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Nitric Oxide xxx (2018) xxx-xxx

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Nitric Oxide

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Effects of long-term nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in male obese type 2 diabetic rats Sevda Gheibia⁠ ,⁠ b⁠ , Sajad Jeddia⁠ , Mattias Carlströmc⁠ , Hanieh Gholamid⁠ , Asghar Ghasemia⁠ ,⁠ ∗⁠ a

Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, Tehran, Iran Neurophysiology Research Center and Department of Physiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran c Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden d Department of Genetics, Tehran Medical Sciences Branch, Islamic Azad University, Tehran, Iran b

ABSTRACT

Keywords: Carbohydrate metabolism Glucose tolerance Inflammation Insulin resistance Nitrate Nitric oxide Oxidative stress Type 2 diabetes

Purpose: Supplementation with inorganic nitrate to boost the nitrate-nitrite-nitric oxide (NO) pathway, may act as a potential therapeutic agent in diabetes. The aim of this study was to determine the effects of nitrate on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in obese type 2 diabetic rats. Methods: Male Wistar rats were divided into 4 groups: Control, control + nitrate, diabetes, and diabetes + nitrate. Diabetes was induced using a high-fat diet and low-dose of streptozotocin. Sodium nitrate (100 mg/L in drinking water) was administered simultaneously for two months. Serum levels of fasting glucose, insulin, and lipid profiles were measured every 2-weeks. Glycated hemoglobin (HbA1c) was measured monthly. Serum thiobarbituric reactive substances (TBARS) level and catalase activity were measured before and after treatment. At the end of the study, glucose, pyruvate, and insulin tolerance tests were done. Glucose-stimulated insulin secretion (GSIS) and insulin content from isolated pancreatic islets were also assessed; mRNA expression of iNOS as well as mRNA expression and protein levels of GLUT4 in insulin-sensitive tissues, and serum IL-1β were determined. Results: Nitrate supplementation in diabetic rats significantly improved glucose tolerance, lipid profiles, and catalase activity as well as decreased gluconeogenesis, fasting glucose, insulin, and IL-1β; although it had no significant effect on GSIS, islet insulin content, HbA1c, and serum TBARS. Compared to the controls, in diabetic rats, mRNA expression and protein levels of GLUT4 were significantly lower in the soleus muscle (54% and 34%, respectively) and epididymal adipose tissue (67% and 41%, respectively). In diabetic rats, nitrate administration increased GLUT4 mRNA expression and protein levels in both soleus muscle (215% and 17%, respectively) and epididymal adipose tissue (344% and 22%, respectively). In diabetic rats, nitrate significantly decreased elevated iNOS mRNA expression in both the soleus muscle and epididymal adipose tissue. Conclusion: Chronic nitrate supplementation in obese type 2 diabetic rats improved glucose tolerance, insulin resistance, and dyslipidemia; these favorable effects were associated with increased mRNA and protein expression of GLUT4 and decreased mRNA expression of iNOS in insulin-sensitive tissues, and with decreased gluconeogenesis, inflammation, and oxidative stress.

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ARTICLE INFO

Abbreviations: AUC, area under the curve; cGMP, cyclic guanosine monophosphate; CV, coefficient of variation; eNOS, endothelial nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GLUT4, glucose transporter type 4; GSIS, glucose-stimulated insulin secretion; GTT, glucose tolerance test; HbA1c, glycated hemoglobin; HDL-C, high-density lipoprotein-cholesterol; HFD, high-fat diet; IL-1β, interleukin-1 beta; iNOS, inducible nitric oxide synthase; I.P, intraperitoneal; ITT, insulin tolerance test; LDL-C, low-density lipoprotein-cholesterol; MDA, malondialdehyde; NO, nitric oxide; NOx, nitrite + nitrate (nitric oxide metabolites); PTT, pyruvate tolerance test; STZ, streptozotocin; TBARS, thiobarbituric reactive substances; TG, triglycerides; TC, total cholesterol. ∗ Corresponding author. Endocrine Physiology Research Center, Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences, No. 24, Parvaneh Street, Velenjak, P.O. Box: 19395-4763, Tehran, Iran. Email address: [email protected] (A. Ghasemi) https://doi.org/10.1016/j.niox.2018.02.002 Received 13 August 2017; Received in revised form 18 December 2017; Accepted 8 February 2018 Available online xxx 1089-8603/ © 2017.

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2.2. Induction of diabetes

Decreased nitric oxide (NO) signaling, due to reduced endogenous formation or increased metabolism, is a risk factor for development of obesity and diabetes and associated comorbidities [1–3]. Thus, restoration of bioavailable NO levels may offer a therapeutic approach for improving insulin resistance and insulin secretion in diabetes. NO is produced via the classical l-arginine-NO pathway, but also from the nitrate-nitrite-NO pathway [4,5]. In an attempt to restore NO levels, administration of inorganic nitrate/nitrite or nitrate-rich diets have yielded many favorable outcomes in type 2 diabetes (T2D) in both human [2,6] and animal studies [4,7,8]. Many of these studies have been short-term, or used supplementation with nitrite [9,10]. Although it is assumed that both nitrate and nitrite can be converted to NO, these inorganic anions have different pharmacokinetic and pharmacodynamic profiles; e.g., the half-life of nitrate (5–8 h) is higher than nitrite (110 s) and nitrate can produces prolonged low-grade nitrite formation [11]; indicating that nitrite administration is more effective in acute states whereas nitrate would be more suitable for long-term interventions. In addition, compared to nitrite, the content of nitrate is much higher in natural foods [12], making nitrate more appropriate for nutrition-based interventions. Nitrate is less toxic than nitrite and there is evidence for carcinogenicity of nitrite in foods [13]. Taken together, these observations warrant further investigations regarding the favorable effects of nitrate supplementation in diabetes mellitus and metabolic syndrome. There are limited studies, if any, on the effects of long-term nitrate administration on carbohydrate metabolism, glucose-stimulate insulin secretion (GSIS), islet insulin content, and gluconeogenesis. We previously showed that nitrite in obese type 2 diabetic rats increases both GSIS and islet insulin content and decreases insulin resistance as well [8]. The aim of this study was to investigate the long-term effects of nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in obese type 2 diabetic rats.

For induction of diabetes, rats were fed with a HFD for two weeks followed by a single intraperitoneal (I.P) injection of streptozotocin (STZ, 25 mg/kg dissolved in 0.1 mM citrate buffer, pH 4.5; Sigma Aldrich, Hamburg, Germany). One week after STZ injection, serum glucose level was measured using glucose oxidase method and rats with fasting glucose levels ≥150 mg/dL were considered diabetic and were included in the study. The rats were fed with their respective diets for two months until the end of the study.

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1. Introduction

2.3. Experimental design

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Rats were randomly divided into 4 groups: i.e. control group (C) fed with regular chow and tap water; control + nitrate group (C + N) fed with regular chow and tap water supplemented with 100 mg/L sodium nitrate; diabetic rats (D) fed with the HFD and tap water; and the diabetic + nitrate group (D + N) fed with the HFD and tap water supplemented with 100 mg/L sodium nitrate. Body weight (A&D Scale, EK-300i, Japan; sensitivity 0.01 g), water consumption (mL/day), food intake (g/day), and calorie intake (kcal/day) were recorded every 3-days. Fasting serum concentrations of glucose, insulin, nitrite + nitrate (NOX⁠ ), nitrite, total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol (HDL-C) were measured and serum nitrate levels were calculated every 2-weeks. Glycated hemoglobin (HbA1c) was determined every month. Serum thiobarbituric reactive substances (TBARS) level and catalase activity were measured before and after nitrate supplementation. Intraperitoneal glucose tolerance test (GTT) and pyruvate tolerance test (PTT) were performed one week after STZ injection, and also at the end of the study. The intraperitoneal insulin tolerance test (ITT) was performed one week after the latest GTT. GSIS from isolated pancreatic islets was measured at 5.6 and 16.7 mM of glucose concentrations; in addition, insulin content of islets was measured. At the end of the study serum interleukin-1 beta (IL-1β) and mRNA expression of inducible nitric oxide synthase (iNOS) as well as mRNA expression and protein concentration of glucose transporter type 4 (GLUT4) in soleus muscle and epididymal adipose tissue were assessed. The experimental design is shown in Fig. 1.

2. Materials and methods 2.1. Animals and diets

Male Wistar rats (190–210 g) were housed under standard conditions (23 ± 2 °C, relative humidity of 50 ± 6%, 12/12-hour light-dark cycle). All experiments were conducted according to the animal care protocol approved by the ethics committee of the Research Institute for Endocrine Sciences (9ECRIES940215), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Rats had free access to water and regular chow, consisting of 72.2% carbohydrates, 22.1% proteins, and 5.7% lipids with a total caloric value of ∼3100 kcal/kg, or a high-fat diet (HFD) containing 27.5% carbohydrates, 14.7% proteins, and 58.8% lipids with a total caloric value of ∼4900 kcal/kg. The composition of the HFD has been described previously [8].

2.4. Biochemical analyses of serum parameters After 12–14 h fasting, blood samples were collected from tail vein and centrifuged at 5000 g for 10 min. Serum concentrations of glucose, TC, TG, LDL-C, and HDL-C were measured using commercial kits (Pars Azmoon, Tehran, Iran). Intra-assay coefficient of variations (CVs) for glucose, TC, TG, LDL-C, and HDL-C were 1.8%, 5.1%, 2.7%, 1.9%, and 2.6%, respectively and inter-assay CVs were 2.7%, 6.9%, 3.1%, 4.8%, and 4.4%, respectively. Serum insulin was measured using a rat ELISA kit (Rat insulin ELISA; Mercodia, Uppsala, Sweden); Intra and inter-assay CVs were 8.2% and 9.9%, respectively. The sensitivity of the assay was ≤0.15 μg/L (26.1 pmol/L). Serum IL-1β con

Fig. 1. Experimental procedures over the course of the study. HFD, high-fat diet; STZ, streptozotocin; GTT, glucose tolerance test; PTT, pyruvate tolerance test; NOX⁠ , nitrite + nitrate; IL-1β, interleukin-1 beta; iNOS, inducible nitric oxide synthase; GLUT4, glucose transporter 4; TBARS, thiobarbituric reactive substances; HbA1c, glycated hemoglobin.

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2.6. Pancreatic islet isolation

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For islet isolation, the Lacy & Kostianovsky method was used with slight modifications in separate groups of rats [17]. In brief, after anesthesia by I.P injection of sodium pentobarbital (60 mg/kg), the abdomen was opened and 10 mL ice-cold Hanks' balanced salt solution containing 0.5 mg/mL of collagenase P (Roche, Germany) was injected through the common bile duct to inflate the pancreas, which was then removed and transmitted to a 50 mL falcon tube and digested in a 37 °C water bath for 15–17 min. Digestion was terminated by adding 30 mL ice-cold Hanks' balanced salt solution, and the tube was shaken for 1 min. After three washes with ice-cold Hanks' balanced salt solution, the tissue suspension was filtered through a 500 μm plastic mesh and islets were hand-picked under a stereomicroscope. Fresh islets were used for insulin secretion and insulin content assessments. 2.7. Glycated hemoglobin measurement

Glycated hemoglobin was measured using a chromatographic-spectrophotometric ion exchange method (BioSystems, Spain) in whole blood, obtained from tip of the tail into the test tubes containing ethylene diaminetetracetic acid (EDTA) (1.8 mg/mL). In brief, after hemolysate preparation, hemoglobins were maintained by cationic exchange resin. After elution, HbA1c was quantified by direct photometric reading at 415 nm and expressed as percent of total hemoglobin.

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centration was determined using a rat ELISA kit (ZellBio GmbH, Germany); intra-assay CV was 2.8% and the sensitivity of the assay was 0.1 pg/mL. Serum TBARS as a product of lipid peroxidation was measured by the method of Satoh with slight modifications [14]. Briefly, 100 μl of the serum was added to a reacting solution containing trichloroacetic acid 20% (v/v) and the thiobarbituric acid reagent 0.67% (w/v) in separate capped plastic tubes and incubated in a boiling water bath for 30 min. Following cooling, 800 μl of n-butanol was added to each tube and after centrifugation, the optical density of the pink color formed was read at 530 nm. Serum TBARS concentration was determined in the samples using a standard calibration curve established by 0–20 μM of 1, 1, 2, 3-tetraethoxypropane as malondialdehyde (MDA) precursor; intra and inter-assay CVs were 4.9% and 7.3%, respectively. Catalase activity was determined by the method of Hadwan [15], in which the decomposition of peroxide is estimated spectrophotometrically by a complex reaction with dichromate/acetic acid reagent with an optimized serum catalase determination. In brief, 20 μl of the serum was added to the test tube containing 200 μl hydrogen peroxide and the control tube containing 200 μl distilled water; the standard tube contained 20 μl distilled water and 200 μl hydrogen peroxide. After incubation at 37 °C for 3 min, 400 μl dichromate acetic acid reagent was added to all tubes and incubated in boiling water for 10 min. Following cooling and centrifugation, the optical density was read at 570 nm and catalase activity was calculated according to the formula: 2.303/t × [log absorbance of standard tube/absorbance of test tube-absorbance of control test] × total volume of reagents in test tube/volume of serum. Serum concentrations of total NOX⁠ and nitrite were determined using the Griess method with slight modifications [16]. Briefly, serum proteins were precipitated with zinc sulfate (15 mg/mL) and NaOH (3.72 M), centrifuged at 10,000 g for 10 min, and supernatants were collected for measurement of NOX⁠ and nitrite levels. For NOX⁠ measurement, 100 μl of the supernatants were dispensed into microplate wells and 100 μl vanadium trichloride (8 mg/mL prepared in 1 M HCl) was added to each well to reduce nitrate to nitrite; then 50 μl sulfanilamide (2%), dissolved in 5% HCl, and 50 μl N-(1-naphthyl) ethylenediamine (0.1%) in ddH2O, were added and incubated for 30 min at 37 °C; absorbance was read at 540 nm by a microplate reader (BioTek, MQX2000R2, USA). NOX⁠ concentrations were determined in the samples using a standard calibration curve established by 2.5–100 μM of sodium nitrate. Nitrite was determined in the same manner, except that samples and nitrite standards (2.5–20 μM of sodium nitrite) were exposed to 1 M HCl instead of vanadium trichloride. Serum levels of nitrate were calculated by subtracting nitrite values from NOX⁠ concentrations. Intra-assay CVs for NOX⁠ and nitrite were 2.4% and 1.7%, respectively and inter-assay CVs were 7.9% and 10.1%, respectively. Results from 5 measurements of Certified Reference Material (CRM) of nitrite and nitrate (Sigma-Aldrich; pre⁠ pared by FLUKA® Analytical; No.74246, and Lot: BCBP0387V for nitrate and No. 67276 and Lot: BCBP4288V for nitrite; purity ≥99.5%) were used for accuracy of the assay evaluation. Mean differences between the measured concentrations of CRM and expected values were +2.2 μM and +3.0 μM for nitrite and nitrate, respectively. Recovery of the NOx assay (Recovery (%) = [expected concentration of analyte/concentration of analyte in spiked sample] × 100) was 94.9 ± 5.3 (n = 5).

2.8. Glucose-stimulated insulin secretion and islet insulin content Batches of five islets were transferred into 1.5 mL plastic tubes containing 1 mL of Krebs–Ringer solution (in mM): NaCl, 115; KCl, 5; CaCl2⁠ , 2.5; MgCl2⁠ , 1; NaHCO3⁠ , 24; and HEPES, 16; supplemented with 5 g/L bovine serum albumin and 5.6 and 16.7 mM of glucose concentrations. Islets were incubated for 60 min in a 37 °C water bath and gassed with 95% O2⁠ –5% CO2⁠ for 5 min at the beginning. The incubation medium was collected and kept at −80 °C for insulin determination. To determine the insulin content, 10 fresh islets from each rat were sonificated (30 s, 40 W) and were extracted overnight at 4 °C in 1 mL of acid–ethanol [0.15 M HCL in 75% (vol/vol) ethanol in water] [18]. 2.9. Protein concentrations of GLUT4 in epididymal adipose tissue and soleus muscle A rat ELISA kit (ZellBio GmbH, Germany) was used for measurement of the protein concentrations of GLUT4 in epididymal adipose tissue and soleus muscle. Frozen tissues were homogenized and sonicated in ice-cold phosphate buffer (100 mM, pH 7.4, 1:100, wt/vol) containing protease inhibitor cocktail (Roche, Germany). After centrifugation the homogenates at 5000 g for 10 min at 4 °C, the supernatant was collected and diluted according to the relevant manufacturer's instructions. Intra-assay CV was 6.0% and the sensitivity for the assay was 0.1 pmol/ mL.

2.5. Intraperitoneal glucose, pyruvate, and insulin tolerance tests

GTT and PTT were performed after 12–14 h fasting; animals were anesthetized with an I.P injection of sodium pentobarbital (60 mg/kg; Sigma Aldrich, Hamburg, Germany), following which 50% glucose solution at a dose of 1 g/kg or pyruvate at a dose of 2 g/kg was injected intraperitoneally. Blood samples for glucose measurement were collected from tip of the tail at 0 min and again 10, 20, 30, 60, and 120 min after glucose or pyruvate administration. For ITT, a bolus of insulin (0.75 U/ kg) was injected and blood samples were obtained for glucose measurement before injection and again at time points of 10, 20, 30, 40, 50, 60, 70, and 80 min post injection.

2.10. RNA extraction, cDNA synthesis and real-time PCR For all groups, the soleus muscle and the epididymal adipose tissue were detached, frozen in liquid nitrogen and then stored at −80 °C for RNA extraction. Total RNA was extracted from soleus muscle, and the epididymal adipose tissue, using the RNX-Plus solution kit (Cinagen Co., Tehran, Iran); extraction was performed according to the kit manufacturer's instructions. The quantity and the purity of RNA samples were measured with a nanodrop spectrophotometer (NanoDrop-1000, Thermo Scientific, USA). cDNA synthesis was done using Thermo Scientific RevertAid Reverse Transcriptase in accordance with manufac-

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the 2

− ΔΔCt

method where ΔCt (cycle threshold) = Ct of target gene- Ct

groups - ΔCt of the target gene in the control group [19].

Statistical analysis was performed using Graph Pad Prism software (Version 6) and all values are presented as mean ± SEM. For analyzing data of GTT, ITT, PTT, water consumption, food and calorie intake, body weight, HbA1c, serum glucose, insulin, lipid profiles, NOX⁠ , nitrite and nitrate levels, two-way mixed (between-within) analysis of variance (ANOVA), followed by Fisher post-hoc test was used. A two-way mixed ANOVA include a repeated measure design with addition of a between subject factor. One-way ANOVA was used for comparing the area under the curves (AUC), IL-1β, and GLUT4 protein levels, TBARS, and catalase activity. For comparing body weight, food and calorie intake, water consumption, HbA1c, serum glucose, insulin, lipid profile, NOX⁠ , nitrite, nitrate, TBARS levels and catalase activity before and after nitrate therapy, paired t-test was used. The Mann-Whitney U test was used for comparing fold changes in mRNA expression of GLUT4 and iNOS between groups. Two sided test, with p value < 0.05 was considered statistically significant.

Table 1 Primers used for real-time PCR analysis.

GLUT4

iNOS

As shown in Fig. 2A, before starting the HFD, there was no significant difference in body weights among the groups (day 0). Feeding on the HFD for 2 weeks induced a significant (p < 0.001) increase in the body weight (day 15), although STZ injection caused a significant (p < 0.001) reduction in the body weights of the HFD-fed rats (day 21). At the initiation of nitrate supplementation the body weights were similar among the groups. During a 2-month observation period (day 24 to day 84), the body weight gain was significantly lower in nitrate-treated diabetic (138.7 ± 3.2 vs. 157.3 ± 3.0, p < 0.001) and control (99.1 ± 2.6 vs. 116.7 ± 2.4, p < 0.001) rats. The differences in body weight gain were significant from day 57 or 54 in nitrate supplemented control (273.2 vs. 283.6 g; p = 0.032) and diabetic (287.6 vs. 299.9 g; p = 0.011) rats, respectively, and remained until the end of the study. As expected, diabetic rats displayed increased amounts of water consumption (Fig. 2B) and calorie intake (Fig. 2C) while food intake (Fig. 2D) was significantly lower than the control group and these parameters were unaffected by nitrate (see Supplementary Table 1). 3.2. Effect of nitrate on serum nitric oxide metabolites

2.11. Statistical analysis

Primer

3.1. Effect of nitrate on body weight, water consumption, food intake, and calorie intake

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of reference gene and ΔΔCt = ΔCt of the target gene in the experimental

3. Results

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was reversed to cDNA using M-MuLV RevertAid Reverse Transcriptase (1 μL of 200 U/μL), random hexamer primers (1 μl of 100 μM), dNTPS (2 μL of 10 mM), and RiboLock RNase-inhibitor (0.5 μL of 40 U/μL), incubated for 10 min at 25 °C, followed by 60 min at 42 °C in a total volume of 20 μL. The reaction was terminated by heating the reactions at 70 °C for 10 min. For Real time PCR, primers were designed using the primer3 and GeneRunner programs; primer sequences are shown in Table 1. Amplifications were performed in a rotor gene 6000 Real time PCR machine (Corbett, Life science, Sydney, Australia). All reactions were set up in 15 μL volumes and contained 1 μL cDNA, 0.5 μL of each forward and reverse primer, 7.5 μL of SYBR Green PCR Master Mix 2X (ThermoFisher, USA), and 5.5 μL nuclease-free water. The following cycling profile was used for PCR reactions: Initial denaturation (10 min at 95 °C) followed by 40 cycles with 45 s at 94 °C, 45 s at 58 °C and 1 min at 72 °C; final extension for 5 min at 72 °C. All samples were run in duplicate and H2⁠ O replaced templates in negative control reactions. Target genes were normalized with reference genes, i.e. β-actin for epididymal adipose tissue and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for the soleus muscle. The relative mRNA level for each target gene was calculated by

Gene bank Accession No. NM_ 012751.1 NM_ 012611.3

β-actin

NM_ 031144.3

GAPDH

NM_ 017008.4

Primer sequence (5´→3′)

Forward: CGCACCACAGAAAGTGATTG Reverse: GGTAGTGAGTGTGCCTTGTG Forward: TGTAACTTCTCAGCCACCTTG Reverse: TCCGTGAGGCTTGTAGTTGA Forward: GCGTCCACCTGCTAGTACAAC Reverse: CGACGACTAGCTCAGCGATA Forward: TGCCGCCTGGAGAAACCTGC Reverse: TGAGAGCAATGCCAGCCCCA

Product length (bp) 100

105

100

172

Sodium nitrate increased serum nitrite, nitrate and total NOX⁠ levels in both control and diabetic rats; however, we observed no significant difference between control and diabetic rats before the treatment (Fig. 3 and Supplementary Table 2). In our study, only on day 42, serum nitrate levels in nitrate-treated diabetic rats was lower than nitrate-treated controls (34.95 ± 3.20 vs. 43.71 ± 5.02 μmol/L) and this difference was marginally significant (p = 0.086). 3.3. Effect of nitrate on serum glucose, insulin concentrations and glycated hemoglobin value Serum glucose (Fig. 4A) and insulin (Fig. 4B) levels were not significantly different between groups on day 0. HFD-fed rats, showed elevated serum levels of glucose and insulin on day 15; STZ injection increased serum glucose (182.20 ± 6.2 in group D vs. 94.0 ± 4.7 mg/dL in group C; p < 0.001) while a decrease in serum insulin (82.0 ± 11.2 in D group vs. 72.1 ± 5.6 pmol/L in C group; p = 0.786) in diabetic rats on day 21. Two months of nitrate supplementation decreased serum glucose level in diabetic rats but not in controls. Compared to non-treated diabetic rats, increase in serum insulin levels was significantly lower in nitrate-treated diabetic rats (see Supplementary Table 3). Compared to control rats, diabetic rats had higher HbA1c level (5.54 ± 0.28% vs. 3.26 ± 0.13%, p < 0.001). Compared to non-treated diabetic rats, sodium nitrate supplementation failed to decrease HbA1c level in nitrate-treated diabetic rats (5.54 ± 0.28% vs. 5.30 ± 0.34%) (Fig. 4C and Supplementary Table 3). 3.4. Effect of nitrate on glucose handling and gluconeogenesis Before starting the nitrate supplementation, diabetic rats had impaired ability to clear glucose during GTT (Fig. 5A); Importantly, nitrate supplementation significantly improved glucose clearance in diabetic rats, whereas this was not changed in control rats (Fig. 5B). Compared to control rats, increases in insulin secretion during GTT, in diabetic rats were lower before treatment (Fig. 5C); 2-months of nitrate supplementation enhanced insulin response to glucose injection (Fig. 5D). To investigate the ability of nitrate treatment to modulate hepatic gluconeogenesis, we performed PTT. Compared to control rats, administration of the gluconeogenic substrate precursor pyruvate caused higher glucose production in diabetic rats before nitrate treatment as measured on day 21 (Fig. 5E). Compared to non-treated diabetic rats, serum glucose concentration during PTT was significantly lower in nitrate-treated diabetic rats as measured

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Fig. 2. Effects of sodium nitrate on body weight (A), water consumption (B), calorie intake (C), and food intake (D) (n = 10). Area under the curves (day 21–84) are shown in columns on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM. ↓STZ, STZ injection; ↓nitrate, start of nitrate supplementation.

Fig. 3. Effects of sodium nitrate on serum nitrite (A), nitrate (B), and NOX⁠ (C) levels in different groups (n = 10). Area under the curves (day 21–84) are shown in columns on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM. ↓STZ, STZ injection; ↓nitrate, start of nitrate supplementation; ↓HFD, start of high fat diet.

on day 84, indicating reduced gluconeogenesis and improved insulin sensitivity (Fig. 5F).

Analysis of GLUT4 protein levels in extracts prepared from control and diabetic rats soleus muscle and epididymal adipose tissue indicated that diabetic rats had lower tissue concentrations of GLUT4 compared to the controls (3.72 ± 0.37 vs. 5.69 ± 0.27 pg/mg protein and 6.95 ± 0.67 vs. 11.85 ± 0.67 pg/mg protein in soleus muscle and epididymal adipose tissue, respectively). Sodium nitrate supplementation in diabetic rats caused a higher GLUT4 concentration in both the soleus muscle (4.82 ± 0.35 vs. 3.72 ± 0.37, p = 0.035) and the epididymal adipose tissue than in non-treated-diabetic rats (9.07 ± 0.51 vs. 6.95 ± 0.67, p = 0.024) (Fig. 8).

3.5. Effect of nitrate on insulin sensitivity

Next, we further investigated the effects of chronic supplementation with nitrate on insulin sensitivity. As shown in Fig. 6 in nitrate-treated diabetic rats, glucose concentration during ITT was significantly lower than in non-treated diabetic rats as measured at the end of the study, suggesting improved insulin sensitivity.

3.7. Effect of nitrate on mRNA expression of iNOS in soleus muscle and epididymal adipose tissue

3.6. Effect of nitrate on mRNA expression and protein level of GLUT4 in soleus muscle and epididymal adipose tissue

As shown in Fig. 9, compared to the controls, mRNA expression of iNOS in diabetic rats was significantly higher in both the soleus muscle (384%, p = 0.001) and the epididymal adipose tissue (231%, p = 0.018). Nitrate supplementation in both control and diabetic rats significantly decreased mRNA expression of iNOS in the soleus muscle (89%, p < 0.001 and 114%, p = 0.001 in control and diabetic rats, respectively) and the epididymal adipose tissue (74%, p = 0.004; and 119%, p = 0.032, in control and diabetic rats, respectively).

As shown in Fig. 7, compared to the controls, mRNA expression of GLUT4 in diabetic rats was significantly lower in both the soleus muscle (46% of control values, p < 0.001) and the epididymal adipose tissue (33% of control values, p < 0.001). Nitrate supplementation in diabetic rats significantly increased mRNA expression of GLUT4 in both the soleus muscle (p = 0.004) and the epididymal adipose tissue near normal values (p < 0.001), but had no significant effect in controls.

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Fig. 4. Effects of sodium nitrate on fasting serum glucose (n = 10) (A) and insulin (n = 6) (B) levels and glycated hemoglobin value (HbA1c) (n = 10) (C). Area under the curves (day 21–84) are shown in columns on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM. ↓STZ, STZ injection; ↓nitrate, start of nitrate supplementation; ↓HFD, start of high fat diet.

3.8. Effect of nitrate on serum lipid concentrations

3.9. Effect of nitrate on serum IL-1β concentration

As shown in Fig. 10 (A–D) and Supplementary Table 2, compared to the controls, diabetic rats showed higher concentrations of TC, TG, LDL-C, and HDL-C. Nitrate supplementation partially restored elevated serum TC and TG levels in diabetic rats while HDL-C and LDL-C concentrations were not significantly affected by nitrate supplementation; the effects of nitrate on TC and TG levels were significant from 4 weeks onwards following treatment.

Compared to the controls, diabetic rats had a significantly higher serum IL-1β concentration (12.09 ± 0.49 vs. 8.73 ± 0.17 pg/mL, p < 0.001), a value which decreased significantly following 2-months of nitrate supplementation in diabetic rats (10.61 ± 0.66 pg/mL) but not in controls (Fig. 11). 3.10. Effect of nitrate on antioxidant enzyme and oxidative stress Next, we investigated the effects of chronic nitrate supplementation on antioxidant enzyme and oxidative stress. As shown in Fig. 12 and Supplementary Table 3, compared to the controls, diabetic rats showed a significant increase in

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Fig. 5. Effects of sodium nitrate on serum glucose concentrations during intraperitoneal glucose tolerance test (GTT) in the different groups before initiating nitrate supplementation (day 21, n = 10) (A) and after nitrate supplementation (day 84, n = 10) (B). Effects of sodium nitrate on serum insulin concentrations during GTT in the different groups before initiating nitrate supplementation (day 21, n = 6) (C) and after nitrate supplementation (day 84, n = 6) (D). Effects of sodium nitrate on serum glucose concentrations during intraperitoneal pyruvate tolerance test (PTT) in the different groups before initiating nitrate supplementation (day 21, n = 10) (E) and after nitrate supplementation (day 84, n = 10) (F). Area under the curves (minutes 0–120) are shown in columns on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM.

Fig. 6. Effect of sodium nitrate on serum glucose during intraperitoneal insulin tolerance test (ITT) in the different groups (n = 10). Area under the curve (minutes 0–80) is shown in column on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM.

serum TBARS concentration as measured on day 21 (7.2 ± 0.79 vs. 4.37 ± 0.76 μmol/L, p = 0.004). However, we observed no significant differences between nitrate-treated and non-treated control and diabetic rats. In diabetic rats, serum catalase activity was significantly lower than controls as measured on day 21 (10.61 vs. 16.68 U/L; p = 0.019). Two months of nitrate supplementation increased catalase activity in diabetic rats but had no significant effect in controls.

3.11. Effect of nitrate on glucose-stimulated insulin secretion and insulin content in isolated islets Isolated islets from diabetic rats had significantly lower basal insulin secretion (in presence of 5.6 mM glucose) (Fig. 13A) and GSIS (in presence of 16.7 mM glucose) (Fig. 13B) compared to the controls. Nitrate supplementation failed to reverse the decreased basal insulin secretion and GSIS in diabetic rats. Islet insulin content in diabetic rats was significantly lower than in controls (58.0 ± 5.3 vs. 89.0 ± 4.1 pmol/mg protein, p < 0.001) (Fig. 13C) which supports the lack of serum insulin during GTT; nitrate was not able to reverse the decreased insulin content of islets.

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Fig. 7. Effects of sodium nitrate on mRNA expression of glucose transporter 4 (GLUT4) in soleus muscle (A) and epididymal adipose tissue (B) in the different groups (n = 8). The Mann-Whitney U test was used for comparing fold changes in mRNA expression. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM.

Fig. 8. Effect of sodium nitrate on protein levels of glucose transporter 4 (GLUT4) in soleus muscle (A) and epididymal adipose tissue (B) in the different groups (n = 8). A significant difference was assessed by one-way ANOVA. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM.

Fig. 9. Effect of sodium nitrate on mRNA expression of inducible nitric oxide synthase (iNOS) in soleus muscle (A) and epididymal adipose tissue (B) in the different groups (n = 8). The Mann-Whitney U test was used for comparing fold changes in mRNA expression. *, Statistically significant difference compared to control group. †, Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM.

trite administration in diabetic mice [10]. Unlike our finding, no change in weight gain has been reported following nitrate administration in diabetic or control rats [4,21,22] or nitrite administration in diabetic rats [23]. Similar to our results, no change in food intake and water consumption has been reported following nitrate [1,4,21,24] or nitrite administration [8,10]. Decrease in food intake and water consumption have however been documented following nitrate [20] or nitrite [25] administration. Variable effects of nitrate or nitrite on weight gain may be related to the dose and duration of administration; a dose dependent decrease in weight gain has been reported following 5 months of nitrate administration (50, 150, and 500 mg/L) in female rats [26] or following 2 years in male (35, 70, and 130 mg/L) and female (40, 80, and 150 mg/L) rats [27], but also related to the total NOx content of the chow. Also decrease in weight gain has been reported following 50 mM nitrite administration in both normotensive and hypertensive male rats, an effect that was not observed at doses of 0.5 or 5 mM [28]. Weight-lowering effects of nitrate/nitrite may be due to browning of white adipose tissues [21,29] and increasing mitochondria biogenesis [30], both of which seem to be related to increased NO production

4. Discussion

The results of this study showed that in obese type 2 diabetic rats, long-term oral nitrate supplementation provides beneficial effects on peripheral carbohydrate metabolism, body weight and metabolic regulation. Improved glucose tolerance and lipid profiles, which may contribute to the favorable effects of nitrate on metabolic regulation, were associated with reduced gluconeogenesis, inflammation and oxidative stress. The glucose-insulin homeostasis effect of nitrate, at least in part, is associated with increase in both mRNA expression and protein levels of GLUT4 as well as decrease in mRNA expression of iNOS in insulin-sensitive tissues. In the current study, HFD-fed diabetic rats displayed increased calorie intake, weight gain, and water consumption. Nitrate supplementation had no effects on calorie intake or water consumption, but decreased weight gain in both control and diabetic rats. In line with our results, nitrate-mediated reduction of body weight gain has been reported in old eNOS-deficient mice with metabolic syndrome [1], in White rabbits [20], and in diabetic rat [8], and following ni

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Fig. 10. Effect of sodium nitrate on serum total cholesterol (A), triglycerides (B), low-density lipoprotein cholesterol (C), and high-density lipoprotein cholesterol (D) in the different groups (n = 10). Area under the curves (day 21–84) are shown in columns on the right. A significant difference was assessed by two-way ANOVA for the left column and by one-way ANOVA for the right column. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM. ↓STZ, STZ injection; ↓nitrate, start of nitrate supplementation; ↓HFD, start of high fat diet.

[21,30]. In the present study, administration of nitrate increased serum nitrate, nitrite, and NOX⁠ concentrations in both control and diabetic rats, demonstrating efficacy of the intervention; No difference was however observed between

control and diabetic rats. Increase in nitrite following nitrate supplementation is dependent on the reduction by commensal bacteria in the mouth [31]; pH in the stomach [32], but the reduction to NO is importantly dependent on functional 10

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In our study, nitrate supplementation decreased elevated serum glucose and insulin, but had no significant impact on HbA1c in diabetic rats. In addition, nitrate improved glucose tolerance, insulin sensitivity, and insulin resistance. Our results are in agreement with previous findings; demonstrating nitrate-mediated, decrease in serum glucose [1,4] and insulin [1,48] and improved glucose tolerance [4], and insulin resistance [22]. In addition, reduction in blood glucose [8,10,23], insulin [8,10,49], and improvement in glucose tolerance [7,8], and insulin resistance [8] has been shown following nitrite administration. Considering the fact that both nitrate and nitrite have favorable effects on peripheral glucose metabolism, it could be speculated that these effects are due to increasing NO production by both anions. We have previously reported that 85 mg/L nitrate for 10 weeks decreased HbA1c level in ⁠ /eNOS− mice [1]. To the best of our knowledge, there is no other report on the favorable effects of nitrate supplementation on HbA1c levels; no change in HbA1c level following 50–100 mg/L nitrite for 8 weeks in obese type 2 diabetic rats has been reported [8,50]; in addition, decreased HbA1c level in ZSF1 diabetic rats has been reported following 85 mg/L nitrite administration for 14 weeks [23], a discrepancy which may be related to the differences in duration of treatment and animal models of studies. In this study, we have shown for the first time that chronic low doses of nitrate improved pyruvate tolerance in diabetic rats, which contrast a previous finding following acute administration of nitrate in a model of metabolic syndrom [51]. There are several possible explanations for these discrepancies, including duration of treatment (acute vs. chronic supplementation), species difference and the model used for obesity and diabetes (HFD + STZ vs. genetic abrogation of the adenosine A2B receptor) [51]. To gain more insight into how nitrate can improve insulin resistance, we measured mRNA expression and protein levels of GLUT4 in insulin-sensitive tissues; nitrate increased mRNA and protein of GLUT4 in both the soleus muscle and epididymal adipose tissue. There is no report on mRNA expression and protein levels of GLUT4 following nitrate administration, although previous studies have reported that administration of 50 [7,8,10,23], 100 [23], and 150 mg/L [7] sodium nitrite in drinking water for 4 [10], 8 [8], 10 [7], and 14 weeks [23] increases glucose uptake by increasing protein expression and translocation of GLUT4 in insulin-sensitive tissues [7,8,10]. In diabetes, ni

Fig. 11. Effect of sodium nitrate on serum interleukin-1 beta (IL-1β) measured at the end of the study (n = 6). A significant difference was assessed by one-way ANOVA. * Statistically significant difference compared to control group. † Statistically significant difference compared to d non-treated diabetic group. Values are mean ± SEM.

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xanthine oxidoreductase activity [33]. Despite decreased eNOS-derived NO in diabetes [10,34], changes in serum nitrate, nitrite, and NOX⁠ levels is a controversial issue and elevated [35–37], no change [38,39] or even decreased levels [4] have been reported. It is still debatable whether circulating nitrite, nitrate, or their sum (NOx) provide an appropriate estimation of NO synthesis [16,40]; indeed, the true contribution of NOS isoforms to circulating nitrite and nitrate has not yet been elucidated [40,41]. In our study, only on day 42, serum nitrate levels in nitrate-treated diabetic rats was lower than nitrate-treated controls; although this observation can not explain from the results of this study, it may be related to alteration in microbiota following nitrate administration. Alteration of mouth and gut microbiota have been reported in diabetes and obesity [42–44]; which may affect nitrate reduction to nitrite and NO. There is no report available addressing the effects of dietary nitrate/nitrite on mouth and gut flora in diabetic rats, it has however been reported that vegetable-based diets increase the ratio of Bacteroidetes to Firmicutes [45]; Bacteroidetes and Firmicutes are two most abundant bacterial phyla in human gut, and their ratio decreases in obesity [46,47].

Fig. 12. Effect of sodium nitrate on serum levels of thiobarbituric reactive substances (TBARS) in the different groups before initiating nitrate supplementation (day 21, n = 10) (A) and after nitrate supplementation (day 84, n = 10) (B). Effect of sodium nitrate on serum catalase activity in the different groups before initiating nitrate supplementation (day 21, n = 10) (C) and after nitrate supplementation (day 84, n = 10) (D). A significant difference was assessed by one-way ANOVA. * Statistically significant difference compared to control group. † Statistically significant difference compared to non-treated diabetic group. Values are mean ± SEM. 11

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Fig. 13. Effect of sodium nitrate on insulin secretion from isolated islets in response to 5.6 (A) and 16.7 (B) mM of glucose concentrations and insulin content of islets (C) in the different groups (n = 10). A significant difference was assessed by one-way ANOVA. * Statistically significant difference compared to control group. Values are mean ± SEM.

trate-nitrite-NO could increase GLUT4 translocation and expression by S-nitrosylation of GLUT4 [10]; insulin-dependent phosphorylation of p85, insulin receptor substrate, and Akt pathway [7]; insulin-independent AMPK phosphorylation [23]; as well as activation of sGC/cGMP/ PKG and cGMP-AMPK dependent pathways [52,53]. In this study, basal insulin secretion, GSIS, and islet insulin content decreased in diabetic rats and nitrate supplementation failed to restore these parameters to normal values. There is no report regarding the effect of nitrate supplementation on insulin secretion and insulin content from isolated islets. We have previously shown that nitrite supplementation increases basal insulin secretion [54], GSIS [8] and insulin content [8] in both control and diabetic rats. Nitrite increases insulin secretion by converting to NO [54]. The likely explanation for the inability of nitrate to increase insulin secretion and content may be due to low capacity of pancreatic islets to reduce nitrate to nitrite and then NO; similar reasoning has previously suggested by Khoo et al. for the ability of nitrite, but not nitrate to stimulate glucose uptake in adipocytes [55]. According to our results, nitrate supplementation partially restored elevated serum TC and TG levels in diabetic rats, while HDL-C and LDL-C concentrations were unaffected; in addition, HDL-C levels were higher in diabetic rats that may be explained by long-time feeding a HFD [49]. Our results demonstrating reduced serum TG levels following nitrate supplementation are in agreement with previous find⁠ /- mice [1] and rats with fructose-induced metabolic ings, using eNOS− syndrome [22], as well as in hypercholesterolemic mice following nitrite administration [56]. We also previously reported nitrate administration in a rat model of hyperglycemia [4] and nitrite administration in obese type 2 diabetic rats [8] reduced both serum TG and TC ⁠ ) for 1 month in patients levels; NO dietary supplement (Neo40 Daily® with elevated serum TG level, reduced this parameter in 72% of patients [57]. Lipid-lowering effects of nitrate on serum TC and TG level may be due to increased fat metabolism or energy utilization and inhibition of acetyl CoA carboxylase [58]. Activity of acetyl CoA carboxylase is controlled by insulin, which regulates fatty acid synthesis and degradation [12,59]. Effect of nitrate/nitrite on the lipid profiles is disputable and decreased in LDL-C concentrations [4,8], no change in

LDL-C [22,56,60], HDL-C [8,56,60], TG [23], and TC [22,56] levels have been reported. In the current study, diabetic rats had higher serum MDA levels and lower CAT activity; nitrate supplementation partially restored decreased CAT activity but had no significant effect on MDA level. Few studies have reported effects of nitrate administration on serum MDA levels and CAT activity; we previously reported that nitrate administration decreased plasma MDA level in hypertensive rats [61] and increased CAT activity in hyperglycemic rats [4]. In addition, it has been reported that nitrite decreases MDA level in rats [62–64]. Previous studies have reported the anti-oxidative effects of nitrate/nitrite on reactive oxygen species [62,63,65–67], bioactive nitrogen oxides [1,61], class VI F2-isoprostanes [61], 8-isoprostane [63], 8-hydroxy-2deoxyguanosine [61,64], total antioxidant capacity [4]. Anti-oxidative effect of nitrate/nitrite is related to both decrease in activity of nicotinamide-adenine-dinucleotide phosphate (NADPH) oxidase [66,68] and increase in anti-oxidante genes [62]. In our study, administration of nitrate in diabetic rats partially restored elevated serum level of IL-1β, which is considered as a key pro-inflammatory cytokine [69]; this finding is in line with our previous studies on nitrate administration in ischemia reperfusion-induced kidney injury in mice [65] and nitrite administration in obese type 2 diabetic rats [8]. Cytotoxic effect of IL-1β involves the induction of iNOS [70]; in our study, nitrate also decreased mRNA expression of iNOS in both the soleus muscle and epididymal adipose tissue. In addition, anti-inflammatory effect of nitrate/nitrite on serum IL-6 [49,65,67], tumor necrosis factor-α [49,60,65], C-reactive protein [56], macrophage migration inhibitory factor [71], macrophage accumulation [60], macrophage infiltration [65], and neutrophil number and recruitment [60] have been reported. Indeed, nitrate-nitrite-NO by decreasing iNOS activity [66] and expression [72], and prevention of leukocyte activation and adhesion [31] exerts anti-inflammatory effects. Decrease in insulin secretion and increase in insulin resistance are the main characteristics of type 2 diabetes [73]. It seems that nitrate/nitrite can reduce the risk of type 2 diabetes [62,66] and provide cardioprotective effects [74–76]. Our results in this study, indicate that although nitrate administration could not

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Acknowledgments

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This article is a part of PhD thesis (No. 407) written by Sevda Gheibi, Faculty of Medicine, Shahid Beheshti University of Medical Sciences and was funded by the Research Institute for Endocrine Sciences (grant No. 766), Shahid Beheshti University of Medical Sciences, Tehran, Iran. The authors wish to thank Mr. Reza Norouzi-rad for his assistance in measurement of antioxidants and also wish to acknowledge Ms. Niloofar Shiva for critical English editing and syntax of the manuscript. Appendix A. Supplementary data

Supplementary data related to this article can be found at https:// doi.org/10.1016/j.niox.2018.02.002. References

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increase insulin secretion, it decreased insulin resistance which is associated with increase in both mRNA expression and protein levels of GLUT4 and decrease in mRNA expression of iNOS in insulin-sensitive tissues, especially in skeletal muscle that is responsible for approximately 80% of insulin-stimulated glucose uptake [77]. Different effects of nitrate and nitrite have been reported for IL-1β [8,24], number of rolling neutrophils [11], glucose uptake [55], HbA1c [1,8], serum insulin [8,24], body weight gain [10,24], and TG [1,23]. Although nitrate/nitrite dose, time of administration, and the animal models of studies are involved in these differences, the different pharmacokinetics of nitrate and nitrite should also be taken into consideration [11]. Regarding strengths of this study, we used the HFD-STZ model of type 2 diabetes, which has been reported to exhibit the metabolic characteristics of human type 2 diabetes [78]. In HFD-STZ model of type 2 diabetes, feeding a HFD causes peripheral insulin resistance while low-dose of STZ injection causes partially destruction and dysfunction of β-cells [79]. Both insulin resistance and β-cell dysfunction are involved in the pathogenesis of type 2 diabetes [79]; feeding a HFD alone without β-cell insufficiency, does not lead to diabetes and administration of STZ is necessary to achieve an animal model of type 2 diabetes [80]. In addition, the dose of nitrate administered in this study is accessible in nitrate-rich diets and has no carcinogenic effect at least in animals; short-term (up to 4500 mg/kg/day for 4 weeks) and long-term (up to 1820 mg/kg/day for 2 years) administrations of high doses of nitrate had no carcinogenic effect in rats [81,82]. This study has some limitations; First, body weight of nitrate-treated diabetic rats were significantly lower than non-treated ones and therefore metabolic improvements in nitrate-treated diabetic rats may at least in part be due to the weight differences; from the data of this study it is not possible to separate effects of weight differential from those of nitrate. Second, metabolic parameters reported in this study were measured under anesthesia state with pentobarbital, which can affect our results [83]. It has however been reported that compared with conscious animals, pentobarbital has no effect on ITT and circulating fasting glucose and insulin [84,85]; moreover, it has been recommended that ITT in anesthetized animals is a simple and authentic assessment method for insulin sensitivity and also decreases animal discomfort and inhibits stress response [84]. Third, other oxidative stress indices and concentration of cGMP that may be related to nitrate supplementation were not measured. Fourth, we cannot differentiate between total and translocated GLUT4 expression. Fifth, compared to the literature, concentrations of serum nitrite in our study seem to be high in non-treated animals. We can not explain the reason underlying these results, it however may be due method for measuring of nitrite (i.e. the Griess method) that is prone to interference and can be influenced by several factors including laboratory ware contamination [16,86]. Sixth, in our study, the amount of fat in the control diet (i.e. 5.7%) is relatively low. Although there is no strict definition, by going through the literature, a diet with 4.5–18% calories from fat has however been considered as standard/ control/regular diet [87–89]. In addition, it has been reported that regular chow and low-fat diet have similar effects on phenotypic, metabolic, and behavioral outcomes in mice and may thus be equally appropriate as controls for an HFD [90]. In conclusion, this study showed that long-term nitrate supplementation in obese type 2 diabetic rats had beneficial effects on peripheral carbohydrate metabolism and lipid homeostasis, although nitrate per se had no effect on GSIS and islet insulin content in the pancreas (in vitro). Improved glucose tolerance and lipid profile as well as decreased gluconeogenesis, inflammation and oxidative stress may contribute to the favorable effects of nitrate on carbohydrate metabolism. These favorable effects were associated with increased expression of GLUT4 and decreased expression of iNOS in insulin-sensitive tissues. Declaration of interest The authors report no conflict of interest.

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