraffin. Sections (8 μm) were stained with hematoxylin and eosin (HE) for light microscopic observation at 200× magnification. The area and size distribution of adipocytes were measured in 500 cells/20 fields/mouse (n = 4/ group) using the Image J software program [12]. Digital images were obtained from a high-resolution digital camera system (Penguin 150CL, Pixera, Los Gatos, CA, USA) linked to a microscope (BX41, Olympus, Tokyo, Japan) and desktop computer (Pentium 4, 2.0 GHz). 2.5. Western blot analysis

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The plasma membrane fraction (used for detecting GLUT4 translocation) and whole tissue lysates (used for detecting PI3/Akt signaling) were prepared from skeletal muscle as described previously [13]. Briefly, tissue samples were homogenized in 10 volumes of ice-cold lysis buffer A (10 mM Tris–HCl at pH 7.8, 10 mM KCl, 1.5 mM MgCl 2 , 1 mM phenylmethylsulfonylfluoride, 0.5 mM dithiothreitol, 5 μg/mL of aprotinin and 10 μg/mL of leupeptin) and lysed with 10 volumes of buffer A with 0.1% Nonidet P-40, and were then passed through a 22-gauge needle three times. The homogenate was spun at 1000 g for 10 min at 4 °C. The plasma membrane fraction was obtained by resuspending the obtained pellet in buffer A containing 1% Nonidet P-40, and centrifuging at 10,000 g for 20 min at 4 °C. To obtain the whole tissue lysate, skeletal muscle was homogenized in 10 volumes of buffer A and lysed with 10 volumes of buffer B (10 mM Tris–HCl at pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.5 mM DTT, 1 mM phenylmethylsulfonylfluoride, 0.5 mM dithiothreitol, 5 μg/mL of aprotinin, and 10 μg/mL of leupeptin). Both the plasma membrane fraction and the whole tissue lysates typically contained about

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Fig. 3. The effects of dietary nitrite on the fasting blood levels of glucose and insulin, and on the insulin sensitivity. (A) The fasting blood glucose level, (B) fasting plasma insulin level and (C) HOMA-IR score in the KKAy diabetic and C57BL/6J control mice with or without dietary nitrite supplementation. The results are shown as the means ± SE (n = 8–10), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L. HOMRIR: The index of homeostasis model assessment of insulin resistance, calculated as the fasting plasma glucose [mmol/L] × fasting plasma insulin [mU/L]/22.5.

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2.4. Histological analysis

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4 mg of protein per milliliter on the basis of the BCA protein assay. Protein samples (20 μg protein/lane) were resolved by SDS–PAGE, electroblotted onto PVDF membranes and immunoblotted with selected antibodies against PI3 kinase p85 (Cell Signaling Technology), p85-Tyr458 (Cell Signaling Technology), Akt (Cell Signaling Technology), Akt-Ser473 (Cell Signaling Technology), GLUT4 (Santa Cruz Biotechnology), and GAPDH (Santa Cruz Biotechnology). Bound antibodies were visualized using the ECL chemiluminescence detection system (SuperSignal West Dura Extended Duration Substrate, Pierce) with HRP-conjugated secondary antibodies (Pierce). The band intensity was quantified using a Bio Imaging System, with Gene Snap and Gene Tools software programs (Syngene Bio Imaging, Cambridge, USA). 2.6. Nitrite and nitrate concentrations in plasma and skeletal muscle

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Fig. 2. The effects of dietary nitrite on the time course of the blood glucose levels and AUC following the IPGTT. (A) The blood glucose concentrations (mg/mL) following the IPGTT, (B) the AUC (mg/dL × min) of the blood glucose concentrations following the IPGTT. ◆: KKAy-0 mg/L, ▲: KKAy-150 mg/L, ◇: C57BL/6J-0 mg/L, △: C57BL/6J-150 mg/L. The results are shown as the means ± SE (n = 8–10), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L. AUC: area under the curve (the area under the blood glucose concentration–time curve following the IPGTT). IPGTT: intraperitoneal glucose tolerance test.

The nitrite and nitrate concentrations in plasma and skeletal muscle were measured using a dedicated HPLC system (ENO-20; EiCom, Kyoto, Japan) [14,15]. This method was based on the separation of nitrite and nitrate by ion chromatography, followed by on-line reduction of nitrate to nitrite, post-column derivatization with Griess reagent, and detection at 540 nm. The proteins in each sample were removed by centrifugation at 10,000 g for 5 min following methanol precipitation (plasma:methanol = 1:1 volume/ volume, muscle:methanol = 1:2 weight/volume, at 4 °C).

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2.7. Statistical analysis All values are expressed as the means ± SE. Data were analyzed by one- or two-way ANOVA, and then the differences among the means were analyzed using the Tukey–Kramer multiple comparison test. A level of P < 0.05 was considered to be significant. 3. Results 3.1. Time course of the changes in body weight, food intake and fasting blood glucose levels In comparison with the C57BL/6J groups, the KKAy groups showed remarkable increases in body weight, food intake and casual blood glucose levels at the designated time points over the course of the study (Fig. 1A–C), and presented characteristic features of type 2 diabetes, such as obesity and hyperglycemia. The nitrite treatment had no impact on the changes in body weight, food intake, or casual blood glucose levels in either the KKAy or C57BL/6J groups. 3.2. Effects of dietary nitrite on the glucose metabolism and insulin sensitivity Fig. 2A shows the time course of the blood glucose levels following the IPGTT performed at the end of the experiment after a 16-hr fast. The blood glucose levels of the KKAy groups were significantly higher

than those in the C57BL/6J groups from 30 min to 120 min after the IPGTT, showing impaired glucose tolerance. However, among the KKAy groups there was a tendency for there to be a decrease in the blood glucose levels in the KKAy-150 group compared with the KKAy-0 group at each time point. Similarly, the AUC (area of the glucose metabolic clearance curve following the IPGTT) of the KKAy-0 group was higher than that of the C57BL/6J control groups, and was apparently reduced by the dietary nitrite in the KKAy-150 group (Fig. 2B). We also measured the HOMA-IR calculated from the fasting plasma levels of glucose and insulin (Fig. 3A and B). Consistent with the results of the IPGTT, the HOMA-IR indicated the presence of insulin resistance in the KKAy-0 group, which was improved by dietary nitrite in both the KKAy-50 and KKAy-150 groups (Fig. 3C). 3.3. Effects of dietary nitrite on the adipose tissue morphology We next investigated the effects of dietary nitrite on the adipose tissue mass and the size of the adipocytes in the KKAy mice. The ratio of the adipose tissue mass to the body weight was significantly higher in the KKAy groups than in the C65BL/6J groups (Fig. 4A), whereas the dietary nitrite had no impact on this ratio in either the KKAy or C65BL/6J groups. On the other hand, histological analyses of fat tissue revealed that the adipocyte size of the KKAy-0 mice was larger than that of the C57BL/6J groups, and was dose-dependently reduced with dietary nitrite exposure in the

Fig. 4. The effects of dietary nitrite on the morphology of adipose tissues. (A) The ratio of the adipose tissue mass to the body weight (%), (B) the area of adipocytes (μm2). The results are shown as the means ± SE (n = 8–10), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L. (C) Representative light microscopic images of HE-stained adipose tissues from KKAy and C57BL/6J mice with or without dietary nitrite, and the distribution histogram showing the adipocyte sizes. The area of adipocyte was traced and measured in 500 cells/mouse (n = 4/group) using the Image J software program.

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KKAy-50 and KKAy-150 groups (Fig. 4B). A qualitative analysis of the adipocyte size revealed a wide distribution of cell frequencies and a tendency to be larger in the KKAy-0 group, whereas the analysis showed an increasing frequency of small adipocytes (area 10,000 μm2) in the KKAy-50 and KKAy-150 groups (Fig. 4C).

3.4. Effects of dietary nitrite on the transcription of proinflammatory cytokines in visceral fat tissues We next examined the relationship between the hypertrophic phenotype changes in adipocytes and the proinflammatory state of fat tissue, and also examined the anti-inflammatory effects of dietary nitrite. We did not observe a significant suppression of the transcription of MCP-1 (Fig. 5A and B), but dietary nitrite obviously reduced the transcriptional level of TNF-α in the adipose tissue (Fig. 5A and C).

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3.5. Sequential phosphorylation of p85/Akt and subsequent expression of GLUT4 on the cell membrane of skeletal muscle We then evaluated the effects of dietary nitrite on the PI3K-AktGLUT4 signaling pathway (an insulin receptor-mediated signaling pathway) in skeletal muscle. In Fig. 6A, the upper panels show Western blots of each signaling protein (the data are representative of three independent experiments). The band intensities were quantified by densitometry, and the ratios of the levels of phosphorylated to total protein were determined as p85-Tyr458 to p85 (Fig. 6B) and Akt-Ser473 to Akt (Fig. 6C), and the ratio of the expression level of GLUT4 to the housekeeping protein, GAPDH, was also determined as the GLUT4 to GAPDH ratio (Fig. 6D). Despite the fact that there were no significant differences in the p85 phosphorylation among the KKAy groups (Fig. 6B), dietary nitrite led to a tendency toward an increase in the insulin-stimulated phosphorylation of the regulatory p85 subunit of PI3K compared with the untreated KKAy-0 group, and this increasing tendency was significantly amplified in the subsequent Akt phosphorylation of the KKAy-50 and -150 groups that received dietary nitrite (Fig. 6C), and which was followed by an increase in GLUT4 expression on the cell membrane of skeletal muscle (Fig. 6D). 3.6. Nitrite and nitrate levels in plasma and skeletal muscle Because it is difficult to measure NO production directly in vivo, we instead measured the plasma (Fig. 7A and B) and skeletal muscle (Fig. 7C and D) levels of nitrite and nitrate (stable NO oxidation products indicative of the level of accumulated NO production under fasting conditions) after a 16-hour fast. Interestingly, the tissue levels of nitrite (the immediate NO oxidation product) in untreated KKAy mice were significantly lower than those in C57BL/6J control mice (Fig. 7C), suggesting that there was reduced NO production and bioavailability in the skeletal muscle of diabetic state. However, consistent with the increase in plasma levels of nitrite following dietary nitrite supplementation (Fig. 7A), dietary nitrite improved the insulin resistance by restoring the tissue levels of nitrite and nitrate (Fig. 7C and D). 4. Discussion

Fig. 5. The effects of dietary nitrite on the transcriptional levels of proinflammatory cytokines in visceral adipose tissues. (A) Representative PCR images of GAPDH, MCP1, and TNF-α of adipose tissues. The data are presented as the values relative to the levels of GAPDH mRNA expression in the adipose tissue specimens that were studied. (B) MCP-1, (C) TNF-α. The results are displayed as means ± SE (n = 6–8), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L.

In this study, we showed diabetic features of insulin resistance (Fig. 3) characterized by glucose intolerance following IPGTT (Fig. 2), fasting hyperglycemia (Fig. 3A) and hyperinsulinemia (Fig. 3B) in KKAy mice, and also showed that dietary nitrite reversed these diabetic features in this model. In order to investigate the mechanism of the therapeutic effect of dietary nitrite on this diabetic mice, we first paid attention to the visceral adipocytes on the grounds that the increased adiposity would be an inflammatory trigger responsible for the subsequent disturbance of insulin signaling and insulin resistance in the skeletal muscle of type 2 diabetes [16]. Adipose tissue is necessary for the normal secretion of adipokines, such as leptin and adiponectin, which enhance insulin sensitivity. Impaired secretion of such adipokines, as is observed in lipodystrophy of humans and mice, leads to increased plasma levels of triglyceride and FFAs, causing insulin resistance [17]. On the contrary, enlarged adipocytes in obese individuals lead to the production of other kinds of adipokines, such as MCP-1 and TNF-α, which increase lipolysis in adipose tissues and release FFAs into the circulation, finally leading to insulin resistance and type 2 diabetes mellitus. In the present study, we showed that, as increased adiposity developed in KKAy diabetic mice (Fig. 4A and B), the transcriptional levels of MCP-1 and TNF-α increased in the adipocytes of these mice compared with C57BL/6J control mice (Fig. 5A–C). We qualitatively analyzed the sizes of adipocytes using a histogram of the cell

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Fig. 6. The sequential phosphorylation of P85/Akt in skeletal muscle and the subsequent expression of GLUT4 on the cell membrane of skeletal muscle. The data in the Western blots are representative of three independent experiments. The band intensities of the phosphorylated targets were quantified by comparison with each of the total protein level. (A) Representative Western blot images of p85-Tyr458, p85, Akt-Ser473, Akt, GLUT4 and GAPDH. Quantitative analyses are shown as the ratios of p85Tyr458 to p85 (B) and Akt-Ser473 to Akt (C). The band intensities of the membrane-associated GLUT4 were also quantified by comparison with a housekeeping protein, GAPDH (D). The results are shown as the means ± SE (n = 6), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L.

Fig. 7. The effects of dietary nitrite on the levels of nitrite and nitrate in plasma and skeletal muscle. The concentrations of nitrite (A and C) and nitrate (B and D) in plasma and skeletal muscle, respectively. The results are shown as the means ± SE (n = 8–10), aP < 0.05 vs. C57BL/6J-0 mg/L, bP < 0.05 vs. KKAy-0 mg/L.

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size distribution, which revealed a wider distribution of sizes and a tendency to spread toward larger sizes in the KKAy-0 group than in C57BL/6J control mice, the cells of which were mainly composed of small adipocytes (Fig. 4C). The phenotypic changes of adipocytes in the diabetic mice were closely associated with the increased transcriptional levels of MCP-1 and TNF-α in adipocytes (Fig. 5A–C). These proinflammatory cytokines and FFAs modulate systemic inflammatory responses through a TLR4-mediated mechanism [18]. It is well-known that TLR4-mediated NF-κB activation is inhibited by NO via S-nitrosylation of the cysteine residues of Ikkβ and NF-κB, which lead to inhibition of IκBα phosphorylation and NF-κB binding to DNA, respectively [19]. Then, this could inhibit the transcription of inflammatory cytokines, including MCP-1 and TNF-α, in adipocytes (Fig. 5A–C). Similarly, in the present study, dietary nitrite supplementation dosedependently shifted the cell frequency to the smaller size distribution in KKAy diabetic mice (Fig. 4C), which was consistent with the decrease in TNF-α transcription in the adipose tissues of the mice with dietary nitrite supplementation. As increased adiposity is supposed to be a cause of subsequent systemic inflammation, it also causes excessive ROS production and an oxidative shift in the intracellular redox environment [20]. Recent evidence has suggested that the defect responsible for insulin resistance lies mostly at the post-receptor level of insulin signaling [21]. Many kinases and phosphatases associated with the insulin signaling pathways are intricately regulated and balanced by protein phosphorylation, dephosphorylation, and in some cases, nitrosylation [22]. The excessive NO production by iNOS S-nitrosylates proteins such as IRS-1 and Akt, which are involved in the early steps of the insulin transduction pathway, results in insulin resistance in skeletal muscle [23] (Fig. 8). On the other hand, physiological levels of NO not only induce microvascular vasodilatation to allow for effective insulin transport to the target cells, but also mediate the S-nitrosylation of protein-tyrosine phosphatase 1B (PTPB1) [4] and enhance the effects of insulin. Because PTPB1 dephosphorylates the insulin receptor and its substrates, attenuating the effects of insulin, its phosphatase activity tends to be suppressed

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Fig. 8. Estimated schema of the effects of nitrite and NO on the insulin-stimulated signaling. Insulin signaling is disturbed by excessive ROS and RNS production. Nitritederived NO suppresses the ROS production in the enlarged adipocytes, and inhibits dephosphorylation activity of PTP1B by S-nitrosylation, then facilitating the phosphorylation of IRS and the subsequent downstream signaling process (vasodilation, GLUT4 translocation and subsequent glucose uptake in the skeletal muscle). ROS: reactive oxygen species, RNS: reactive nitrogen species, GLUT 4: glucose transporter 4, IRS: insulin receptor substrate, PI3K: phosphatidylinositol-3 kinase, PKB: protein kinase B, eNOS: endothelial nitric oxide synthase, PTP1B: protein–tyrosine phosphatase 1B, ET-1: endothelin-1.

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by eNOS-mediated S-nitrosylation. In contrast, once the vascular eNOS activity is impaired, as occurs in individuals with diabetes and metabolic syndrome, PTPB1 suppresses the downstream signaling to PKB/ Akt, leading to insulin resistance. Therefore, NO might act as a regulatory factor for the downstream signaling linking it to GLUT4 translocation and glucose uptake [24,25] (Fig. 8). In addition, Jiang recently reported that a GLUT4-mediated mechanism was involved in dietary nitrite-induced improvement of insulin resistance, and they demonstrated that NO-dependent nitrosylation of GLUT4 itself facilitates GLUT4 translocation to the membrane for glucose uptake, and thereby improves insulin resistance [26]. In the present study, we did not examine the nitrosative modification of these signal-related proteins, and the evidence concerning these issues so far remains limited, but this might be a possible mechanism by which dietary nitrite leads to the recovery of the insulin-stimulated phosphorylation of p85 and Akt, as well as the expression of GLUT4 and glucose metabolism in the skeletal muscles (Fig. 6A–D). In addition to the NO-mediated mechanisms mentioned earlier, NO has also been reported to play important roles in suppressing the development of insulin resistance at various levels and by affecting various processes, including the mitochondrial function [27], insulin secretion [28], insulin transport [4], glucose uptake [29], and modulation of inflammation [30]. Recent data have indicated that a loss of NO bioavailability might be a pivotal molecular mechanism underlying the development of insulin resistance [8,31]. In agreement with these proposals, the present study also showed a significant decrease in the nitrite levels in the skeletal muscle of KKAy diabetic mice compared with C57BL/6J control mice (Fig. 7C), suggesting that there was a decrease in the NO bioavailability in the skeletal muscle of the diabetic mice. Dietary nitrite supplementation increased the nitrite levels of skeletal muscle, thus contributing to the subsequent improvements of the metabolic and histological features of KKAy diabetic mice. To apply this strategy in a clinical setting, it would be necessary to consider the human dietary sources of nitrate/nitrite. According to a report by Hord (2009) [37], in which the daily nitrate intake was calculated using the vegetable and fruit components of the Dietary Approaches to Stop Hypertension (DASH) dietary pattern (Lin PH 2003) [38], the daily nitrate intake easily exceeds 1200 mg/ day. Approximately 25% of the ingested nitrate is secreted in saliva, and 20% of the secreted nitrate in saliva is converted to nitrite by commensal bacteria on the tongue (Lundberg JO, 1994) [39], and they also indicated that about 5% of the originally ingested nitrate is swallowed and enters the stomach [32]. Thus, on the DASH diet containing 1200 mg nitrate, 46 mg of nitrite (reduced from 60 mg nitrate) would be swallowed per day. In the present study, the amounts of nitrite the mice consumed totaled approximately 0.1 and 0.3 mg/day per mouse (for the 50 mg/L and 150 mg/L doses of nitrite in the drinking water, respectively, based on an assumption that mouse would drink 2 mL of water/day). These doses of nitrite (2 and 6 mg/kg/day, calculated based on 50 g body weight/ mouse) could be orally achievable by increasing the consumption of nitrate/nitrite-rich foods and vegetables. With respect to the side effects of dietary nitrite, we did not measure the methemoglobin levels and blood pressure in the present study, because we previously reported a mouse model of experimental colitis treated with 25 mmol/L sodium nitrite in drinking water (approximately 35- and 12-fold more concentrated nitrite compared with the doses used in the present study), and no measurable effects on the methemoglobin levels and blood pressure were observed even at those levels [15]. Carcinogenesis is another concern attributed to nitrite/nitrate consumption; however, no causative link between nitrite/nitrate exposure and cancer, including stomach cancer, has so far been documented in humans [33]. Taken together, dietary nitrite supplementation might provide a possible therapeutic strategy for diabetic patients.

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In conclusion, the present results suggest that an endogenous NO defect might underlie the development of insulin resistance. Therefore, dietary nitrite may provide an alternative source of NO, and subsequently improve the insulin-mediated signaling pathways and the metabolic and histological features of diabetes.

Acknowledgment This work was supported by a research grant from the Japan Food Chemical Research Foundation to Dr. Ohtake.

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