Chronic Inorganic Nitrate Administration Increases the Expression of Genes Involved in the Browning of Gonadal Adipose Tissue in Ovariectomized Rats.

BACKGROUND AND OBJECTIVE: Nitrate, as nitric oxide (NO) donor, has been suggested as a nutrition-based treatment for decreasing the risk of menopause-related obesity. This study aimed to specify the effects of chronic inorganic nitrate administration on uncoupling protein-1 (UCP-1), peroxisome proliferator-activated-receptor-947; (PPAR-947;) coactivator-1945; (PGC-1945;), and PPAR-947; expression in gonadal adipose tissue (GAT) of ovariectomized (OVX) rats. METHODS: Female rats were assigned to 3 groups: Control, OVX, and OVX+nitrate (n=7/group), which consumed water containing inorganic nitrate (100 mg/L) for 9 months. At month 9, GAT was used for the measurement of NO metabolites (NOx), mRNA levels of NO synthases (endothelial (eNOS), inducible (iNOS), neuronal (nNOS)), and mRNA and protein levels of UCP-1, PGC-1945;, and PPAR-947;. RESULTS: OVX rats had lower NOx concentration (45%) and eNOS (38%) and nNOS (30%) expression in GAT that was restored to normal values following nitrate administration. OVX rats had significantly lower mRNA and protein levels of UCP-1 (83% and 30%), PGC-1945; (65% and 39%), and PPAR-947; (66% and 34.5%) in GAT. Chronic inorganic nitrate administration in OVXrats increased mRNA and protein levels of UCP-1 (128% and 34%), PGC-1945; (115% and 43%), and PPAR-947; (236% and 38%), respectively. CONCLUSION: In OVX rats, chronic nitrate administration increased gene and protein levels of UCP-1, PGC-1945;, and PPAR-947; in GAT, indicating the anti-obesity effects of nitrate are partially mediated by the white adipose tissue (WAT) browning. Moreover, the stimulatory effect of inorganic nitrate on the WAT browning in OVX rats was associated with blunting the OVXinduced NO deficiency in GAT.

Effect of long-term inorganic nitrate administration on myocardial ischemia-reperfusion injury in ovariectomized rats.

Introduction: Menopause is associated with reduced nitric oxide (NO) bioavailability and lower tolerance against myocardial ischemia-reperfusion (IR) injury. This study investigated whether long-term nitrate administration provides resistance against myocardial IR injury in ovariectomized (OVX) rats. Method: After ovariectomy, female rats were assigned to the OVX and the OVX + nitrate groups (n = 14/group); the latter group consumed nitrate (100 mg/L) for 9 months. At month 9, each group was divided into two subgroups (n = 7/subgroup), of which one subgroup was exposed to myocardial IR (IR+ hearts) and the other was not exposed (IR- hearts). The hearts of rats were isolated, and NO metabolite (NOx), oxidative stress indices, and mRNA expressions of endothelial (eNOS), inducible (iNOS), and neuronal (nNOS) NO synthases, as well as markers of apoptosis, were measured in the IR- and IR+ hearts. In the IR+ hearts, cardiac function indices (CFI) and the infarct size were also measured. Results: Nitrate increased catalase activity (97%) and eNOS expression (2.94-fold) in the IR- hearts. In the IR+ hearts, nitrate reduced left ventricular (LV) end-diastolic pressure (11.6%) and infarct size (26.2%) and increased recovery of LV developed pressure (44.0%) and peak rate of positive (28.9%) and negative (15.4%) changes in LV pressure. In addition, in the IR+ hearts, nitrate increased eNOS and B-cell lymphoma-2 (Bcl-2) as well as decreased iNOS, Bcl-2 associated X protein (Bax), caspase-3, caspase-8, caspase-9, and tumor necrosis factor-α (TNF-α) expression. Nitrate increased total antioxidant capacity (TAC) and catalase (CAT) activity and decreased malondialdehyde (MDA) levels at month nine in serum and IR+ hearts. Conclusion: The favorable effects of nitrate against IR injury were associated with higher eNOS and Bcl-2 expression, CAT activity, TAC, and lower iNOS, Bax, caspase-3, caspase-8, caspase-9 and TNF-α expression, and MDA in the heart tissue. Nitrate preconditioning alleviated IR-induced myocardial injury in OVX rats; this effect was associated with eNOS upregulation before IR and the blunting of OVX-induced eNOS downregulation, iNOS upregulation, apoptosis, and oxidative stress in heart tissue after IR.

Dissolving sodium hydrosulfide in drinking water is not a good source of hydrogen sulfide for animal studies.

Hydrogen sulfide (H2S) has many physiological and pathological roles in the human body. Sodium hydrosulfide (NaHS) is widely used as a pharmacological tool for assessing H2S effects in biological experiments. Although H2S loss from NaHS solution is a matter of minutes, some animal studies use NaHS in solution as an H2S-donating compound in drinking water. This study addresses whether 30 µM NaHS in drinking water prepared in rat/mouse water bottles remains stable for at least 12-24 h, as presumed by some authors. NaHS solutions (30 µM) were prepared in drinking water and immediately transferred to rat/mice water bottles. Samples were obtained from the tip of water bottles and from inside of the bottles at 0, 1, 2, 3, 4, 5, 6, 12, and 24 h for sulfide measurement using the methylene blue method. Furthermore, NaHS (30 µM) was administered to male and female rats for two weeks, and serum sulfide concentrations were measured every other day in the first week and at the end of the second week. NaHS solution was unstable in the samples obtained from the tip of water bottles; it declined by 72% and 75% after 12 and 24 h, respectively. In the samples obtained from the inside of the water bottles, the decline in the NaHS was not significant until 2 h; however, it decreased by 47% and 72% after 12 and 24 h, respectively. NaHS administration did not affect serum sulfide levels in male and female rats. In conclusion, NaHS solution prepared in drinking water can not be used for H2S donation as the solution is unstable. This route of administration exposes animals to variable and lower-than-expected amounts of NaHS.

Long-term Ovariectomy Reduces Tolerance of Rats to Myocardial Ischemia-reperfusion Injury.

Background: The harmful impact of ovariectomy on myocardial ischemia-reperfusion (M/IR) injury has been established in the short term. Objectives: In this study, we aimed to investigate the long-term effects of ovariectomy on M/IR injury. Methods: Two methods involving dorsolateral skin incisions were used to induce the ovariectomized (OVX) rat model. The rats were divided into 2 groups: Control and OVX (n = 6). At the end of the study, the hearts were isolated and subjected to global ischemia using the Langendorff apparatus. Cardiac function indices (CFIs) were recorded, including left ventricular end-diastolic pressure (LVEDP), peak rates of positive (+dp/dt) and negative (-dp/dt) changes in LV pressure, and LV-developed pressure (LVDP). At the end of the reperfusion period, the hearts were used to measure the size of the infarct, levels of nitric oxide metabolites (NOx), and mRNA expression of NO synthase (NOS) enzymes, including endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). Results: Compared to controls, OVX rats had larger infarct size by 51%, higher LVEDP by 29%, and lower recovery of +dp/dt, -dp/dt, and LVDP by 29%, 22%, and 35%, respectively. Furthermore, in heart tissue, rats that underwent OVX had significantly higher concentrations of nitrate, nitrite, and NOx by 79%, 82%, and 83%, respectively. Additionally, these rats had lower mRNA levels of eNOS by 38% and higher mRNA levels of iNOS by 71%. Conclusions: The long-term deficiency of estrogen increased the expression of iNOS and decreased the expression of eNOS in the heart tissue of OVX rats. Imbalanced NOS expressions were associated with exacerbated responses to M/IR injury in OVX rats.

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.

Protective effects of long-term nitrate administration against ovariectomy-induced kidney dysfunction in rats.

BACKGROUND: Menopause is associated with higher risks of chronic kidney disease. We determined the effect of nitrate on ovariectomy-induced kidney dysfunction METHODS: Control, ovariectomized (OVX), control + nitrate, and OVX + nitrate female Wistar rats (n = 10/group); sodium nitrate (100 mg/L) administered in drinking water for 9 months. Glomerular filtration rate (GFR) and albumin excretion rate (AER) were calculated from serum and urine parameters. At month 9, serum and kidney levels of nitric oxide (NO) metabolites (NOx), oxidative stress indices, and mRNA expression of endothelial NO synthase (eNOS) were measured; with histological analyses of the kidney. RESULTS: Compared to controls, OVX rats had lower GFR (31%, p = 0.0079), higher glomerular tuft volume (30%, p = 0.0402), and Bowman's capsule space (39%, p = 0.0224). OVX rats had lower serum NOx (33%, p = 0.0061) and kidney eNOS mRNA expression (34%, p = 0.0368). Nitrate administration to: (i) control rats increased serum NOx (59%, p < 0.0001), with no effect on other parameters; (ii) OVX rats increased serum (85%, p < 0.0001) and kidney (106%, p = 0.0008) NOx values, and restored kidney eNOS expression to normal value. Nitrate administration to OVX rats increased GFR (36%, p = 0.0361) and restored glomerular tuft volume and Bowman's capsule space to normal values. In OVX rats, it also increased serum catalase (CAT) activity, serum and kidney total antioxidant capacity (TAC), and decreased serum malondialdehyde (MDA). CONCLUSIONS: Low-dose long-term nitrate administration protects against ovariectomy-induced kidney dysfunction in rats. This effect is associated with reducing ovariectomy-induced oxidative stress and restoring eNOS-derived NO deficiency in systemic circulation and the kidney.

Altered sialin mRNA gene expression in type 2 diabetic male Wistar rats: implications for nitric oxide deficiency.

Nitrate therapy has been suggested to boost nitric oxide (NO) levels in type 2 diabetes (T2D); however, little is known about nitrate transport across the membranes. This study aimed to assess changes in the mRNA expression of sialin, as a nitrate transporter, in the main tissues of rats with T2D. Rats were divided into two groups (n = 6/group): Control and T2D. A high-fat diet combined with a low dose of streptozotocin (STZ, 30 mg/kg) was used to induce T2D. At month 6, samples from the main tissues of rats were used to measure the mRNA expression of sialin and levels of NO metabolites. Rats with T2D had lower nitrate levels in the soleus muscle (66%), lung (48%), kidney (43%), aorta (30%), adrenal gland (58%), epididymal adipose tissue (eAT) (61%), and heart (37%) and had lower nitrite levels in the pancreas (47%), kidney (42%), aorta (33%), liver (28%), eAT (34%), and heart (32%). The order of sialin gene expression in control rats was: soleus muscle > kidney > pancreas > lung > liver > adrenal gland > brain > eAT > intestine > stomach > aorta > heart. Compared to controls, rats with T2D had higher sialin mRNA expressions in the stomach (2.1), eAT (2.0), adrenal gland (1.7), liver (8.9), and soleus muscle (3.4), and lower sialin expression in the intestine (0.56), pancreas (0.42), and kidney (0.44), all P values < 0.05. These findings indicate altered sialin mRNA expression in the main tissues of male T2D rats and may have implications for future NO-based treatment of T2D.

Long-term inorganic nitrate administration protects against ovariectomy-induced osteoporosis in rats.

The risk of osteoporotic fractures increases in women after menopause. This study aims at determining the effects of long-term inorganic nitrate administration against ovariectomy-induced osteoporosis in rats. Rats were divided into 4 groups (n=6/group): Control, control+nitrate, ovariectomized (OVX), and OVX+nitrate. Sodium nitrate (100 mg/L in drinking water) was administered for 9 months. Trabecular bone quality in the proximal tibia was measured using a Micro-Computed Tomography (micro-CT) scanner at months 0, 1, 3, and 9. Levels of nitric oxide (NO) metabolites (NOx) and oxidative stress indices, and mRNA expression of endothelial NO synthase (eNOS) were measured at month 9 in the proximal tibia. Compared to controls, OVX rats had lower NOx levels by 47 %, eNOS mRNA expression by 55 %, catalase activity (CAT) by 45 %, total antioxidant capacity (TAC) by 70 %, and higher malondialdehyde (MDA) levels by 327 % in the bone tissue at month 9. OVX rats, compared to controls, had lower bone volume/tissue volume (BV/TV), trabecular number (Tb.N.), and trabecular thickness (Tb.Th.) by 32 %, 58 %, and 17 %, respectively, and higher trabecular separation (Tb.Sp.) by 123 %, at month 9. Nitrate administration to control rats increased TAC by 46 % in the bone tissue at month 9 but did not significantly affect other parameters in serum and bone tissue. Nitrate in OVX rats significantly increased NOx levels by 86 %, eNOS expression by 2.14-fold, CAT activity by 75 %, TAC by 170 %, and decreased MDA levels by 36 % at month 9 in the bone tissue. Nitrate-treated OVX rats at month 9 had higher BV/TV (42 %) and Tb.N. (61 %) and lower Tb.Sp. (15 %). Long-term inorganic nitrate administration at a low dose has protective effects against OVX-induced osteoporosis in rats; this effect is associated with increasing eNOS-derived NO and decreasing oxidative stress in the bone tissue.

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.

Role of nitric oxide in type 1 diabetes-induced osteoporosis.

Type 1 diabetes (T1D)-induced osteoporosis is characterized by decreased bone mineral density, bone quality, rate of bone healing, bone formation, and increased bone resorption. Patients with T1D have a 2-7-fold higher risk of osteoporotic fracture. The mechanisms leading to increased risk of osteoporotic fracture in T1D include insulin deficiency, hyperglycemia, insulin resistance, lower insulin-like growth factor-1, hyperglycemia-induced oxidative stress, and inflammation. In addition, a higher probability of falling, kidney dysfunction, weakened vision, and neuropathy indirectly increase the risk of osteoporotic fracture in T1D patients. Decreased nitric oxide (NO) bioavailability contributes to the pathophysiology of T1D-induced osteoporotic fracture. This review discusses the role of NO in osteoblast-mediated bone formation and osteoclast-mediated bone resorption in T1D. In addition, the mechanisms involved in reduced NO bioavailability and activity in type 1 diabetic bones as well as NO-based therapy for T1D-induced osteoporosis are summarized. Available data indicates that lower NO bioavailability in diabetic bones is due to disruption of phosphatidylinositol 3­kinase/protein kinase B/endothelial NO synthases and NO/cyclic guanosine monophosphate/protein kinase G signaling pathways. Thus, NO bioavailability may be boosted directly or indirectly by NO donors. As NO donors with NO-like effects in the bone, inorganic nitrate and nitrite can potentially be used as novel therapeutic agents for T1D-induced osteoporosis. Inorganic nitrites and nitrates can decrease the risk for osteoporotic fracture probably directly by decreasing osteoclast activity, decreasing fat accumulation in the marrow cavity, increasing osteoblast activity, and increasing bone perfusion or indirectly, by improving hyperglycemia, insulin resistance, and reducing body weight.

Long Term Sodium Nitrate Administration Positively Impacts Metabolic and Obesity Indices in Ovariectomized Rats.

BACKGROUND: In postmenopausal women, nitric oxide (NO) deficiency is associated with obesity and type 2 diabetes (T2D). This study aims at determining the long-term effects of low-dose nitrate administration on metabolic and obesity indices in ovariectomized (OVX) rats. METHODS: OVX rat model was induced using the two dorsolateral skin incision method. Two months after ovariectomy, rats were divided into three groups (n = 10/group): Control, OVX, and OVX+nitrate, and the latter received sodium nitrate at a dose of 100 mg/L in their drinking water for nine months. Fasting serum glucose and lipid profile were measured every month. A glucose tolerance test was performed at months 1, 3, and 9 (the end of the study). Obesity indices were calculated, and histological analyses were performed on the gonadal adipose tissues at month 9. RESULTS: OVX rats had impaired fasting glucose, glucose intolerance, and dyslipidemia with higher obesity indices at month 9. Nitrate improved glucose and lipid metabolism in OVX rats and decreased body weight (6.9%), body mass index (12.5%), Lee index (5.4%), adiposity index (23.9%), abdominal circumference (10.5%), and thoracic circumference (17.1%). Also, nitrate decreased adipocyte area by 49% and increased adipocyte density by 193% in gonadal adipose tissue. CONCLUSION: Long-term low-dose nitrate administration improves glucose and lipid metabolism in OVX rats in association with decreasing OVX-induced adiposity, increasing adipocyte density, and decreasing adipocyte area. These findings provide support for a potential therapeutic role of nitrate in postmenopausal women with some features of metabolic syndrome.

Diabetoporosis: Role of nitric oxide.

Diabetoporosis, diabetic-related decreased bone quality and quantity, is one of the leading causes of osteoporotic fractures in subjects with type 2 diabetes (T2D). This is associated with lower trabecular and cortical bone quality, lower bone turnover rates, lower rates of bone healing, and abnormal posttranslational modifications of collagen. Decreased nitric oxide (NO) bioavailability has been reported within the bones of T2D patients and can be considered as one of the primary mechanisms by which diabetoporosis is manifested. NO donors increase trabecular and cortical bone quality, increase the rate of bone formation, accelerate the bone healing process, delay osteoporosis, and decrease osteoporotic fractures in T2D patients, suggesting the potential therapeutic implication of NO-based interventions. NO is produced in the osteoblast and osteoclast cells by three isoforms of NO synthase (NOS) enzymes. In this review, the roles of NO in bone remodeling in the normal and diabetic states are discussed. Also, the favorable effects of low physiological levels of NO produced by endothelial NOS (eNOS) versus detrimental effects of high pathological levels of NO produced by inducible NOS (iNOS) in diabetoporosis are summarized. Available data indicates decreased bone NO bioavailability in T2D and decreased expression of eNOS, and increased expression and activity of iNOS. NO donors can be considered novel therapeutic agents in diabetoporosis.

Long-term nitrate administration increases expression of browning genes in epididymal adipose tissue of male type 2 diabetic rats.

Expression of browning genes are lower in both humans and animals with type 2 diabetes (T2D). This study aims at determining effects of long-term nitrate administration on protein and mRNA levels of uncoupling protein 1 (UCP1), peroxisome proliferator activated receptor gamma (PPAR-γ), and PPAR-γ coactivator 1 alpha (PGC1-α) in epididymal adipose tissue (eAT) of rats with T2D. Male Wistar rats were divided into 4 groups (n = 6/group): Control, diabetes, control + nitrate (CN), and diabetes + nitrate (DN). T2D was induced using high fat diet combined with a low-dose of streptozotocin (30 mg/kg body weight). Sodium nitrate was administrated at a dose of 100 mg/L for 6 months in nitrate-treated rats. Fasting serum glucose and insulin concentrations were measured at months 0 (i.e. at start of the protocol), 3, and 6. At month 6, protein and mRNA levels of UCP1, PPAR-γ, and PGC1-α were measured in eAT samples. In addition, tissue concentration of cyclic guanosine monophosphate (cGMP) was measured and histological analyses were done at month 6. In rats with T2D, 6-month administration of nitrate decreased serum glucose and insulin concentrations by 13% and 23%, respectively and increased cGMP level by 85%. Rats with T2D had lower mRNA and protein levels of PPAR-γ (62%, P < 0.0001 and 18%, P = 0.0472), PGC1-α (49%, P = 0.0019 and 21%, P = 0.0482), and UCP1 (35%, P = 0.0613 and 30%, P = 0.0031) in eAT; 6-month nitrate administration restored these decreased levels to near control values. In addition, nitrate increased adipocyte density by 193% and decreased adipocyte area by 53% in rats with T2D. In conclusion, long-term low-dose nitrate administration increased mRNA and protein expressions of browning genes in white adipose tissue of male rats with T2D; these findings partly explain favorable metabolic effects of nitrate administration in diabetes.

Impaired Cardiovascular Function in Male Rats with Hypo- and Hyperthyroidism: Involvement of Imbalanced Nitric Oxide Synthase Levels.

BACKGROUND AND OBJECTIVE: All three isoforms of nitric oxide (NO) synthase (NOS) are targets for thyroid hormones in the cardiovascular system. The aim of this study was to assess the effects of hypo- and hyperthyroidism on inducible (iNOS), endothelial (eNOS), and neural (nNOS) NOS levels in aorta and heart tissues of male rats. METHODS: Rats were divided into control, hypothyroid, and hyperthyroid groups; hypo- and hyperthyroidism were induced by adding propylthiouracil (500 mg/L) and L-thyroxine (12 mg/L) to drinking water for a period of 21 days. On day 21, systolic blood pressure, heart rate, left ventricular developed pressure (LVDP), peak rate of positive and negative (±dp/dt) changes in left ventricular pressure as well as NO metabolites (NOx) and iNOS, eNOS, and nNOS protein levels in aorta and heart, were all measured. RESULTS: Compared to controls, LVDP and ±dp/dt were lower in both hypo- and hyperthyroid rats. Compared to controls, heart rate and systolic blood pressure were lower in hypothyroid and higher in hyperthyroid rats. NOx levels in the heart of hypothyroid rats were lower (53%), whereas that in hyperthyroid rats were higher (56% and 40%) than controls. Compared to controls, hypothyroid rats had lower levels of eNOS, iNOS, and nNOS in the aorta (16%, 34%, and 15%, respectively) and lower iNOS and higher nNOS in heart tissue (27% and 46%). In hyperthyroid rats, eNOS levels were lower (54% and 30%) and iNOS were higher (63%, and 35%) in the aorta and heart while nNOS was lower in the aorta (18%). CONCLUSION: Hypothyroidism increased while hyperthyroidism decreased the ratio of eNOS/iNOS in aorta and heart; these changes of NOS levels were associated with impaired cardiovascular function.

Ovariectomized rat model of osteoporosis: a practical guide.

Osteoporosis affects about 200 million people worldwide and is a silent disease until a fracture occurs. Management of osteoporosis is still a challenge that warrants further studies for establishing new prevention strategies and more effective treatment modalities. For this purpose, animal models of osteoporosis are appropriate tools, of which the ovariectomized rat model is the most commonly used. The aim of this study is to provide a 4-step guideline for inducing a rat model of osteoporosis by ovariectomy (OVX): (1) selection of the rat strain, (2) choosing the appropriate age of rats at the time of OVX, (3) selection of an appropriate surgical method and verification of OVX, and (4) evaluation of OVX-induced osteoporosis. This review of literature shows that (i) Sprague-Dawley and Wistar rats are the most common strains used, both responding similarly to OVX; (ii) six months of age appears to be the best time for inducing OVX; (iii) dorsolateral skin incision is an appropriate choice for initiating OVX; and (iv) the success of OVX can be verified 1-3 weeks after surgery, following cessation of the regular estrus cycles, decreased estradiol, progesterone, and uterine weight as well as increased LH and FSH levels. Current data shows that the responses of trabecular bones of proximal tibia, lumbar vertebrae and femur to OVX are similar to those in humans; however, for short-term studies, proximal tibia is recommended. Osteoporosis in rats is verified by lower bone mineral density and lower trabecular number and thickness as well as higher trabecular separation, changes that are observed at 14, 30, and 60 days post-OVX in proximal tibia, lumbar vertebrae and femur, respectively.

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 Cardiac Myosin Heavy Chain Expression in Male Rats / O Efeito de Hipotireoidismo Fetal na Expressão da Miosina Cardíaca de Cadeia Pesada em Ratos Macho

Abstract Background: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. Objective: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and β-MHC isoforms. Methods: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and β-MHC were measured by qPCR. Results: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, β-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. Conclusion: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of β-MHC to α- MHC ratio in the heart.

Resumo Fundamento: Deficiência de hormônio da tireoide durante vida fetal pode afetar a função cardíaca no futuro. O mecanismo subjacente dessa ação em hipotireoidismo fetal (HF) em ratos ainda não tem explicação. Objetivo: O objetivo desse estudo é avaliar o efeito de HF na função cardíaca em ratos macho e determinar a contribuição da α-miosina de cadeia pesada (α-MCP) e de isoformas β-MCP. Métodos: Seis ratos fêmea gestantes foram aleatoriamente divididas em dois grupos. O grupo do hipotireoidismo recebeu água contendo 6-propil-2-tiouracil durante a gestação, e os ratos no grupo de controle receberam água de torneira. Os filhotes dos ratos foram testados quando atingiram idade adulta. O coração dos ratos HF e controle foram isolados e submetidos a perfusão pelo método de Langendorff para medição de parâmetros hemodinâmicos. Também foram medidas as expressões de mRNA do coração de α-MCP e β-MCP por qPCR. Resultados: PVED de base (74,0 ± 3,1 vs. 92,5 ± 3,2 mmHg, p < 0,05) e pressão arterial (217 ± 11 vs. 273 ± 6 batidas/min, p < 0,05) mostraram-se mais baixas em ratos HF do que em ratos controle. Além disso, esses resultados mostraram a mesma significância em ±dp/dt. Em ratos HF, a expressão de β-MCP foi mais alta (201%) e a de α-MCP foi mais baixa (47%) do que em ratos controle. Conclusão: Deficiência de hormônio da tireoide durante a vida fetal pode enfraquecer funções cardíacas normais em ratos adultos, efeito devido em parte à expressão aumentada de β-MCP em relação a α-MCP no coração.

Effect of Fetal Hypothyroidism on Cardiac Myosin Heavy Chain Expression in Male Rats.

BACKGROUND: Thyroid hormone deficiency during fetal life could affect the cardiac function in later life. The mechanism underlying this action in fetal hypothyroidism (FH) in rats has not been elucidated thus far. OBJECTIVE: The aim of this study is to evaluation the effect of FH on cardiac function in male rats and to determine the contribution of α-myosin heavy chain (MHC) and ß-MHC isoforms. METHODS: Six pregnant female rats were randomly divided into two groups: The hypothyroid group received water containing 6-propyl-2-thiouracil during gestation and the controls consumed tap water. The offspring of the rats were tested in adulthood. Hearts from the FH and control rats were isolated and perfused with langendroff setup for measuring hemodynamic parameters; also, the heart mRNA expressions of α- MHC and ß-MHC were measured by qPCR. RESULTS: Baseline LVDP (74.0 ± 3.1 vs. 92.5 ± 3.2 mmHg, p < 0.05) and heart rate (217 ± 11 vs. 273 ± 6 beat/min, p < 0.05) were lower in the FH rats than controls. Also, these results showed the same significance in ±dp/dt. In the FH rats, ß-MHC expression was higher (201%) and α- MHC expression was lower (47%) than control. CONCLUSION: Thyroid hormone deficiency during fetal life could attenuate normal cardiac functions in adult rats, an effect at least in part due to the increased expression of ß-MHC to α- MHC ratio in the heart.

Deregulation of NF-кB-miR-146a negative feedback loop may be involved in the pathogenesis of diabetic neuropathy.

The current study was designed to explore whether microRNA-146a and its adapter proteins (tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1)) are involved in the pathogenesis of diabetes neuropathy. Twelve male Sprague Dawley rats were randomized into control and diabetic groups (n = 6). Diabetes was induced by a single-dose injection of nicotinamide (110 mg/kg; i.p.), 15 min before injection of streptozotocin (50 mg/kg; i.p.) in 12-h-fasted rats. Diabetic neuropathy was evaluated by hot plate and tail emersion tests, 2 months after the injection of streptozotocin. The gene expression level of microRNA-146a (miR-146a), IRAK1, TRAF6, and nuclear factor kappa B (NF-κB) was measured in the sciatic nerve of rats using the real time-PCR method. Moreover, the activity of NF-κB and the concentration of pro-inflammatory cytokines were determined by the ELISA method. In comparison with the control group, a threefold increase in the expression of miR-146a and NF-κB, and a twofold decrease in the expression of TRAF6 were observed in the sciatic nerve of diabetic rats. Furthermore, the NF-κB activity and the concentration of TNF-α, interleukin 6 (IL-6), and interleukin 1ß (IL-1ß) in the sciatic nerve of diabetic rats were higher than in those of control counterparts. These results suggest that a defect in the NF-кB-miR-146a negative feedback loop may be involved in the pathogenesis of diabetic neuropathy.

Deregulation of NF-kB–miR-146a negative feedback loop may be involved in the pathogenesis of diabetic neuropathy

The current study was designed to explore whether microRNA-146a and its adapter proteins (tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) and interleukin-1 receptor-associated kinase 1 (IRAK1)) are involved in the pathogenesis of diabetes neuropathy. Twelve male Sprague Dawley rats were randomized into control and diabetic groups (n = 6). Diabetes was induced by a single-dose injection of nicotinamide (110 mg/kg; i.p.), 15 min before injection of streptozotocin (50 mg/kg; i.p.) in 12-h-fasted rats. Diabetic neuropathy was evaluated by hot plate and tail emersion tests, 2 months after the injection of streptozotocin. The gene expression level of microRNA-146a (miR-146a), IRAK1, TRAF6, and nuclear factor kappa B (NF-κB) was measured in the sciatic nerve of rats using the real time-PCR method. Moreover, the activity of NF-κB and the concentration of pro-inflammatory cytokines were determined by the ELISA method. In comparison with the control group, a threefold increase in the expression of miR-146a and NF-κB, and a twofold decrease in the expression of TRAF6 were observed in the sciatic nerve of diabetic rats. Furthermore, the NF-κB activity and the concentration of TNF-α, interleukin 6 (IL-6), and interleukin 1β (IL-1β) in the sciatic nerve of diabetic rats were higher than in those of control counterparts. These results suggest that a defect in the NF-кB–miR-146a negative feedback loop may be involved in the pathogenesis of diabetic neuropathy