The organic nitrate NDBP (2)

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Journal of Functional Foods 104 (2023) 105526
1756-4646/© 2023 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The organic nitrate NDBP promotes cardiometabolic protection in type 1 
diabetic mice 
Francineide Fernandes-Costa a, Rayanelle Tissiane Gomes da Silva b, 
Atalia Ferreira de Lima Flôr b, Maria Cláudia R. Brandão b, Petrônio F. Athayde-Filho b, 
Maria do Socorro França-Falcão b, Valdir de Andrade Braga b, Gustavo Jorge dos Santos c, 
Mattias Carlstrom d, Josiane de Campos Cruz b,* 
a Graduate Program in Bioactive Synthetic and Natural Products, Center for Health Sciences, Federal University of Paraíba, João Pessoa, Brazil 
b Biotechnology Center, Federal University of Paraíba, João Pessoa, Brazil 
c Multicenter Graduate Program in Physiological Sciences, Department of Physiological Sciences, Center for Biological Sciences, Federal University of Santa Catarina – 
UFSC, Brazil 
d Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden 
A R T I C L E I N F O 
Keywords: 
Diabetes 
Nitric oxide 
Aorta 
Blood pressure 
A B S T R A C T 
Studies from our laboratory have shown that the nitric oxide (NO) donor 2-nitrate-1,3-dibutoxypropan (NDBP) 
lowers blood pressure in hypertension. However, the role of NDBP in other cardiometabolic diseases such as 
diabetes remains unknown. Our study aimed to evaluate the cardiometabolic effect of NDBP treatment in type 1 
diabetes (T1DM) mice. T1DM was induced by streptozotocin (i.p. 50 mg/kg, 5 days) and confirmed by fasting 
glucose (≥200 mg/dL). Following induction of T1DM, mice were treated with NDBP (40 mg/kg/day i.p.) or 
vehicle (cremophor in saline) for 14 days. Our data revealed that NDBP treatment in T1DM mice reduced hy-
perglycemia, water consumption, urine volume, and oxidative stress in the liver and pancreas and increased 
glucose tolerance. Furthermore, NDBP treatment improved vascular function, although it was not able to 
attenuate hypertension. In conclusion, our study results indicate that NDBP promotes beneficial metabolic effects 
and improves endothelium-independent vascular function in a mouse model of T1DM. 
1. Introduction 
Diabetes mellitus (DM) is a metabolic disorder that is characterized 
by chronic hyperglycemia due to absolute insulin deficiency (type 1 
diabetes mellitus, T1DM) and/or insulin resistance (type 2 diabetes 
mellitus, T2DM). Streptozotocin (STZ) is a drug that destroys pancreatic 
islet β cells (Junod et al., 1967) and induces signs of insulin deficiency, 
hyperglycemia, polyuria, and symptoms characteristic of T1DM (Kolb, 
1987). Additionally, STZ-induced diabetes promotes blood pressure 
dysfunction, body weight decrease, and elevated serum glucose levels 
(Westermann et al., 2007). 
Nitric oxide (NO) is an important molecule that modulates endo-
thelial function and contributes to the maintenance of vascular tone. A 
decrease in NO bioavailability is related to the endothelial dysfunction 
that is observed in cardiovascular diseases such as hypertension (Por-
pino et al., 2016; Forte et al., 1997; Moncada and Higgs, 1993; Hermann 
et al., 2006; Lundberg et al., 2015). NO molecules also appear to be 
important in modulating insulin/glucose homeostasis, as a decrease in 
NO availability was observed in T1DM (Keyhanmanesh et al., 2018). 
The therapeutic value of different NO donors has been studied in dia-
betic animals. Studies by Lundberg et al. (Lundberg et al., 2018) revealed 
that inorganic NO donor sodium nitrate (NaNO3) treatment prevented 
hypertension and hyperglycemia in STZ-induced diabetic rats. Further-
more, Oghbaei et al. (Oghbaei et al., 2020) and Gheibi et al. (Gheibi 
et al., 2018) revealed that NaNO3 supplementation in drinking water for 
two months decreased blood glucose and increased serum insulin con-
centration in T1DM and T2DM mice. Additionally, Tian et al. (Tian et al., 
2020) observed an improvement in acetylcholine-mediated vascular 
relaxation in the aortas of T1DM mice after chronic NaNO3 treatment. 
The compound 2-nitrate-1,3-dibutoxypropan (NDBP) is a new 
organic nitrate that is derived from glycerin and is considered a “res-
idue” of the biodiesel production process (dos Santos, 2009). 
* Corresponding author at: Address: Centro de Biotecnologia, Universidade Federal da Paraíba, João Pessoa, PB 58051-900, Brazil. 
E-mail address: josianecruz@cbiotec.ufpb.br (J.C. Cruz). 
Contents lists available at ScienceDirect 
Journal of Functional Foods 
journal homepage: www.elsevier.com/locate/jff 
https://doi.org/10.1016/j.jff.2023.105526 
Received 13 October 2022; Received in revised form 10 March 2023; Accepted 25 March 2023 
mailto:josianecruz@cbiotec.ufpb.br
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Journal of Functional Foods 104 (2023) 105526
2
Interestingly, it was observed that, unlike other organic nitrates, NDBP 
did not induce long-term tolerance (Porpino et al., 2016; França-Silva 
et al., 2012). Previous results from our group have demonstrated that 
NDBP treatment promotes vasorelaxation and hypotension and in-
creases the bioavailability of NO in hypertensive rats (Porpino et al., 
2016; França-Silva et al., 2012; Queiroz et al., 2014). However, the 
metabolic and vascular effects induced by NDBP in the context of dia-
betes mellitus have not yet been investigated. We hypothesized that 
chronic treatment with organic nitrate NDBP promotes beneficial car-
diometabolic effects by reducing fasting blood glucose and insulin 
resistance, improving glucose tolerance and vascular function, and 
lowering blood pressure and oxidative stress in T1DM mice. Thus, our 
goal was to evaluate the vascular and metabolic effects of NDBP treat-
ment on STZ-induced T1DM in mice. 
2. Materials and methods 
2.1. Animals 
Male C57BL/6 mice (7 weeks old) were housed under controlled 
room temperature (21 ± 1 ◦C) with a 12 h light–dark cycle and access to 
water and feed ad libitum (Labina Presence, SP, Brazil). The protocols 
were approved by the Ethics Committee on the Use of Animals at the 
Biotechnology Center (CEUA-CBiotec no. 9387160419) of the Federal 
University of Paraiba, Brazil that were carried out by the ARRIVE 
guidelines and National Research Council’s Guide for the Care and Use 
of Laboratory Animals. 
2.2. Diabetes induction 
Diabetes was induced by intraperitoneal (i.p.) injection of strepto-
zotocin (STZ, 50 mg/kg dissolved in 0.1 M citrate buffer, pH 4.5, Sigma 
Aldrich, Hamburg, Germany) once a day in the morning for five 
consecutive days (Brosius, 2009). The control group received an equal 
volume of vehicles (i.p. citrate buffer; 0.1 M, pH 4.5) following the same 
schedule. Mice fasted for 2 h. One week after the STZ-induced diabetes 
protocol, blood glucose was measured to confirm diabetes mellitus in-
duction (3 blood glucose readings greater than 200 mg/dL on 3 
consecutive days after a week of the last dose of STZ). 
2.3. Synthesis and preparation of the 2-nitrate-1,3-dibuthoxypropan 
(NDBP) 
NDBP was synthesized at the Department of Chemistry at the Federal 
University of Paraiba (dos Santos, 2009). Its synthesis was divided into 
three steps as described by França-Silva et al. (2012) (França-Silva et al., 
2012): initially, halohydrin (1,3-dichloro-propan-2-ol) was obtained by 
the reaction between glycerin and dry hydrochloric acid (HCl). Then 
1,3-dibutoxipropan-2-ol was obtained, for that, the halohydrin was 
added dropwise into a solution of sodium alkoxide, the latter obtained 
by the reaction between metallic sodium and anhydrous alcohol. As a 
product of this second phase, 1,3-dibutoxipropan-2-ol and sodium 
chloride were obtained. Finally, 2-nitrate-1,3-dibutoxypropan (NDBP) 
wasobtained by the nitration of 1,3-dibutoxipropan-2-ol (NDBP syn-
thesis details in supplementary material). NDBP was stored at room 
temperature (21 ± 1 ◦C) until the end of the experiments (approximately 
6 months). 
NDBP preparation: the compound was solubilized in a mixture of 
cremophor at a ratio of 1:1 and diluted in saline (0.15 M NaCl) to a 
concentration of 40 mg/mL (10 min before beginning the experiments) 
(França-Silva et al., 2012; Queiroz et al., 2014; França-Silva et al., 
2012). No effect was observed when cremophor was administered alone 
to control animals using the same dose (Table S1 – supplementary 
material). 
2.4. Experimental design 
30 animals were used in this study, the mice were divided into four 
groups: the control group treated with vehicle (cremophor in 0.9% sa-
line i.p., once daily for 14 days) (n = 8); mice treated with NDBP diluted 
in cremophor (40 mg/kg i.p., once daily for 14 days) (Porpino et al., 
2016) (NDBP) (n = 7); diabetic mice treated with vehicle (cremophor in 
0.9% saline i.p., once daily for 14 days) (Diabetes) (n = 8); diabetic mice 
treated with NDBP diluted in cremophor (40 mg/kg i.p., once daily for 
14 days) (Porpino et al., 2016) (Diabetes/NDBP) (n = 7). 
Before and after treatment, body weight (g) and 2 h fasting blood 
glucose (mg/dL) levels were assessed. The water (mL) and feed (g) 
consumption were both evaluated one day after finishing the treatment, 
and the mice were housed in individual boxes for 24 h. The next day, the 
mice were housed in individual metabolic cages for 4 h after i.p. injec-
tion of 0.9% saline (2 mL) to evaluate urine volume frequency. Glucose 
tolerance test (GTT), and tail-cuff blood pressure (mmHg) results were 
measured on the 3rd and 6th days after treatment. Then the animals 
were euthanized and the vascular reactivity of the thoracic aorta was 
evaluated. Additionally, the concentration of malondialdehyde (MDA) 
in the plasma, liver, and pancreas was evaluated using the thiobarbituric 
acid reactive substances (TBARS) method. The experimental design is 
illustrated in Fig. 1. 
2.5. Water and food consumption 
After 14 days of treatment, mice were housed in individual boxes at 
8:00 a.m. with access to water and food in a known quantity, and after 
24 h, the consumption of food (g) and water (mL) was verified. The data 
were normalized to 10 g of body weight for the statistical analysis. 
2.6. Urinary volume 
The mice received an i.p. injection of 0.9% saline (2 mL) at room 
temperature and were gradually injected into the lower abdomen 
(Meijer et al., 2005). After the injection, the animals were housed in 
individual metabolic cages without access to food or water. Urinary 
volume analysis was performed every 1 h for 4 h. 
2.7. Intraperitoneal glucose tolerance test 
All glycemic tests were performed on conscious animals based on the 
consideration that anesthesia changes blood flow and induces hyper-
glycemia in mice (Pomplun et al., 2004). Thus, for the GTT test after 
12–14 h of fasting, mice were gently and safely immobilized, and a cut 
on the tip of the tail was then made with sharp scissors (1–2 mm) to 
collect the blood sample. Blood glucose was measured using a com-
mercial glucometer (ACCU-CHEK® Active, Roche Diagnostica, Brazil 
Ltda) immediately before and at 15, 30, 60, and 90 min after i.p. in-
jection of 25% glucose in a 0.9% sterile saline at a dose of 2 g/kg (Ayala 
et al., 2010; Bowe et al., 2014). 
2.8. Oxidative stress 
The concentration of MDA (nmol/mL) was evaluated to assess 
oxidative stress using the TBARS technique. Briefly, the mice were 
anesthetized, and an incision was made in the thoracoabdominal region. 
The lung was sectioned to collect blood (1 mL) from the thoracic cavity 
with the help of a volumetric pipette (1000 μL), and it was then stored in 
an Eppendorf tube and centrifuged (7000 rpm for 15 min) for plasma 
extraction (250 µL). After blood collection, the liver and pancreas were 
extracted, weighed, and homogenized with 10% KCl solution, and 250 
µL of each sample was collected. The samples were placed in a dry bath 
for 1 h at 37 ◦C, 200 µL of 7% perchloric acid was added and vortexed for 
1 min. Subsequently, the samples were centrifuged (14000 rpm, 4 ◦C, 20 
min), 250 µL of the precipitate was collected, and 200 µL of the 
F. Fernandes-Costa et al. 
Journal of Functional Foods 104 (2023) 105526
3
thiobarbituric acid solution was added to each sample. Then, the sam-
ples were placed in a rapid vortex (20 s) and then in a dry bath for 1 h at 
100 ◦C. Finally, the samples were assessed on a spectrophotometer 
(Chem Well T automatic analyzer) to read the absorbance (532 nm) 
(Ohkawa et al., 1979). 
2.9. Vascular reactivity 
The mice were anesthetized, and thoracotomy surgeries and careful 
extraction of the thoracic aortic artery were performed. The artery was 
immersed in a Krebs nutrient solution (pH 7.4) contained in a Petri dish, 
and this solution was composed of mM NaCl (118 mM), KCl (4.5 mM), 
CaCl2 (1.8 mM), MgSO4 (2.7 mM), KH2PO4 (1.0 mM), NaHCO3 (25 mM), 
and glucose (11 mM) and constantly aerated with a carbogenic mixture 
(95% O2 and 5% CO2) and maintained at 37 ◦C. The connective and 
adipose tissues were removed, and the aorta was divided into three or 
four rings (~3 mm in length). Each ring was coupled through the 
vascular lumen to two steel triangles in a parallel orientation. The rings 
were suspended vertically by cotton threads inside the myograph tanks 
(containing 10 mL of Krebs solution aerated with carbogen and main-
tained at 37 ◦C) that were connected to an isometric voltage transducer 
coupled to a data acquisition system (ADInstruments/Panlab Organ 
Bath Systems, Australia) and a computer for registration. After a 60 min 
stabilization period at basal tension (0.50 g), aortic rings were exposed 
to KCl (125 mM) (Balarini et al., 2013) to evaluate their maximal ten-
sion. The integrity of the rings was evaluated by acetylcholine (ACh)- 
induced relaxation (10 μM) after pre-contraction with phenylephrine 
(PHE, 10 μM). The endothelium was considered functional when there 
was a relaxation that was greater than 50% of the maximum contraction 
reached with Phe and considered without functional endothelium when 
the relaxation was less than 10%. Finally, after washing and stabilizing, 
concentration–response curves were obtained with cumulative concen-
trations of PHE (100 pM–20 µM, aorta with and without endothelium), 
ACh (100 pM–20 µM, aorta with endothelium), and sodium nitroprus-
side (SNP, 10 pM–20 µM, aorta without endothelium) at a 1 min interval 
between each dose. The concentration–response curves of ACh and SNP 
pre-contraction with PHE (10 µM) were obtained. 
2.10. Non-invasive evaluation of arterial blood pressure 
The arterial blood pressure of the mice was measured using a non- 
invasive tail-cuff method (Insight, v. 2.11, Ribeirão Preto/SP) accord-
ing to the manufacturer’s protocol. Mice were adapted to the procedure 
for two consecutive days, and this adaptation consisted of restraining the 
mice in a cylindrical container for 5 min. The experiments were con-
ducted in a quiet area (22 ± 2 ◦C) where the animals were acclimated for 
1 h before the start of the experiments. The mice were encouraged to 
walk into the restraint tubes, and the tube end holders were adjusted to 
prevent excessive movement. The occlusion cuff was placed at the base 
of the tail, and the sensor cuff was placed adjacent to the occlusion cuff. 
Thermal bags were preheated, and the mice were warmed for 5 min 
before and during blood pressure recordings. To measure blood pres-
sure, the occlusion cuff was inflated to 300 mmHg and then deflated 
over 60 s. The sensor cuff detects changes in tail volume as blood returnsto the tail during occlusion cuff deflation. The diastolic blood pressure 
marker (DBP) was adjusted to the highest point of the first pulse wave, 
and the systolic blood pressure marker (SBP) was adjusted to the point 
immediately before the beginning of the first pulse wave as instructed by 
the manufacturer. An average of 10 readings for SBP, DBP, and mean 
arterial pressure (MAP) were recorded. Blood pressure measurements 
were performed for two consecutive days before the day of the experi-
ment to acclimatize the animals. 
2.11. Chemical compounds 
1. Streptozotocin - (S0130 - Sigma-Aldrich). 
2. 2-nitrate-1,3-dibuthoxypropan (NDBP) – (Laboratory of Prof. Dr. 
Petrônio Filgueiras de Athayde Filho, Department of Chemistry, Federal 
University of Paraíba). 
3. D-(+)-Glucose – (ALPHA1731 – ALPHATEC). 
4. 2-Thiobarbituric acid – (T5500 - Sigma-Aldrich®). 
5. Potassium chloride (KCl) – (01006 – NEON). 
6. Acetylcholine chloride (ACh) – (A6625 - Sigma® Life Science). 
7. L - Phenylephrine (PHE) – (Sigma-Aldrich®). 
8. Sodium nitroprusside dihydrate (SNP) – (8496 J – MP 
Biomedicals). 
2.12. Statistical analysis 
The analyses were performed using Graph Pad Prism software 
(Version 6), and all values are presented as mean ± standard error of the 
mean. One-way analysis of variance (ANOVA) followed by Tukey’s post- 
test and unpaired t-tests were used to analyze data detailing fasting 
blood glucose, body weight, water, and feed consumption, total urinary 
volume, SBP, DBP, MAP, MDA concentration, area under the curve, and 
vascular reactivity (pEC50 and Emax). EC50 values are presented as 
negative logarithms (pEC50). Two-way ANOVA followed by Tukey’s 
post-test and multiple t-tests were used to analyze data detailing GTT, 
cumulative urinary volume, and vascular reactivity (mean). Statistical 
significance was set at p less than 0.05. 
3. Results 
3.1. Fasting blood glucose 
Fig. 2 presents the fasting blood glucose measurements after STZ 
treatment to induce diabetes. STZ-diabetic mice exhibited higher blood 
glucose levels than did non-diabetic controls (354.42 ± 19.19 vs 122.28 
± 2.35 mg/dL, n = 8) or NDBP (354.42 ± 19.19 vs 136.85 ± 3.27 mg/ 
dL, n = 8), before starting treatment. Additionally, NDBP treatment 
Fig. 1. Experimental design. Squares correspond to 1 week, and the dark squares indicate the weeks with NDBP or vehicle treatments. STZ = streptozotocin, GTT =
glucose tolerance test. 
F. Fernandes-Costa et al. 
Journal of Functional Foods 104 (2023) 105526
4
reduced glycemia in diabetic mice (305.85 ± 36.42 vs 498.42 ± 21.18 
mg/dL, n = 7). 
3.2. Body weight 
Fig. 3 presents the body weight (g) before (pre-treatment) and after 
(post-treatment) NDBP treatment of control and diabetic mice. Diabetic 
conditions prevented body weight gain in mice after 14 days of analysis 
(21.25 ± 0.65 vs 20.75 ± 0.62 g, n = 8). Moreover, NDBP treatment did 
not induce this response (24.29 ± 1.02 vs 24.29 ± 0.52 g, n = 7). The 
graphs on the right side of Fig. 3 detail the variation in body weight (Δ) 
between post- and pre-treatment weights. We observed that diabetes 
resulted in decreased body weight gain compared to that of the control 
(0.5 ± 0.75 vs 4.0 ± 1.25 g, n = 8) or NDBP (0.5 ± 0.75 vs 4.28 ± 0.68 
g, n = 8) groups. Moreover, NDBP treatment did not prevent body 
weight loss in mice (0.0 ± 0.61 vs 0.5 ± 0.75 g, n = 7). 
3.3. Water and food consumption 
Fig. 4 presents the water (Fig. 4, A) and feed (Fig. 4, B) consumption 
during the 24 h analysis. Diabetes resulted in increased water intake 
(11.56 ± 0.78 vs 3.47 ± 0.47 mL/10 g/24 h, n = 8), and this intake was 
reduced by NDBP treatment (7.46 ± 1.18 vs 11.56 ± 0.78 mL/10 g/24 
h, n = 8) (Fig. 4, A). Diabetes increased food intake compared to that of 
the control (3.15 ± 0.20 vs 1.86 ± 0.16 g/10 g/24 h, n = 8), and NDBP 
treatment reduced food intake in diabetic mice (2.11 ± 0.31 vs 3.15 ±
0.20 g/10 g/24 h, n = 8) (Fig. 4, B). 
3.4. Urinary volume 
Fig. 4 panels C and D present the cumulative and total urinary vol-
umes, respectively. We observed that NDBP increased the urinary vol-
ume of non-diabetic mice (0.31 ± 0.02 vs 0.14 ± 0.02 mL/10 g/4h, n =
6). Furthermore, diabetes resulted in increased urinary volume (0.53 ±
0.05 vs 0.14 ± 0.02 mL/10 g/4h, n = 8) that was attenuated by NDBP 
treatment (0.29 ± 0.07 vs 0.53 ± 0.05 mL/10 g/4h, n = 7). 
3.5. Glucose tolerance test 
Fig. 5 and Table 1 present the blood glucose curve (mg/dL) after an 
overload with glucose (i.p.) and also the respective area under the curve 
(AUC). Diabetic mice possessed a lower glucose tolerance than did the 
control mice at 15, 30, 60, and 90 min. NDBP treatment increased 
glucose tolerance in diabetic mice at 60 and 90 min. Additionally, non- 
diabetic animals that were treated with NDBP exhibited an improve-
ment in glucose intolerance compared to that of the control at 30 min. 
3.6. Oxidative stress 
Fig. 6 indicates the MDA concentrations (nmol/mL) in the plasma, 
liver, and pancreas. Diabetes resulted in increased oxidative stress in 
plasma (23.98 ± 2.7 vs 15.64 ± 1.55 mmol/mL, n = 8). Additionally, 
NDBP did not alter plasma MDA concentrations in diabetic mice (18.78 
± 2.49 vs 23.98 ± 2.7 mmol/mL, n = 6) (Fig. 6, A). Diabetes resulted in 
increased MDA concentration in the liver (22.03 ± 1.61 vs 15.67 ± 1.92 
mmol/mL, n = 7), and NDBP treatment reduced oxidative stress in the 
diabetic mice livers (10.93 ± 1.6 vs 22.03 ± 1.61 mmol/mL, n = 6) 
(Fig. 6, B). Finally, diabetes resulted in increased pancreas MDA levels 
(12.35 ± 1.16 vs 6.65 ± 0.82 mmol/mL, n = 8), and these levels were 
reduced by NDBP treatment (7.66 ± 0.98 vs 12.35 ± 1, 16 mmol/mL, n 
= 7) (Fig. 6, C). 
3.7. Vascular reactivity in thoracic aortas of diabetic mice 
3.7.1. Vascular response to phenylephrine (PHE) 
Fig. 7 presents the concentration–response curve of PHE in aortic 
artery rings isolated from mice. PHE (100 pM–20 µM) produced 
concentration-dependent contractions. Moreover, diabetes resulted in 
Fig. 2. Effects of the STZ-induced diabetes mellitus (50 mg/kg) and NDBP (40 mg/kg) treatment on fasting blood glucose. *p <.05 compared to control and NDBP, 
#p <.05 compared to diabetes. Values are mean ± SEM. 
Fig. 3. Body weight (g) analysis before and after NDBP treatment (40 mg/kg). 
&p <.05 compared to pre-treatment. *p <.05 compared to control and NDBP. Δ 
(post-treatment minus pre-treatment). Values are mean ± SEM. 
F. Fernandes-Costa et al. 
Journal of Functional Foods 104 (2023) 105526
5
increased maximum effect (Emax) and potency (pEC50) for PHE in aortas 
with functional or denuded endothelium. NDBP treatment prevents 
diabetes by decreasing the maximum effect (Emax) and potency (pEC50) 
of PHE in aortic rings in the presence of functional endothelium (Fig. 7, 
A) and in endothelium-denuded aorta rings (Fig. 7, B). Table 2 sum-
marizes the pEC50 and Emax values for all groups. 
Fig. 4. Effect of NDBP (40 mg/kg) on water (A) and food (B) intake during a 24 h period, on cumulative (C) and total (D) urinary volume during a 4 h period. + p 
<.05 compared to control group, *p <.05 compared to control and NDBP, #p <.05 compared to diabetes. Values are mean ± SEM. 
Fig. 5. Effect of NDBP (40 mg/kg) on blood glucose during intraperitoneal glucose tolerance tests (GTT). The area under the curve is presented in the right column. 
+ p <.05 compared to control group, *p <.05 compared to control and NDBP, #p <.05 compared to diabetes. Values are mean ± SEM. 
F. Fernandes-Costa et al.Journal of Functional Foods 104 (2023) 105526
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3.7.2. Vascular response to acetylcholine (ACh) and sodium nitroprusside 
(SNP) 
Fig. 7 panels C and D present the concentration–response curves for 
ACh and SNP in aortic artery rings with functional or denuded endo-
thelium. ACh (100–20 µM) produced concentration-dependent relaxa-
tion in phenylephrine pre-contracted aortic artery rings. Moreover, 
diabetes resulted in decreased maximum effect (Emax) and potency 
(pEC50) of ACh in the presence of functional endothelium. NDBP treat-
ment did not alter the decrease of the Emax and pEC50 for ACh induced by 
diabetes (Fig. 7, C. SNP (10–20 µM) also produced concentration- 
dependent relaxation in phenylephrine pre-contracted aortic artery 
rings. Additionally, diabetes resulted in decreased Emax for SNP in 
endothelium-denuded aortic rings. NDBP treatment prevented diabetes 
by increasing Emax for SNP (Fig. 7, D). Table 2 summarizes the pEC50 and 
Emax values for all groups. 
3.8. Arterial blood pressure 
Fig. 8 presents the systolic (SBP, mmHg), diastolic (DBP, mmHg), 
and mean arterial pressure (MAP, mmHg) in diabetic mice treated with 
NDBP. Diabetes resulted in increased SBP and DBP; however, NDBP 
treatment did not alter the diabetes-induced increase in blood pressure 
(Fig. 8, A and B). Furthermore, diabetes resulted in increased MAP 
compared to that of control (113.9 ± 1.01 vs 97.7 ± 0.83 mmHg, n = 8) 
and non-diabetic NDBP (113.9 ± 1.01 vs 97.79 ± 2.30 mmHg, n = 8) 
mice. However, NDBP treatment did not alter the diabetes-induced in-
crease in MAP (113.7 ± 2.00 vs 113.9 ± 1.01 mmHg, n = 6) (Fig. 8, C). 
4. Discussion 
The effects of NO3– treatment in diabetic animals may be due to the 
conversion of NO3– into NO metabolites (Keyhanmanesh et al., 2018; 
Oghbaei et al., 2020; Gheibi et al., 2018). Previous studies from our 
laboratory have demonstrated that NDBP increases plasma levels of NO2– 
and NO3– in mice with angiotensin II-induced hypertension (Porpino 
et al., 2016). 
We observed that NDBP decreased STZ diabetes-induced hypergly-
cemia. In agreement with our results, nitrate-mediated reduction of 
fasting blood glucose has recently been reported in diabetic rats and 
mice treated with organic and inorganic nitrates (Oghbaei et al., 2020; 
Gheibi et al., 2018; Tian et al., 2020; Schuhmacher et al., 2011; Xie et al., 
2020). Inorganic nitrate supplementation in combination with NaNO3 
has been demonstrated to reduce blood glucose in models of T2DM 
(Gheibi et al., 2018; Tian et al., 2020) and T1DM (Keyhanmanesh et al., 
2018; Oghbaei et al., 2020). Additionally, oral treatment for 8 weeks 
with organic nitrate pentaerithrityl tetranitrate (PETN) induced a 
decrease in blood glucose in T1DM rats (Schuhmacher et al., 2011). 
T1DM is normally characterized by a loss of body weight, even in 
diabetic mice induced by STZ (Westermann et al., 2007; Oghbaei et al., 
2020; Schuhmacher et al., 2011; Howarth et al., 2005; Khalifi et al., 
2015). In our study, we did not observe a significant decrease in body 
weight in diabetic mice, and the NDBP did not change this outcome. This 
is possibly due to the relatively short follow-up time and shorter treat-
ment time compared to those of other studies (Oghbaei et al., 2020; 
Gheibi et al., 2018). Interestingly, previous studies have revealed that 
treatment with the organic nitrate PETN (56 days) did not prevent 
weight loss in T1DM rats (Schuhmacher et al., 2011). In contrast, a study 
by Oghbaei et al. (Oghbaei et al., 2020) observed that the NaNO3 (60 
days) prevented the weight loss of T1DM rats only 40 days after STZ 
injection. 
T1DM is typically associated with polyphagia, as glucose is not uti-
lized efficiently by tissues due to low insulin production that promotes 
the release of hormones such as leptin and orexin that activate the feed 
center in the hypothalamus (Adinortey, 2017). Indeed, we observed an 
increase in food intake in STZ-diabetic mice, and this was reduced by 
NDBP. Khalifa et al. (Khalifi et al., 2015) revealed an increase in food 
intake in T2DM rats (induced by STZ and nicotinamide) that was not 
Table 1 
Effect of NDBP treatment on glucose tolerance. 
Group Mean ± SEM (mg/dL) n 
0 15 30 60 90 
Control 89.62 ±
4.17 
467 ±
27.14 
408.5 ±
34.77 
236.12 
± 14.19 
152.37 
± 13.28 
8 
NDBP 121 ±
10.73 
363.14 
± 30.44 
301.43 
±
42.77a 
188.71 
± 16.91 
163.28 
± 11.64 
7 
Diabetes 301.62 
±
31.29b 
598.12 
± 1.87a 
600 ±
0b 
567.37 
±
16.32b 
559.5 ±
18.15b 
8 
Diabetes/ 
NDBP 
192.5 ±
17.83b,c 
476.17 
± 72.28 
475 ±
68.84 
412.5 ±
53.61b,c 
376.83 
±
14.24b,c 
6 
a p <.05 compared to the control group. 
b p <.05 compared to control and NDBP (40 mg/kg). 
c p <.05 compared to diabetes. 
Fig. 6. Effect of NDBP (40 mg/kg) on MDA’s concentration (nmol/mL) in the plasma (A), liver (B) and pancreas (C). + p <.05 compared to control group, *p <.05 
compared to control and NDBP, #p <.05 compared to diabetes. Values are mean ± SEM. MDA = malondialdehyde. 
F. Fernandes-Costa et al. 
Journal of Functional Foods 104 (2023) 105526
7
altered by NaNO3. Diabetes also produces polyuria, as hyperglycemia 
promotes greater urinary glucose excretion that increases the osmotic 
pressure of urine, inhibits renal water reabsorption, and increases urine 
excretion with loss of water and electrolytes, ultimately leading to the 
stimulation of thirst (Adinortey, 2017). We observed an increase in 
water intake in STZ-diabetic animals, and this was reduced by NDBP. A 
study by Khalifi et al. (Khalifi et al., 2015) revealed an increase in water 
intake in T2DM rats, while Gheibi et al. (Gheibi et al., 2018) observed an 
increase in water intake by T2DM animals; however, both outcomes 
were not altered by NaNO3. 
Previous studies with hypertensive rats (2K1C) treated with L-argi-
nine and healthy humans treated with inhaled NO, demonstrated that 
nitric oxide, by a still uncertain mechanism, appears to act in the renal 
tubules by increasing sodium reabsorption along the nephron and 
leading to an increase in renal water excretion (Wraight and Young, 
2001; Gouvêa et al., 2003). Corroborating these findings, we observed 
that NDBP promoted an increase in urinary volume in control animals. 
However, NDBP decreased urine volume in diabetic mice. Since diabetic 
animals have osmotic diuresis, the reduction in blood glucose may have 
contributed to the antidiuretic effect (Adinortey, 2017). Corroborating 
our findings, other studies have demonstrated increased urinary volume 
in animals with diabetes induced by STZ (T1DM), STZ + nicotinamide 
(T2DM) (Kaikini et al., 2020); and STZ + high-fat diet (T2DM) (De 
Magalhães et al., 2019); and no previous study has evaluated nitrate 
treatment for diabetes mellitus. 
We observed that NDBP increased glucose tolerance in STZ-diabetic 
mice. Similar results were observed after NaNO3 (Gheibi et al., 2018; 
Gheibi et al., 2017) or NaNO2 (Ohtake et al., 2015) oral supplementation 
in T2DM animals based on the performances of the intraperitoneal 
glucose tolerance test. A previous study revealed an increase in serum 
insulin levels in T1DM diabetic rats after NaNO3 chronic treatment (8 
weeks) (Keyhanmanesh et al., 2018), thus indicating that nitrate 
increased insulin release by the pancreas. This may explain the 
improvement in glucose tolerance observed in our study. Additionally, 
NaNO2 increases blood flow in the pancreatic islets of rats, and this 
contributes to increased insulin secretion (Nyström et al., 2012). 
Furthermore, a study in T2DM rats treated with NaNO3 revealed an 
increase in the mRNA and protein levels of GLUT4 in the soleus muscle 
Fig. 7. Concentration-response curve of phenylephrine (100 pM–20 µM) in the aorta ofmice in the presence of functional endothelium (A) and in endothelium- 
denuded (B) mice, and of acetylcholine (100 pM–20 µM) and sodium nitroprusside (10 pM–20 µM) in the aorta of mice in the presence of functional endothe-
lium (C) and in endothelium-denuded (D) mice, respectively. PHE = phenylephrine, ACh = acetylcholine, SNP = sodium nitroprusside, +p <.05 compared to control 
group, *p <.05 compared to control and NDBP, #p <.05 compared to diabetes. Values are mean ± SEM. 
Table 2 
Pec50 and Emax obtained from the concentration–response curve to PHE (10-7-10-2 M), ACh (100 pM–20 µM) and SNP (10 pM–20 µM) in aortas with functional or 
denuded endothelium. 
Phenylephrine (E + ) Phenylephrine (E-) Acetylcholine (E + ) Sodium Nitroprusside (E-) 
pEC50 Emax (%) n pEC50 Emax (%) n pEC50 Emax (%) n pEC50 Emax (%) n 
Control 6.98 ± 0.07 83.52 ± 4 8 7.29 ± 0.1 107.8 ± 5.93 9 6.69 ± 0.13 107.3 ± 9.07 8 7.51 ± 0.21 158.9 ± 4 9 
NDBP 7.04 ± 0.11 84.55 ± 7.63 6 7.21 ± 0.12 113.1 ± 5.15 5 6.71 ± 0.16 100.1 ± 12.38 5 7.41 ± 0.22 139.6 ± 3.11a 5 
Diabetes 7.93 ± 0.24b 102.6 ± 2.88b 8 8.33 ± 0.1b 149.4 ± 7.95b 9 6.12 ± 0.17b 73.89 ± 5.12b 8 7.88 ± 0.41 104 ± 3.57b 9 
Diabetes/ 
NDBP 
6.8 ± 0.19c 62.23 ± 6.64a,c 5 7.1 ± 0.21c 85.09 ± 8.89b,c 9 5.66 ± 0.41b 90.54 ± 6.31 5 7.23 ± 0.23 161.3 ± 16.04c 5 
pEC50 = potency, Emax = maximum effect, E+ = functional endothelium aorta, E- = endothelium denuded aorta. 
a p <.05 compared to control group. 
b p <.05 compared to control and NDBP (40 mg/kg). 
c p <.05 compared to diabetes. 
F. Fernandes-Costa et al. 
Journal of Functional Foods 104 (2023) 105526
8
and adipose tissue that ultimately resulted in favorable effects regarding 
peripheral glucose absorption (Gheibi et al., 2018). There is evidence 
that the use of the NO donor sodium nitroprusside (SNP) increased 
GLUT4 on the cell surface in rat skeletal muscle through activation of the 
cGS/cGMP pathway, and this was independent of PI3-K (Etgen et al., 
1997). Additionally, NO can induce S-nitrosylation of GLUT-4, thus 
stimulating the translocation of this protein to the plasma membrane in 
skeletal muscle and adipose tissue (Jiang et al., 2014; Bahadoran et al., 
2015). 
Furthermore, NO inhibits protein tyrosine phosphatase 1B (PTP1B) 
which is related to dephosphorylation of the insulin receptor substrate, 
thus improving insulin signaling (Ohtake et al., 2015; Wang et al., 
2013). It was observed in a previous study that oral supplementation 
with inorganic nitrite dampened the production of reactive oxygen 
species (ROS) in adipocytes of T2DM mice, and this led to improved 
signaling via the insulin transduction pathway with a consequent in-
crease in GLUT-4 translocation and glucose absorption (Ohtake et al., 
2015). Our results revealed that NDBP decreased oxidative stress in the 
pancreas and liver in T1DM mice. However, there was no reduction in 
the malondialdehyde (MDA) concentration in plasma, thus corrobo-
rating the results of a previous study revealing no change in MDA levels 
in the serum of diabetic rats treated with NaNO3 (Gheibi et al., 2018). In 
contrast, 3,4-Dihydroxyphenethyl nitrate (HT-ONO2) demonstrated 
antioxidant activity in T1DM mice by increasing plasma superoxide 
dismutase (SOD) levels and reducing plasma MDA levels (Xie et al., 
2020). These differences may be related to the dose, as we used a dose of 
40 mg/kg that was lower than the 65 mg/kg dose used by Xie et al. (Xie 
et al., 2020). Additionally, although both compounds are organic ni-
trates, they may differ in their biological activities and antioxidant ef-
fects. Other studies have demonstrated that nitrite reduces MDA 
concentration in the myocardium of rats subjected to hypoxia (Singh 
et al., 2012), in the plasma of DOCA-salt hypertensive rats (Amaral et al., 
2015), and in the kidneys of STZ-diabetic mice (Ohtake et al., 2007). 
It is established that the antioxidant effect of nitrate/nitrite includes 
a decrease in NADPH oxidase activity (Schuhmacher et al., 2011; 
Amaral et al., 2015; Gao et al., 2014; Yang et al., 2015), and a recent 
study from our laboratory revealed that NDBP dampened oxidative 
stress as evidenced by a reduction in NADPH oxidase activity and su-
peroxide levels in the heart and renal cortex of mice with Ang II-induced 
hypertension (Porpino et al., 2016). Furthermore, it was observed in a 
previous study that NaNO3 and PETN decrease NADPH oxidase activity 
and oxidative stress in isolated aortic rings from T2DM mice (Tian et al., 
2020) and T1DM rats (Schuhmacher et al., 2011), respectively. 
Hyperglycemia is frequent in diabetes mellitus and is known to 
induce vascular damage and increase vascular smooth muscle contrac-
tility (Kizub et al., 2014). NDBP did not promote improvement in vessel 
relaxation in response to ACh, and this was possibly due to the inability 
of this drug to improve the endothelial dysfunction present in STZ- 
diabetic mice. Our results suggest that NDBP acts directly on vascular 
smooth muscle to promote relaxation (França-Silva et al., 2012), that is, 
it has an endothelium-independent action. Additionally, by mechanisms 
that possibly increase the availability of NO in the vessel (França-Silva 
et al., 2012), NDBP promotes a lower tendency towards vasoconstric-
tion, and this may explain why there was less intensity in the contractile 
response to PHE in the aorta of diabetic animals treated with NDBP. No 
reports have demonstrated that NDBP affects the vascular reactivity of 
the aorta in diabetic animals. However, it was previously observed that 
NDBP promoted concentration-dependent relaxation in the mesenteric 
artery of wild mice that were pre-contracted with PHE (França-Silva 
et al., 2012). A previous study revealed that chronic oral treatment with 
the organic nitrate PETN increased endothelium-dependent and-inde-
pendent vasodilation in aortic rings in T1DM diabetic rats (Schuh-
macher et al., 2011). Mechanistically, using in vitro analyses of rat 
mesenteric arteries, we demonstrated that NDBP acts via the NO/sGC/ 
cGMP pathway and directly activates K+ channels in the vascular 
smooth muscle, thus promoting vasodilation (França-Silva et al., 2012). 
Inorganic and organic nitrates have been demonstrated to increase 
vasorelaxation in the aortas of T1DM and T2DM mice, likely by reducing 
the activity of NADPH oxidase (Tian et al., 2020; Schuhmacher et al., 
2011; Xie et al., 2020). As NDBP modulates the activity of NADPH ox-
idase (Porpino et al., 2016), it is possible that the improvement of 
vascular function, observed in our study, could be coupled to the 
dampening of vascular oxidative stress. Our previous studies also 
observed that in mesenteric arteries isolated from rats, NDBP does not 
cause tolerance (Porpino et al., 2016; França-Silva et al., 2014). Addi-
tionally, studies from our group have revealed that NDBP treatment 
lowers blood pressure in mice with angiotensin II-induced hypertension 
(Porpino et al., 2016). Furthermore, França-Silva et al. (França-Silva 
et al., 2012) observed that acute intravenous administration of NDBP 
induced hypotension that was associated with bradycardia in sponta-
neously hypertensive rats. The authors noted that the muscarinic 
blocker atropine abolished bradycardia and attenuated NDBP-induced 
hypotension in SHRs and WKY rats. Similar effects were observed 
when bilateral vagotomy was developed, thus suggesting that NDBP 
Fig. 8. Effect of NDBP (40 mg/kg) on systolic (A) and diastolic (B) blood pressure and mean arterial pressure (C). *p <.05 compared to control and NDBP. Values are 
mean ± SE. 
F. Fernandes-Costa et al.Journal of Functional Foods 104 (2023) 105526
9
may also act by increasing vagal stimulation to the heart. This will 
reduce cardiac output and consequently lead to hypotension (França- 
Silva et al., 2012). Nevertheless, we observed an increase in the ex vivo 
vasorelaxation of smooth muscle cells in the aorta that was not corre-
lated with a decrease in blood pressure in our T1DM mice. However, we 
assessed blood pressure using a non-invasive technique (tail-cuff pleth-
ysmography), and we used the T1DM mouse model. França-Silva et al. 
(França-Silva et al., 2012) recorded intra-aortic blood pressure in hy-
pertensive rats. The treatment time can also influence the results. For 
example, in a previous study using the DOCA-salt hypertensive model, 
oral treatment with NaNO2 did not change the SBP following 2 weeks of 
treatment (Amaral et al., 2015). However, there was a reduction in SBP 
from the third week onward (Amaral et al., 2015). In contrast to our 
findings, Tian et al. (Tian et al., 2020) observed a reduction in SBP after 
7 weeks of treatment in T2DM mice, and Khalifi et al. (Khalifi et al., 
2015) observed a decrease in SBP after 8 weeks of treatment in T2DM 
rats after NaNO3 supplementation. 
Several nutritional supplements containing nitrate have recently 
been introduced into the consumer market, mainly in the sports com-
munity, as a supplement to improve physical performance (Todorovic 
et al., 2021; Jurado-Castro et al., 2022; McMahon et al., 2017). A recent 
study used a nitrate-producing formulation (MagNOVOx™) as a dietary 
supplement for healthy men and observed improvement in cardiore-
spiratory endurance and muscle fitness (Todorovic et al., 2021). 
Furthermore, classical studies from Webb et. (Webb et al., 2008) used a 
nutritional supplement rich in nitrate, beet juice, which observed a 
reduction in blood pressure, inhibition of platelet aggregation, and 
prevention of endothelial dysfunction in healthy volunteers. Another 
study using beetroot juice observed a reduction in diastolic blood 
pressure to a greater degree in older adults, while systolic blood pressure 
was reduced in older and younger adults (Stanaway et al., 2019). The 
natural and organic juice market is booming and selective nutrition for 
health outcomes is not just a trend but is the focal point for consumers 
(Clements et al., 2014). In that regard, our pre-clinical study showed 
that NDBP treatment induces anti-diabetic effects, suggesting a potential 
to this compound to be a nutritional supplementation, inducing health 
benefits for individuals with type 1 diabetes, which has to be com-
plemented by future additional clinical studies. 
5. Conclusion 
Our studies reveal that the newly developed organic nitrate NDBP 
exerts favorable metabolic effects in an STZ model of T1DM and im-
proves the vascular function of the aorta independently of the endo-
thelium. Mechanistically, these cardiometabolic changes were 
associated with the dampening of oxidative stress in the pancreas and 
liver. Our results supported future studies to evaluate an NDBP as a 
nutritional supplementation in addition to the treatment of type 1 dia-
betes individuals. 
6. Funding sources 
VAB received research support from the National Council for the 
Development of Science and Technology (CNPq, 429767/2016–1 and 
304718/2011–4) and the Paraiba State Research Foundation (FAPESQ, 
ID: 007/2019 FAPESQ-PB-MCT/CNPq). 
JCC received research support from CNPq (4086117/2018–7) and 
CAPES/STINT (88881.304749/2018–01). 
MK received research support from the Swedish Research Council 
(2020–01645), the Swedish Heart and Lung Foundation (20180568), 
and Novo Nordisk (2019#0055026). 
CRediT authorship contribution statement 
Francineide Fernandes-Costa: Data curation, Methodology, 
Writing – original draft, Investigation. Rayanelle Tissiane Gomes da 
Silva: Data curation, Methodology, Investigation. Atalia Ferreira de 
Lima Flôr: Data curation, Methodology. Maria Cláudia R. Brandão: 
Methodology. Petrônio F. Athayde-Filho: Methodology. Maria do 
Socorro França-Falcão: Methodology, Conceptualization, Supervision. 
Valdir de Andrade Braga: Writing – review & editing, Supervision, 
Resources. Gustavo Jorge dos Santos: Writing – review & editing, 
Conceptualization, Supervision. Mattias Carlstrom: Writing – review & 
editing, Conceptualization, Supervision, Resources. Josiane de Campos 
Cruz: Data curation, Conceptualization, Writing – original draft, Su-
pervision, Resources. 
Declaration of Competing Interest 
The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper. 
Data availability 
No data was used for the research described in the article. 
Appendix A. Supplementary data 
Supplementary data to this article can be found online at https://doi. 
org/10.1016/j.jff.2023.105526. 
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	The organic nitrate NDBP promotes cardiometabolic protection in type 1 diabetic mice
	1 Introduction
	2 Materials and methods
	2.1 Animals
	2.2 Diabetes induction
	2.3 Synthesis and preparation of the 2-nitrate-1,3-dibuthoxypropan (NDBP)
	2.4 Experimental design
	2.5 Water and food consumption
	2.6 Urinary volume
	2.7 Intraperitoneal glucose tolerance test
	2.8 Oxidative stress
	2.9 Vascular reactivity
	2.10 Non-invasive evaluation of arterial blood pressure
	2.11 Chemical compounds
	2.12 Statistical analysis
	3 Results
	3.1 Fasting blood glucose
	3.2 Body weight
	3.3 Water and food consumption
	3.4 Urinary volume
	3.5 Glucose tolerance test
	3.6 Oxidative stress
	3.7 Vascular reactivity in thoracic aortas of diabetic mice
	3.7.1 Vascular response to phenylephrine(PHE)
	3.7.2 Vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP)
	3.8 Arterial blood pressure
	4 Discussion
	5 Conclusion
	6 Funding sources
	CRediT authorship contribution statement
	Declaration of Competing Interest
	Data availability
	Appendix A Supplementary data
	References