Efeitos metabólicos e vasculares do mononitrato de isossorbida e do nitrato de sódio em camundongos diabéticos tipo 1

Abstract

Diabetes is associated with several cardiometabolic comorbidities that may be associated with endothelial dysfunction and reduced bioavailability of nitric oxide (NO). Furthermore, there is evidence that dietary supplementation with nitrates, NO donors, promotes antidiabetogenic effects. In previous studies, it was observed that inorganic sodium nitrate (NaNO3) promoted a reduction in blood glucose, an increase in pancreatic blood flow and a reduction in oxidative stress in diabetic animals. However, the antidiabetic effects of organic nitrates are poorly known. Among the commercially available organic nitrates, isosorbide mononitrate (ISMN) stands out as it has greater systemic bioavailability compared to others, such as nitroglycerin and isosorbide dinitrate. In this study, our objective was to evaluate the metabolic and vascular effects of ISMN and compare them to the effects of NaNO3 in mice with type 1 diabetes mellitus (T1DM). For this purpose, T1DM was induced through the injection of streptozotocin (STZ i.p. 50 mg/Kg, 5 days) in male C57BL6 mice and confirmed by fasting blood glucose > 250 mg/dL. The animals received ISMN (10 mg/Kg, p.o., gavage), NaNO3 (85 mg/L, p.o., diluted in drinking water) or water without supplementation for 14 days. Then, blood and urinary glucose, food and water intake, urinary volume, serum insulin concentration, serum and urinary nitrite concentration, insulin sensitivity and glucose tolerance, non-invasive blood pressure were evaluated by tail cuff plethysmography, aortic vascular reactivity in response to phenylephrine (PHE), sodium nitroprusside (SNP) and acetylcholine (ACh). Finally, the bioavailability of reactive oxygen species (ROS) was evaluated by fluorescence assay with dihydroethidium (DHE, 5 µM) and NO with 4-Amino-5-Methylamino-2',7'-Difluorofluorescein diacetate (DAFFM, 10 µM) in thoracic aortas. The data were analyzed using the Two-way test, Graphpad Prism software (v. 6.0), p < 0.05. The protocols were approved by CEUA/UFPB under nº 7236120121 and nº 9387160419. The results showed that ISMN (276.5 ± 33.10 vs 498.43±21.18 mg/dL, n=7) and NaNO3 treatments (372.43 ± 24.30 vs 498.43±21.18 mg/dL, n=7) decreased blood glucose in T1DM mice, but this reduction was more pronounced after ISMN treatment. Both nitrates, ISMN (29.18 ± 13.72 vs 91.64 ± 5.52 mmol/dL, n=6) and NaNO3 (42.37 ± 12.45 vs 91.64 ± 5.52 mmol/dL, n=5) similarly reduced urinary glucose in T1DM mice. Furthermore, diabetes/STZ increased water intake (11.35 ± 0.87 vs 4.39 ± 0.49 ml/10g/24h, n=7) which was reduced after both ISMN (7.48 ± 1.22 ml/10g/24h, n=7) or NaNO3 treatments (6.75 ± 1.64 ml/10g/24h, n=7). Diabetes/STZ also increased food intake (3.66 ± 0.16 vs 1.55 ± 0.12 g/10g/24h, n=7) which was reduced after both ISMN (2.74 ± 0.18 g/10g/24h, 17 n=7) or NaNO3 treatments (2.63 ± 0.35 g/10g/24h, n=7). Diabetes/STZ increased urinary volume (0.54 ± 0.06 vs 0.15 ± 0.02 mL/10g/4h, n=7) which was reduced after both ISMN (0.30 ± 0. 03 mL/10g/4h, n=6) or NaNO3 treatments (0.36 ± 0.06 mL/10g/4h, n=7). Diabetes/STZ increased glucose intolerance (p < 0.0001) which was similarly reduced after both ISMN (p < 0.0001) or NaNO3 treatments (p = 0.02). Diabetes/STZ did not develop insulin resistance (p = 0.74), but both treatments with ISMN (p = 0.01) or NaNO3 (p = 0.03) increased insulin sensitivity in T1DM mice. Diabetes/STZ reduced serum nitrite concentration (37.04 ± 7.84 vs 68.13 ± 6.87 mM, n=5) which was increased after both ISMN (87.64 ± 8.98 mM , n=7) or NaNO3 treatments (81.79 ± 7.84 mM, n=7). Diabetes/STZ also reduced urinary nitrite concentration (25.39 ± 7.79 vs 68.70 ± 9.29 mM, n=5) which was increased after both ISMN (68.21 ± 6.18 mM, n=7) or NaNO3 treatments (57.98 ± 12.30 mM, n=7). Diabetes/STZ reduced serum insulin concentration (65.04 ± 3.14 vs 99.90 ± 7.34 µIU/mL, n=5) which was increased after both ISMN (82.47 ± 3. 80 µIU/mL, n=5) or NaNO3 treatments (89.63 ± 8.46 µIU/mL, n=6). ISMN or NaNO3 treatments also reduced vasoconstriction in response to PHE (p = 0.003 and 0.002 respectively) and increased vasorelaxation in response to SNP (p = 0.007 and 0.0003 respectively), but not to ACh (p = 0.69 and 0.48 respectively) in aortas from T1DM mice. Furthermore, both treatments with ISMN or NaNO3 reduced the fluorescence intensity at DHE (ROS) (p < 0.0001 and p = 0.001 respectively) and increased the fluorescence at DAF-FM (NO) (p = 0.012 and 0.016 respectively) in aortas from T1DM mice. Finally, diabetes/STZ increased mean arterial pressure (114.5 ± 0.90 vs 97.91 ± 1.09 mmHg, n=7) which was reduced after both ISMN (106.7 ± 1. 07 mmHg, n=6) or NaNO3 treatments (108.8 ± 1.10 mmHg, n=7). In conclusion, ISMN exhibited a more pronounced antihyperglycemic effect relative to NaNO3, both conferred similar metabolic and vascular benefits, promoting hypotensive and antioxidant effects in T1DM mice.