Long-term inorganic nitrate administration protects against myocardial ischemia-reperfusion injury in female rats.

BACKGROUND: The favorable effects of nitrate against myocardial ischemia-reperfusion injury (MIRI) have primarily focused on male rats and in short term. Here we determine the impact of long-term nitrate intervention on baseline cardiac function and the resistance to MIRI in female rats. METHODS: Female Wistar rats were randomly divided into untreated and nitrate-treated (100 mg/L sodium nitrate in drinking water for 9 months) groups (n = 14/group). At intervention end, levels of serum progesterone, nitric oxide metabolites (NOx), heart NOx concentration, and mRNA expressions of NO synthase isoforms (NOS), i.e., endothelial (eNOS), neuronal (nNOS), and inducible (iNOS), were measured. Isolated hearts were exposed to ischemia, and cardiac function indices (CFI) recorded. When the ischemia-reperfusion (IR) period ended, infarct size, NO metabolites, eNOS, nNOS, and iNOS expression were measured. RESULTS: Nitrate-treated rats had higher serum progesterone (29.8%, P = 0.013), NOx (31.6%, P = 0.035), and higher heart NOx (60.2%, P = 0.067), nitrite (131%, P = 0.018), and eNOS expression (200%, P = 0.005). Nitrate had no significant effects on baseline CFI but it increased recovery of left ventricular developed pressure (LVDP, 19%, P = 0.020), peak rate of positive (+ dp/dt, 16%, P = 0.006) and negative (-dp/dt, 14%, P = 0.014) changes in left ventricular pressure and decreased left ventricular end-diastolic pressure (LVEDP, 17%, P < 0.001) and infarct size (34%, P < 0.001). After the IR, the two groups had significantly different heart nitrite, nitrate, NOx, and eNOS and iNOS mRNA expressions. CONCLUSIONS: Long-term nitrate intervention increased the resistance to MIRI in female rats; this was associated with increased heart eNOS expression and circulating progesterone before ischemia and blunting ischemia-induced increased iNOS and decreased eNOS after MIRI.

Hydrogen sulfide potentiates the protective effects of nitrite against myocardial ischemia-reperfusion injury in type 2 diabetic rats.

Decreased heart levels of nitric oxide (NO) and hydrogen sulfide (H2S) in type 2 diabetes (T2D) are associated with a higher risk of mortality following ischemia-reperfusion (IR) injury. This study aimed to determine the effects of co-administration of sodium nitrite and sodium hydrosulfide (NaSH) on IR injury in the isolated heart from rats with T2D. Two-month-old male rats were divided into 5 groups (n = 7/group): Control, T2D, T2D + nitrite, T2D + NaSH, and T2D + nitrite + NaSH. T2D was induced using a high-fat diet and a single low dose streptozotocin (30 mg/kg) in intraperitoneal injection. Nitrite (50 mg/L in drinking water) and NaSH (0.28 mg/kg, daily intraperitoneal injection) were administrated for 9 weeks. At the end of the study, hemodynamic parameters were recorded, and infarct size and mRNA expression of H2S- and NO-producing enzymes were measured in the isolated hearts. Nitrite administration to rats with T2D improved recovery of left ventricular developed pressure (LVDP) and the peak rates of positive and negative changes in LV pressure (±dp/dt) by 30%, 17%, and 7.9%, respectively, and decreased infarct size by 18.4%. Co-administration of nitrite and NaSH resulted in further improve in recovery of LVDP, +dp/dt, and -dp/dt by 8.3% (P = 0.0478), 8.4% (P = 0.0085), and 9.0% (P = 0.0004), respectively, and also further decrease in infarct size by 24% (P = 0.0473). Nitrite treatment decreased inducible and neuronal NO synthases (iNOS, 0.4-fold; nNOS, 0.4-fold) and cystathionine ß-synthase (CBS, 0.1-fold) expression in the isolated heart from rats with T2D. Co-administration of nitrite and NaSH further increased cystathionine γ-lyase (CSE, 2.8-fold) and endothelial NOS (eNOS, 2.0-fold) expression and further decreased iNOS (0.4-fold) expression. In conclusion, NaSH at a low dose potentiates the favorable effects of inorganic nitrite against myocardial IR injury in a rat model of T2D. These anti-ischemic effects, following co-administration of nitrite and NaSH, were associated with higher CSE-derived H2S and eNOS-derived NO as well as lower iNOS-derived NO in the diabetic hearts.

Quantitative aspects of nitric oxide production in the heart.

Nitric oxide (NO) has essential roles in heart physiology, including the regulation of myocardial contractility and coronary blood flow, and in heart pathophysiology, particularly in the ischemic heart. NO is produced by both NO synthase (NOS)-dependent and NOS-independent pathways in the heart. This review summarizes quantitative aspects of NO production in the heart; the contribution of cardiomyocytes, endothelial cells (ECs), red blood cells (RBCs), and neurons are also discussed. Based on the available data, under normal conditions, the human heart produces about 50-70 µmol NO per day, primarily attributed to eNOS activity; ECs produce the highest amount of NO compared to other cell types in the heart. On the other hand, during ischemic conditions, NOS-independent NO production participates a significant role in the heart NO production that can exceed NOS-dependent NO generation. These data are relevant as most cardiovascular disorders are associated with NO dysfunction, and increasing NO bioavailability and signaling is a potential therapeutic approach for cardiovascular diseases.

Association between serum hydrogen sulfide concentrations and dysglycemia: a population-based study.

BACKGROUND AND AIM: Hydrogen sulfide (H2S), a signaling gasotransmitter, is involved in carbohydrate metabolism. Here, we aimed to assess the potential association between serum H2S and dysglycemia in the framework of a population-based study. METHODS: Adults men and women with completed data (n = 798), who participated in the Tehran Lipid and Glucose Study (2014-2017) were included in the study. Medians of fasting serum H2S concentration were compared across the glycemic status of the participants, defined as type 2 diabetes mellitus (T2DM), isolated impaired fasting glucose (IIFG), isolated impaired glucose tolerance (IIGT), combined IFG-IGT, and normal glycemia [i.e., those with both normal fasting glucose (NFG) and normal glucose tolerance (NGT)]. Multinomial logistic regression was used to assess potential associations between serum H2S and the defined glycemic status. RESULTS: Mean age of the participants was 45.1 ± 14.0 y, and 48.1% were men. Prevalence of T2DM, IIFG, IIGT, and combined IFG-IGT was 13.9, 9.1, 8.1, and 4.8% respectively. No significant difference was observed in serum H2S concentrations between the groups. Lower serum H2S (< 39.6 µmol/L) was associated with an increased chance of having IIGT (OR = 1.96, 95% CI = 1.15-3.34) in the adjusted model. CONCLUSION: Reduced serum H2S level may be associated with impaired glucose tolerance.

Chronic nitrate administration increases the expression the genes involved in the browning of white adipose tissue in female rats.

Nitrate, a nitric oxide (NO) donor, has antiobesity effect in female rats. This study hypothesized that the antiobesity effect of nitrate in female rats is due to the browning of white adipose tissue (WAT). Female Wistar rats (aged 8 months) were divided into two groups (n = 10/group): the control group received tap water and the nitrate group received water containing 100 mg/L of sodium nitrate for 9 months. At months 0, 3, 6, and 9, obesity indices were measured. At month 9, gonadal adipose tissue was used to measure messenger RNA (mRNA) and protein levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), PPAR-γ coactivator 1-α (PGC1-α), uncoupling protein 1 (UCP1), and adipocyte density and area. After the 9-month intervention, nitrate-treated rats had lower body weight, body mass index, thoracic circumference, and abdominal circumference by 6.4% (p = .012), 9.1% (p = .029), 6.0% (p = .056), and 5.7% (p = .098), respectively. In addition, nitrate-treated rats had higher PPAR-γ (mRNA: 1.78-fold, p = .016 and protein: 19%, p = .076), PGC1-α (mRNA: 1.69-fold, p = .012 and protein: 68%, p = .001), and UCP1 (mRNA: 2.50-fold, p = .001 and protein: 81%, p = .001) in gonadal adipose tissue. Nitrate also reduced adipocyte area by 35% (p = .054) and increased adipocyte density by 31% (p = .086). In conclusion, antiobesity effect of nitrate in female rats is associated with increased browning of gonadal adipose tissue as indicated by higher expression of PPAR-γ, PGC1-α, and UCP1 and reduced adipocyte area and increased adipocyte density.

Effect of inorganic nitrate on metabolic parameters in patients with type 2 diabetes: A 24-week randomized double-blind placebo-controlled clinical trial.

AIM: In this randomized placebo-controlled clinical trial, effect of oral inorganic nitrate (NO3-) on metabolic parameters was assessed in patients with type 2 diabetes mellitus (T2DM). METHODS: Seventy-four eligible patients with T2DM were randomly assigned to NO3--rich beetroot powder (5 g/d contains ~250 mg NO3-) and placebo groups to complete intervention over a 24-week period. Blood HbA1c, fasting serum glucose, insulin, C-peptide, as well as lipid profile were assessed at baseline and again at weeks 4, 12, and 24; indices of insulin resistance were also calculated. To assess safety of long-term oral NO3-, liver and renal function tests were measured. An intention-to-treat approach was used for data analysis. To compare effect of intervention over time between the groups (time×group), repeated measures generalized estimating equation (GEE) linear regression models were used. RESULTS: Mean age of the participants was 54.0 ± 8.5 (47.9% were male) and mean duration of diabetes was 8.5 ± 6.1 years. A total of 64 patients (n = 35 in beetroot group and n = 29 in placebo group) completed at least two visits and were included in the analyses. No significant difference was observed between the groups for glycemic and lipid parameters over time. Liver and renal function tests, as safety outcome measures, showed no undesirable changes during the study follow-up. CONCLUSION: Supplementation with inorganic NO3- had no effect on metabolic parameters in patients with T2DM.

Acidified Nitrite Accelerates Wound Healing in Type 2 Diabetic Male Rats: A Histological and Stereological Evaluation.

Impaired skin nitric oxide production contributes to delayed wound healing in type 2 diabetes (T2D). This study aims to determine improved wound healing mechanisms by acidified nitrite (AN) in rats with T2D. Wistar rats were assigned to four subgroups: Untreated control, AN-treated control, untreated diabetes, and AN-treated diabetes. AN was applied daily from day 3 to day 28 after wounding. On days 3, 7, 14, 21, and 28, the wound levels of vascular endothelial growth factor (VEGF) were measured, and histological and stereological evaluations were performed. AN in diabetic rats increased the numerical density of basal cells (1070 ± 15.2 vs. 936.6 ± 37.5/mm3) and epidermal thickness (58.5 ± 3.5 vs. 44.3 ± 3.4 µm) (all p < 0.05); The dermis total volume and numerical density of fibroblasts at days 14, 21, and 28 were also higher (all p < 0.05). The VEGF levels were increased in the treated diabetic wounds at days 7 and 14, as was the total volume of fibrous tissue and hydroxyproline content at days 14 and 21 (all p < 0.05). AN improved diabetic wound healing by accelerating the dermis reconstruction, neovascularization, and collagen deposition.

Acidified nitrite improves wound healing in type 2 diabetic rats: Role of oxidative stress and inflammation.

PURPOSE: Decreased nitric oxide bioavailability in skin contributes to impaired wound healing in type 2 diabetes (T2D). This study aims at determining effects of acidified nitrite on wound closure as well as inflammatory and antioxidants markers in wound tissue of rats with T2D. MAIN METHODS: Skin wound was made on the back of rats 28 days after the induction of T2D (high-fat diet/low-dose of streptozotocin). Control and diabetic rats were subdivided into two subgroups: Untreated control (C), acidified nitrite-treated control (CN), untreated diabetes (D), and acidified nitrite-treated diabetes (DN). Acidified nitrite was applied once daily from day 3 to day 28 and the wounds were photographed for macroscopic changes. On days 3, 7, 14, 21, and 28 after wounding, wound levels of inflammatory and antioxidant markers were measured. RESULTS: Half closure time (CT50%) was significantly lower in acidified nitrite-treated diabetic rats compared to untreated ones (5.1 vs. 8.0 days, P < 0.001). Inflammatory response was delayed in diabetic rats and persistent inflammatory response was observed at day 14 after wounding. Acidified nitrite application restored the inflammatory response and antioxidant levels to control values. CONCLUSIONS: Acidified nitrite accelerated wound healing in rats with T2D by restoring delayed inflammatory response and augmentation of antioxidant defense.

Dose-Dependent Effects of Long-Term Administration of Hydrogen Sulfide on Myocardial Ischemia-Reperfusion Injury in Male Wistar Rats: Modulation of RKIP, NF-κB, and Oxidative Stress.

Decreased circulating levels of hydrogen sulfide (H2S) are associated with higher mortality following myocardial ischemia. This study aimed at determining the long-term dose-dependent effects of sodium hydrosulfide (NaSH) administration on myocardial ischemia-reperfusion (IR) injury. Male rats were divided into control and NaSH groups that were treated for 9 weeks with daily intraperitoneal injections of normal saline or NaSH (0.28, 0.56, 1.6, 2.8, and 5.6 mg/kg), respectively. At the end of the study, hearts from all rats were isolated and hemodynamic parameters were recorded during baseline and following IR. In isolated hearts, infarct size, oxidative stress indices as well as mRNA expression of H2S-, nitric oxide (NO)-producing enzymes, and inflammatory markers were measured. In heart tissue following IR, low doses of NaSH (0.28 and 0.56 mg/kg) had no effect, whereas an intermediate dose (1.6 mg/kg), improved recovery of hemodynamic parameters, decreased infarct size, and decreased oxidative stress. It also increased expression of cystathionine γ-lyase (CSE), Raf kinase inhibitor protein (RKIP), endothelial NO synthase (eNOS), and neuronal NOS (nNOS), as well as decreased expression of inducible NOS (iNOS) and nuclear factor kappa-B (NF-κB). At the high dose of 5.6 mg/kg, NaSH administration was associated with worse recovery of hemodynamic parameters and increased infarct size as well as increased oxidative stress. This dose also decreased expression of CSE, RKIP, and eNOS and increased expression of iNOS and NF-κB. In conclusion, chronic treatment with NaSH has a U-shaped concentration effect on IR injury in heart tissue. An intermediate dose was associated with higher CSE-derived H2S, lower iNOS-derived NO, lower oxidative stress, and inflammation in heart tissue following IR.

Altered gene expression of hydrogen sulfide-producing enzymes in the liver and muscles tissues of hyperthyroid rats.

Thyroid hormones have a role in the regulation of hydrogen sulfide (H2 S) biosynthesis. In this study, we determined the effects of hyperthyroidism on H2 S levels in various tissues and messenger RNA (mRNA) expression of cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in the liver and muscles of the rat. Sixteen male Wistar rats were divided into the hyperthyroid and the control groups. Hyperthyroidism was induced by adding l-thyroxine (12 mg/L) to drinking water for a period of 21 days. H2 S concentrations in serum, liver, aorta, heart, and soleus muscles, as well as mRNA expressions of CBS, CSE, and 3-MST in these tissues were measured at Day 21. Hyperthyroid rats had lower H2 S levels in the serum compared with controls (14.7 ± 1.4 vs. 25.7 ± 1.6 µmol/L, p < 0.001). Compared with controls, hyperthyroid rats had lower levels of H2 S in the aorta (89%), heart (80%), and soleus (103%) muscles, but higher levels in the liver (35%). Hyperthyroidism decreased the ratio of CBS/CSE mRNA expression in the liver and the CSE/CBS mRNA expression in the muscles by decreasing CBS levels in liver (34% cf. controls) and CSE levels in the aorta, heart, and soleus muscles (respectively, 51%, 7%, and 52% cf.). In addition, hyperthyroidism decreased the mRNA expression of 3-MST in the liver (51%) and aorta (33%), and increased it in the heart (300%) and soleus muscle (182%). In conclusion, hyperthyroidism increased H2 S levels in the liver and decreased it in muscles; these effects are at least in part due to increases and decreases in expression of CSE in the liver and muscles, respectively. These data indicate an association between thyroid hormone status and gene expression of the H2 S-producing enzymes in the rat.

Hydrogen sulfide potentiates the favorable metabolic effects of inorganic nitrite in type 2 diabetic rats.

OBJECTIVE: Decreased nitric oxide (NO) bioavailability and hydrogen sulfide (H2S) deficiency have been linked with the pathophysiology of type 2 diabetes (T2D). Restoration of NO levels by nitrite have been associated with favorable metabolic effects in T2D. Moreover, H2S can potentiate the effects of NO in the cardiovascular system. The aim of this study was to determine the effects of long-term co-administration of sodium nitrite and sodium hydrosulfide (NaSH) on carbohydrate metabolism in type 2 diabetic rats. METHODS: T2D was induced using chronic high fat diet (HFD) feeding combined with low dose streptozotocin (STZ) regimen. Rats were divided into 5 groups (N = 10/group): Control, T2D, T2D + nitrite, T2D + NaSH, and T2D + nitrite + NaSH. Nitrite (50 mg/L in drinking water) and NaSH (0.28 mg/kg, daily i. p. injection) were administered for 9 weeks. Fasting serum glucose, insulin, lipid profile, liver function tests, and oxidative stress indices were measured. Intraperitoneal glucose tolerance test (GTT) was performed at the end of the eighth week, and three days later, intraperitoneal pyruvate tolerance test (PTT) was done. Protein levels and mRNA expression of glucose transporter type 4 (GLUT4) in soleus muscle and epididymal adipose tissue as well as mRNA expression of H2S-producing enzymes in the liver, soleus muscle, and epididymal adipose tissue were measured at the end of the study. RESULTS: Compared to the controls, HFD and STZ treated rats developed metabolic dysfunction. Nitrite treatment improved carbohydrate metabolism, liver function, and oxidative stress indices whereas NaSH treatment per se had no significant effects. However, co-administration of NaSH and nitrite resulted in further improvement in serum insulin level, GTT, PTT, liver function, oxidative stress, protein level and mRNA expression of GLUT4, as well as mRNA expression of H2S-producing enzymes in diabetic rats. CONCLUSION: Low dose of NaSH per se had no effect on carbohydrate metabolism while it potentiated the favorable metabolic effects of inorganic nitrite in type 2 diabetic rats. These favorable effects were associated with decreased oxidative stress and increased GLUT4 expression in insulin-sensitive tissues as well as improvement of liver function.

Effect of long-term sodium nitrate administration on diabetes-induced anemia and glucose homeostasis in obese type 2 diabetic male rats.

PURPOSE: Anemia is common in patients with type 2 diabetes. This aims at determining long-term effects of nitrate administration on diabetes-induced anemia in obese type 2 diabetic rats. METHODS: Male Wistar rats were divided into 4 groups: Control, control + nitrate, diabetes, and diabetes + nitrate. Type 2 diabetes was induced using high-fat diet followed by injection of streptozotocin (30 mg/kg). Sodium nitrate (100 mg/L in drinking water) was administered for six months. After overnight fasting, levels of glucose and erythropoietin (EPO) and complete blood cell count (CBC) were measured at month 0, month 3, and month 6. At month 6, serum iron, and testosterone as well as EPO protein levels and hypoxia-inducible factor-1 (HIF-1) mRNA levels in kidney and liver were measured. RESULTS: Nitrate administration decreased serum glucose in diabetic rats by 10% and 15% at months 3 and 6, respectively. Nitrate restored decreased red blood cells count, hemoglobin concentration, and hematocrit to control levels in diabetic rats; in addition, nitrate restored decreased serum, kidney, and liver EPO levels to near normal values. Nitrate also increased HIF-1 mRNA levels in both kidney and liver of diabetic rats. Diabetic rats had lower serum testosterone (37%) and iron (20%) and nitrate restored these parameters to near normal values. CONCLUSION: Long-term and low dose of nitrate had beneficial effects against anemia in obese type 2 diabetic rats; these effects were associated with increased EPO and HIF-1 levels in kidney and liver as well as increased circulating EPO, testosterone, and iron.

Effects of Hydrogen Sulfide on Carbohydrate Metabolism in Obese Type 2 Diabetic Rats.

Hydrogen sulfide (H2S) is involved in the pathophysiology of type 2 diabetes. Inhibition and stimulation of H2S synthesis has been suggested to be a potential therapeutic approach for type 2 diabetes. The aim of this study was therefore to determine the effects of long-term sodium hydrosulfide (NaSH) administration as a H2S releasing agent on carbohydrate metabolism in type 2 diabetic rats. Type 2 diabetes was established using high fat-low dose streptozotocin. Rats were treated for 9 weeks with intraperitoneal injections of NaSH (0.28, 0.56, 1.6, 2.8, and 5.6 mg/kg). Serum glucose was measured weekly for one month and then at the end of the study. Serum insulin was measured before and after the treatment. At the end of the study, glucose tolerance, pyruvate tolerance and insulin secretion were determined and blood pressure was measured. In diabetic rats NaSH at 1.6⁻5.6 mg/kg increased serum glucose (11%, 28%, and 51%, respectively) and decreased serum insulin, glucose tolerance, pyruvate tolerance and in vivo insulin secretion. In controls, NaSH only at 5.6 mg/kg increased serum glucose and decreased glucose tolerance, pyruvate tolerance and insulin secretion. Chronic administration of NaSH in particular at high doses impaired carbohydrate metabolism in type 2 diabetic rats.

Effects of long-term nitrate supplementation on carbohydrate metabolism, lipid profiles, oxidative stress, and inflammation in male obese type 2 diabetic rats.

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-1ß 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-1ß; 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.

Role of inducible nitric oxide synthase in myocardial ischemia-reperfusion injury in sleep-deprived rats.

INTRODUCTION: REM sleep deprivation (SD) decreases tolerance of the rat heart to ischemia-reperfusion (IR) injury; the underlying mechanisms, however, are unknown. This study aimed at determining whether changes in iNOS, Bax, and Bcl-2 gene expression are involved in this detrimental effect. METHOD: SD was induced by flowerpot technique for a period of 4 days. This method is simple and able to induce sleep fragmentation which occurs as one of the sleep disorder symptoms in clinical conditions. The hearts of control and SD rats were perfused in Langendorff apparatus and subjected to 30 min ischemia, followed by 90 min reperfusion. The hemodynamic parameters (left ventricular developed pressure (LVDP), and ± dp/dt), NOx (nitrite + nitrate) level, infarct size, and mRNA expression of iNOS, Bax, and Bcl-2 were measured after IR. RESULTS: SD rats had lower recovery of post-ischemic LVDP (32.8 ± 2.5 vs. 51.5 ± 2.1 mmHg; P < 0.05), + dp/dt (1555 ± 66 vs. 1119.5 ± 87 mmHg/s; P < 0.05) and - dp/dt (1437 ± 65 vs. 888 ± 162 mmHg/s; P < 0.05). SD rats also had higher NOx levels (41.4 ± 3.1 vs. 22.4 ± 3.6 µmol/L; P < 0.05) and infarct size (64.3 ± 2.3 vs. 38.3 ± 1.6%; P < 0.05) after IR, which along with LVDP, ± dp/dt restored to near normal status in the presence of aminoguanidine, a selective iNOS inhibitor. Following IR, expression of iNOS and Bax increased and Bcl-2 decreased (502, 372, and 54%, respectively) in SD rats; whereas in the presence of aminoguanidine, expression of iNOS and Bax significantly decreased and Bcl-2 increased (165, 168, and 19%, respectively). CONCLUSION: Higher expression of iNOS and subsequent increase in apoptosis in the hearts after IR may contribute to less tolerance to myocardial IR injury in SD rats.

Transient Congenital Hypothyroidism Alters Gene Expression of Glucose Transporters and Impairs Glucose Sensing Apparatus in Young and Aged Offspring Rats.

BACKGROUND/AIMS: Transient congenital hypothyroidism (TCH) could disturb carbohydrate metabolism in adulthood. Aging is associated with increased risk of type 2 diabetes. This study aims to address effects of TCH on mRNA expressions of glucose transporters (GLUTs) and glucokinase (GcK) in islets and insulin target tissues of aged offspring rats. METHODS: The TCH group received water containing 0.025% 6-propyl-2-thiouracil during gestation. Offspring from control and TCH groups (n=6 in each group) were followed until month 19. Gene expressions of GLUTs and GcK were measured at months 3 and 19. RESULTS: Compared to controls, aged TCH rats had higher GLUT4 expression in heart (4.88 fold) and soleus (6.91 fold), while expression was lower in epididymal fat (12%). In TCH rats, GLUT2 and GcK expressions in islets were lower in young (12% and 10%, respectively) and higher in aged (10.85 and 8.42 fold, respectively) rats. In addition, liver GLUT2 and GcK expressions were higher in young (13.11 and 21.15 fold, respectively) and lower in aged rats (44% and 5%, respectively). CONCLUSION: Thyroid hormone deficiency during fetal period impaired glucose sensing apparatus and changed glucose transporter expression in insulin-sensitive tissues of aged offspring rats. These changes may contribute to impaired carbohydrate metabolism.

Anti-obesity and anti-diabetic effects of nitrate and nitrite.

Prevalence of obesity is increasing worldwide and type 2 diabetes to date is the most devastating complication of obesity. Decreased nitric oxide bioavailability is a feature of obesity and diabetes that links these two pathologies. Nitric oxide is synthesized both by nitric oxide synthase enzymes from l-arginine and nitric oxide synthase-independent from nitrate/nitrite. Nitric oxide production from nitrate/nitrite could potentially be used for nutrition-based therapy in obesity and diabetes. Nitric oxide deficiency also contributes to pathogeneses of cardiovascular disease and hypertension, which are associated with obesity and diabetes. This review summarizes pathways for nitric oxide production and focuses on the anti-diabetic and anti-obesity effects of the nitrate-nitrite-nitric oxide pathway. In addition to increasing nitric oxide production, nitrate and nitrite reduce oxidative stress, increase adipose tissue browning, have favorable effects on nitric oxide synthase expression, and increase insulin secretion, all effects that are potentially promising for management of obesity and diabetes. Based on current data, it could be suggested that amplifying the nitrate-nitrite-nitric oxide pathway is a diet-based strategy for increasing nitric oxide bioavailability and the management of these two interlinked conditions. Adding nitrate/nitrite to drugs that are currently used for managing diabetes (e.g. metformin) and possibly anti-obesity drugs may also enhance their efficacy.

Nitrite increases glucose-stimulated insulin secretion and islet insulin content in obese type 2 diabetic male rats.

PURPOSE: Reduced bioavailability of nitric oxide (NO) is associated with pathogenesis of type 2 diabetes. Nitrite can act as a substrate for generation of systemic NO. The aim of this study was to examine the effects of nitrite administration on glucose-stimulated insulin secretion (GSIS) and islet insulin content in obese type 2 diabetic rats. METHODS: Male rats were divided into 4 groups: Control, control + nitrite, diabetes, and diabetes + nitrite. Sodium nitrite (50 mg/L in drinking water) was administered for 8 weeks. Diabetes was induced using high-fat diet and low-dose of streptozotocine. Serum levels of fasting glucose, insulin, and lipid profile were measured and the insulin resistance/sensitivity indices were calculated every 2 weeks. Glycated hemoglobin (HbA1C) was measured every month. At the end of the study, tissue levels of glucose transporter 4 (GLUT4) protein and serum interleukin-1 beta (IL-1ß) were measured as well as glucose and insulin tolerance test were done. GSIS from isolated pancreatic islets and islet insulin content were also determined. RESULTS: Nitrite administration significantly increased insulin secretion in both control and diabetic rats in presence of 16.7 mM glucose. Nitrite also significantly increased islet insulin content by 27% and 39% in both control and diabetic rats, respectively. Nitrite decreased elevated serum IL-1ß in diabetic rats (4.0 ± 0.2 vs. 2.9 ± 0.2 pg/mL, P = 0.001). In diabetic rats, nitrite also significantly increased tissue levels of GLUT4 by 22% and 26% in soleus muscle and epididymal adipose tissue, respectively. In addition, nitrite significantly improved glucose and insulin tolerance, insulin sensitivity, lipid profile, and decreased fasting glucose and insulin, but had no effect on HbA1C. CONCLUSIONS: Long-term nitrite administration increased both insulin secretion and insulin content in obese type 2 diabetic rats. In addition, nitrite therapy had favorable effects on glucose tolerance, insulin resistance, inflammation, and dyslipidemia in type 2 diabetic rats.

Effect of fetal hypothyroidism on MyomiR network and its target gene expression profiles in heart of offspring rats.

Thyroid hormone deficiency during fetal life (fetal hypothyroidism) causes intrauterine growth restriction (IUGR). Fetal hypothyroidism (FH) could attenuate normal cardiac functions in the later life of the offspring rats. The aim of this study was to evaluate the contribution of myomiR network and its target gene expression in cardiac dysfunction in fetal hypothyroid rats. Six Pregnant female rats were divided into two groups: Control consumed tap water, and the hypothyroid group received water containing 0.025% 6-propyl-2-thiouracil during gestation. Hearts from male offspring rats in adulthood (month 3) were tested with Langendorff apparatus for measuring hemodynamic parameters. Expressions of miR-208a, -208b, and -499 and its target genes including thyroid hormone receptor 1 (Thrap1), sex-determining region Y-box 6 (Sox6), and purine-rich element-binding protein ß (Purß) were measured by qPCR. FH rats had lower LVDP (%20), +dp/dt (%26), -dp/dt (%20), and heart rate (%21) than controls. FH rats at month 3 had a higher expression of ß-MHC (190%), Myh7b (298%), and lower expression of α-MHC (36%) genes in comparison with controls. FH rats at month 3 had a higher expression of miR-499 (520%) and miR-208b (439%) and had lower expression of miR-208a (74%), Thrap1 (47%), Sox6 (49%), and Purß (45%) compared with controls. Our results showed that thyroid hormone deficiency during fetal life changes the pattern of gene expression of myomiR network and its target genes in fetal heart, which, in turn, resulted in increased ß-MHC expression and associated cardiac dysfunction in adulthood.

Effect of fetal hypothyroidism on tolerance to ischemia-reperfusion injury in aged male rats: Role of nitric oxide.

Aging is associated with increased prevalence of cardiovascular disease. Thyroid hormone deficiency during fetal life decreases myocardial tolerance to ischemia-reperfusion (IR) injury in later life. The long-term effects of fetal hypothyroidism (FH) on response to IR injury in aged rats have not been well documented. The aim of this study was therefore to compare the effect of FH on tolerance to IR injury in young and aged male rats and to determine contribution of iNOS (inducible nitric oxide synthase), Bax, and Bcl-2. Pregnant female rats were divided into two groups: The FH group received water containing 0.025% 6-propyl-2-thiouracil during gestation and the controls consumed tap water. Isolated perfused hearts from young (3 months) and aged (12 months) rats were subjected to IR. Hemodynamic parameters, infarct size, and heart NOx (nitrite+nitrate) levels were measured; in addition, mRNA expression of iNOS, Bax, and Bcl-2 and their protein levels in heart were measured. Recovery of post-ischemic LVDP and ±dp/dt were lower and infarct sizes were higher than controls in aged FH rats (68.38 ± 6.7% vs. 50.5 ± 1.7%; P < 0.05). Aged FH rats had higher heart NOx values than controls (74.3 ± 2.6 vs. 47.6 ± 2.5 µmol/L, P < 0.05). After IR, in FH rats, mRNA expression of iNOS and Bax were higher and Bcl-2 was lower in both the young (350 and 240% for iNOS and Bax, respectively and 51% for Bcl-2) and aged rats (504 and 567% for iNOS and Bax, respectively and 67% for Bcl-2). Compared to controls, in FH rats protein levels of iNOS (37% for young and 45% for aged rats) and Bax (94% for young and 118% for aged rats) were higher while for Bcl-2 (36% for young and 62% for aged rats) were lower. After IR, in FH rats, aminoguanidine, a selective iNOS inhibitor, decreased mRNA expression of iNOS and Bax and increased expression of Bcl-2 in both young (65% and 58% for iNOS and Bax, respectively and 152% for Bcl-2) and aged rats (76% and 64% for iNOS and Bax, respectively and 222% for Bcl-2). In addition, in the heart of FH rats, aminoguanidine decreased protein levels of iNOS (47% for young and 60% for aged rats) and Bax (57% for young and 80% for aged rats) and increased protein levels of Bcl-2 (124% for young and 180% for aged rats). In conclusion, thyroid hormone deficiency during fetal life decreases tolerance to IR injury in aged rats; this effect is at least in part, due to increased expression of iNOS and Bax-to-Bcl-2 ratio in the heart and is restored by iNOS inhibition.