Neonatal Streptozotocin-induced Diabetic Rats Research Articles

Antidiabetes of Combination of Fractionated-extracts of Andrographispaniculata and Centellaasiatica in Neonatal Streptozotocin-induced Diabetic Rats

Many medicinal plants are widely grown in South- and Southeast Asia countries. Some of them are traditionally used for treatment of diabetes mellitus such as Andrographispaniculata and Centellaasiatica. In the study, we provided fractionated-extracts of A. paniculata and C. asiatica to increase the concentration of their active compounds and eliminate unexpected substances. The aim of the study was to evaluate the antidiabetes effect of the combination of their fractionated-extracts in male neonatal streptozotocin (STZ)-induced diabetic rats. In the study, diabetes was injected intraperitoneally 90mg.kg-1 BWSTZ to two day-old rats. At the age of three months, the rats were administered with the combination of both fractionated-extracts for 14 consecutive days. We evaluated antidiabetes with parameters of blood glucose levels, morphology of pancreatic islet, β-cell density as well as immunohistochemical pancreatic insulin. The levels of MDA, SOD and GPx were also determined about oxidative stress change after treatment with the combination. In the study, the combination succeeded to lower the blood glucose level in neonatal STZ-induced diabetic rats. Inline with fact, improvement of rat pancreatic islets and β cells density, as well as moderate restoration of pancreatic insulin, were observed after treatment with the combination. The substance could decrease the level of MDA, and increase the levels of SOD and GPx. In conclusion, the combination of fractionated-extracts of A. paniculata and C. asiatica exhibited potential antidiabetic effect to its antioxidative effect in male neonatal STZ-induced diabetes rats.

Proinflammation in maternal and fetal livers and circulating miR-122 dysregulation in a GDM rat model induced by intrauterine programming

In gestational diabetes mellitus (GDM) pregnancies, a compromised fetal liver may impact offspring's metabolic health. Here, we aimed to address prooxidant, proinflammatory and profibrotic markers in the livers from GDM rats and their fetuses, and to analyze the expression of miR-122 (a relevant microRNA in liver pathophysiology) in fetal and maternal plasma of GDM rats, as well as in the fetal livers of neonatal streptozotocin-induced (nSTZ) diabetic rats, the rats that generate GDM through intrauterine programming. GDM and nSTZ rats were evaluated on day 21 of pregnancy. We found increased nitric oxide production and lipoperoxidation in the livers from GDM rats and their fetuses compared to controls. Livers from GDM fetuses also showed increased levels of connective tissue growth factor and matrix metalloproteinase-2. The expression of miRNA-122 was downregulated in the plasma from GDM rats and their male fetuses, as well as in the livers from male fetuses of nSTZ diabetic rats. miR-122 levels were regulated both in vitro through PPARγ activation and in vivo through a maternal diet enriched in PPAR ligands. Our findings revealed a prooxidant/proinflammatory environment in the livers from GDM rats and their fetuses and a dysregulation of miR-122, likely relevant in the programming of offspring's diseases.

Maternal diets enriched in olive oil regulate lipid metabolism and levels of PPARs and their coactivators in the fetal liver in a rat model of gestational diabetes mellitus

In a rat model of gestational diabetes mellitus (GDM) programmed in the offspring of neonatal streptozotocin-induced (nSTZ) diabetic rats, lipids are accumulated in the fetal liver in a sex-dependent way. Here, we evaluated whether maternal diets enriched in olive oil in rats that will develop GDM ameliorate lipid metabolic impairments in the fetal livers. Pregnant offspring of control and nSTZ diabetic rats (F0) were fed a 6% olive oil-supplemented diet throughout the F1 gestation. We evaluated maternal metabolic parameters as well as lipid content, expression of lipid metabolizing enzymes and protein expression of PLIN2, PPARs and PPAR coactivators in the fetal livers. The offspring of nSTZ diabetic rats developed GDM regardless of the maternal treatment. Hypertriglyceridemia in GDM rats was prevented by the olive oil-enriched maternal treatment. In the livers of male fetuses of GDM rats, the maternal olive oil-supplemented diet prevented lipid overaccumulation and prevented the increase in PPARγ and PPARδ levels. In the livers of female fetuses of GDM rats, the maternal olive oil supplementation prevented the increase in PPARδ levels and the reduction in PGC1α levels, but did not prevent the reduced lipid content. Control and GDM rats showed a reduction of lipid metabolic enzymes in the fetal livers, which was associated with reduced levels of the PPAR coactivators PGC-1α and SRC-1 in males and of SRC-1 in females. These results suggest powerful effects of a maternal olive oil-supplemented diet in the fetal liver, possibly providing benefits in the fetuses and offspring from GDM rats.

Antiallodynic interaction and motor performance of the pregabalin/thioctic acid and pregabalin/α-tocopherol combinations in neonatal streptozotocin-induced diabetic rats.

Painful peripheral neuropathy can be associated with nerve damage caused by diabetes mellitus. Although pregabalin is the first-line therapy for peripheral neuropathy, it shows substantial discontinuation rates, mainly because of nervous system side effects as motor incoordination. Multimodal therapy may improve the motor side effect profile of pregabalin. The aim of this study was to evaluate the interaction of pregabalin + thioctic acid or pregabalin + α-tocopherol on allodynia and motor performance in neonatal streptozotocin-induced diabetic rats. Efficacy of drugs separately or in combination was tested by tactile allodynia using von Frey filaments. Isobolographic and interaction index analysis were used to determine the antiallodynic interaction between pregabalin and either thioctic acid or α-tocopherol. Motor performance was measured using a rotarod test. Pregabalin, thioctic acid, and α-tocopherol reduced, in a dose-dependent fashion, tactile allodynia. Pregabalin + thioctic acid and pregabalin + α-tocopherol combinations also dose-dependently reduced allodynic behavior in diabetic rats. Isobolographic analysis revealed an additive interaction for both combinations. Consistently, the interaction indices confirmed the additive effect between pregabalin + thioctic acid and pregabalin + α-tocopherol. In addition, the administration of either combination improved motor incoordination induced by pregabalin. Data suggests that thioctic acid or α-tocopherol could positively impact the therapeutic profile of pregabalin, because they might be useful for reducing motor incoordination associated to pregabalin in patients with peripheral neuropathy.

Antidiabetic activity of two aqueous extracts of two cucurbitaceae: Citrullus colocynthis and Bryonia dioica

The aim of the present study was to evaluate the antidiabetic activity of the aqueous extract of Citrullus colocynthis L Schrad. (colocynth) fruits, CCFaq and Bryonia dioica Jacq. (White bryony) roots, BDRaq in neonatal streptozotocin-induced diabetic rats (n5-STZ diabetic rats). Experimental type 2 diabetes mellitus was induced by neonatal injection of streptozotocin (90 mg/kg i.p.) to pups of rats on the 5th day of birth. After 9 weeks, experimental type 2 diabetes mellitus was confirmed by polyphagia, polydipsia, polyuria, glucose intolerance, and fasting blood glucose levels > 1.50 g/L. Antidiabetic effect of CCFaq (20 mg/kg, i.p.) and BDRaq (30 mg/kg, i.p.) extracts as well as standard glibenclamide (10 mg/kg, i.p.) was evaluated on acute (3 h) and subacute (21 days) administration. Variations of body and some organs’ weight (pancreas, liver, kidney, and spleen) were recorded. Biochemical estimations of glucose, total cholesterol, triglycerides and urea levels were carried out during the experimentation. Intraperitoneal administration of CCFaq and BDRaq extracts as well as standard glibenclamide for 21 days to n5-STZ diabetic rats significantly (P < 0.01) decreased the level of serum glucose by –51%,–64% and–58% reductions, respectively. The body weight as well as serum levels of total cholesterol, triglycerides, and urea were markedly reversed secondary to subacute administration of aqueous extracts. In addition, changes in the weight of internal organs were restored to normal by the prolonged effect of BDRaq extract treatment. When compared with the CCFaq extract, the BDRaq extract appeared to have maximum antidiabetic activity in normalizing all studied parameters during the acute and subacute treatments. This study reveals the interesting antidiabetic effect of C. colocynthis and B. dioica and supports their traditional use for the treatment of diabetes mellitus.

Antidiabetic and antihypertensive effect of Virgin Argan Oil in model of neonatal streptozotocin-induced diabetic and l-nitroarginine methylester (l-NAME) hypertensive rats

The goal of this study was to examine the effect of Virgin Argan Oil (VAO) obtained from the fruit of Argania spinosa in a model of type 2 diabetes and hypertensive rats. Neonatal diabetes was induced by a single i.p. injection of streptozotocin (90 mg/kg) 2 days after birth. To induce NO-deficient hypertension, the adult diabetic animals were treated with l-nitroarginine methylester (l-NAME) (30 mg/kg/day) given orally for 21 days. Following treatment with VAO (21 days), the hyperglycemia decreased to 1.3 ± 0.07 g/l compared with 1.92 ± 0.09 g/l (p < 0.01) in the untreated diabetic-hypertensive rats. The simultaneous administration of VAO with l-NAME prevented the increase in blood pressure during the 3 weeks of treatment. Blood pressure remained constant at 131 ± 1 mm Hg after 21 days - vs 157 ± 0.64 mm Hg in untreated animals (p < 0.001). The treatment with VAO to diabetic-hypertensive rats caused a significant increase of hepatic glycogen levels (13.3 ± 1.8 vs 6.34 ± 0.75 mg/g tissue in untreated diabetic-hypertensive control group; p < 0.01). In conclusion, the overall findings indicate that VAO possesses antidiabetic and antihypertensive activity in n-stz/l-NAME rats. This effect may be related to its high content of tocopherols, phenolic compounds, and unsaturated fatty acids.

Antidiabetic and Antioxidant Effects of Newly Synthesized Pyrimido[1,6-A] Pyrimidine Derivatives in Neonatal Streptozotocin-Induced Diabetic Rats

Synthesis of some novel pyrimido[1,6-a]pyrimidine derivatives 4, 8 and 10 was described through the respective reactions of sodium salts of formyl ketones 1, 7 and 9 with 6-aminothiouracil. The characterization of the reaction products was confirmed by using the available elemental analysis and spectral data. One of these derivatives ( 4b), 1-thioxo-1,2,7,8,9,10-hexahydro-3H-pyrimido[1,6-a]quinazolin-3-one, was tested using sublethal dose level (10 mg/kg b. w./day for 3 weeks) and was found to have potent antihyperglycemic, antihyperlipidemic and antioxidant properties in neonatal streptozotocin-induced (n-STZ) diabetic male and female albino rats.

Constitutively active heat shock factor 1 enhances glucose-driven insulin secretion

Weak pancreatic β-cell function is a cause of type 2 diabetes mellitus. Glucokinase regulates insulin secretion via phosphorylation of glucose. The present study focused on a system for the self-protection of pancreatic cell by expressing heat shock factor (HSF) and heat shock protein (HSP) to improve insulin secretion without inducing hypoglycemia. We previously generated a constitutively active form of human HSF1 (CA-hHSF1). An adenovirus expressing CA-hHSF1 using the cytomegalovirus promoter was generated to infect mouse insulinoma cells (MIN6 cells). An adenovirus expressing CA-hHSF1 using a human insulin promoter (Ins-CA-hHSF1) was also generated to infect rats. We investigated whether CA-hHSF1 induces insulin secretion in MIN6 cells and whether Ins-CA-hHSF1 can improve blood glucose and serum insulin levels in healthy Wister rats and type 2 diabetes mellitus model rats. CA-hHSF1 expression increased insulin secretion 1.27-fold compared with the overexpression of wild-type hHSF1 in MIN6 cells via induction of HSP90 expression and subsequent activation of glucokinase. This mechanism is associated with activation of both glucokinase and neuronal nitric oxide synthase. Ins-CA-hHSF1 improved blood glucose levels in neonatal streptozotocin-induced diabetic rats. Furthermore, Ins-CA-hHSF1 reduced oral glucose tolerance testing results in healthy Wister rats because of an insulin spike at 15 minutes; however, it did not induce hypoglycemia. CA-hHSF1 induced insulin secretion both in vitro and in vivo. These findings suggest that gene therapy with Ins-CA-hHSF1 will be able to be used to treat patients with type 2 diabetes mellitus and impaired glucose tolerance without causing hypoglycemia at fasting.

3-Aryl-2-{4-[4-(2,4-dioxothiazolidin-5-ylmethyl)phenoxy]-phenyl}-acrylic acid alkyl ester: synthesis and antihyperglycemic evaluation

A number of 2,4-thiazolidinedione derivatives of aryl-substituted cinnamic acid were synthesized and studied for their antihyperglycemic activity in neonatal streptozotocin-induced diabetic Wister male rats. Substitution of the aryl ring resulted in higher activity. 3-(2,4-Dimethoxyphenyl)-2-{4-[4-(2,4-dioxothiazolidin-5-ylmethyl)phenoxy]-phenyl}-acrylic acid methyl ester (Thz7), was found to be most active compound in this series which showed that disubstitutions on aryl ring by electron releasing groups were found suitable for promising antihyperglycemic activity. Increase in the ester carbon chain length decreased the activity.

Sergliflozin etabonate, a selective SGLT2 inhibitor, improves glycemic control in streptozotocin-induced diabetic rats and Zucker fatty rats

The low-affinity sodium glucose cotransporter (SGLT2) is responsible for most of the glucose reabsorption in the kidney and has been highlighted as a novel therapeutic target for the treatment of diabetes. We discovered sergliflozin etabonate, a novel selective SGLT2 inhibitor, and found that selective inhibition of SGLT2 increased urinary glucose excretion and consequently decreased plasma glucose levels. In this report, we examined the antihyperglycemic effects of sergliflozin etabonate in normal and diabetic rats in comparison with those of a sulfonylurea (gliclazide) and an α-glucosidase inhibitor (voglibose). Sergliflozin etabonate increased urinary glucose excretion in a dose-dependent manner, and inhibited the increase in plasma glucose after sucrose loading independently of insulin secretion in normal rats. Sergliflozin etabonate also improved postprandial hyperglycemia in neonatal streptozotocin-induced diabetic rats; whereas gliclazide did not improve it. In rats with mild or moderate streptozotocin-induced diabetes, the degree of the antihyperglycemic effects of sergliflozin etabonate correlated with the severity of the diabetic condition. Sergliflozin etabonate did not affect the plasma glucose level of normal rats as seen with gliclazide. Chronic treatment with sergliflozin etabonate reduced the levels of glycated hemoglobin and fasting plasma glucose, and improved the glycemic response after glucose loading in Zucker fatty rats. In addition, sergliflozin etabonate did not affect the body weight or food intake. These data indicate that sergliflozin etabonate could improve glycemic control without its use resulting in insulin secretion, hypoglycemia, and body weight gain, and may provide a unique approach to the treatment of diabetes.

The allergic inflammatory reaction in neonatal streptozotocininduced diabetic rats: evidence of insulin resistance and microvascular dysfunction

To investigate the allergic reaction in neonatal streptozotocin (nSTZ)-induced diabetes mellitus. Male newborn Wistar rats were made diabetic by the injection of streptozotocin (160 mg/kg, i. p.) and used 8 weeks thereafter. Animals were sensitized against ovalbumin (OA, 50 microg and Al(OH)3, 5 mg, s. c.) and challenged 14 or 21 days thereafter. OA-induced airway inflammation and OA-induced pleurisy models were used to investigate leukocyte migration (total and differential leukocyte counts) and lung vascular permeability (Evans blue dye extravasation). nSTZ-diabetic rats presented glucose intolerance and insulin resistance. Relative to controls, nSTZ rats exhibited a 30% to 50% reduction in lung vascular permeability. Leukocyte infiltration in both models of allergen-induced inflammation, and number of pleural mast cells did not differ between groups. Data suggest that the reduction of allergic inflammatory reactions in nSTZ rats is restricted to microvascular dysfunctions and associated, probably, with insulin resistance in lung microvascular endothelium.

Chlorpropamide treatment restores the reduced carrageenan-induced paw edema and pleural exudate volume in diabetic rats

Knowing that hyperglycemia is a hallmark of vascular dysfunction in diabetes and that neonatal streptozotocin-induced diabetic rats (n-STZ) present reduced inflammatory response, we decided to evaluate the effect of chlorpropamide-lowered blood glucose levels on carrageenan-induced rat paw edema and pleural exudate in n-STZ. Diabetes was induced by STZ injection (160 mg/kg, ip) in neonates (2-day-old) Wistar rats. n-STZ diabetic rats were treated with chlorpropamide (200mg/kg, 15d, by gavage) 8 weeks after STZ injection. Carrageenan-induced paw edema and pleural exudate volumes were assessed concomitantly with peripheral and exudate leukocyte count. We also evaluated the expression of inducible nitric oxide synthase (iNOS) in lungs of all experimental groups. Chlorpropamide treatment improved glucose tolerance, beta-cell function (assessed by HOMA-beta), corrected paw edema, and pleural exudate volume in n-STZ. Neither leukocyte count nor iNOS expression were affected by diabetes or by chlorpropamide treatment. Chlorpropamide treatment by restoring beta-cell function, reducing blood sugar levels, and improving glucose tolerance might be contributing to the correction of the reduced inflammatory response tested as paw edema and pleural exudate in n-STZ diabetic rats.

Selective Small-Molecule Agonists of G Protein–Coupled Receptor 40 Promote Glucose-Dependent Insulin Secretion and Reduce Blood Glucose in Mice

OBJECTIVE— Acute activation of G protein–coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40.RESEARCH DESIGN AND METHODS— We developed a series of GPR40-selective small-molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests in wild-type and GPR40 knockout mice (GPR40−/−).RESULTS— Small-molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improved glucose tolerance in wild-type mice but not in GPR40−/− mice. While a 72-h exposure to FFAs in tissue culture significantly impaired GDIS in islets from both wild-type and GPR40−/− mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from wild-type mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats and improved glucose levels in mice with high-fat diet–induced obesity acutely and chronically.CONCLUSIONS— GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial for overall glucose control in patients with type 2 diabetes.

뽕잎 함유 대두발효물이 신생 당뇨유도쥐에 미치는 혈당강하효과

당뇨 환자를 위한 기존의 뽕잎 건강식품의 기능을 보완할 목적으로 뽕잎분말과 DNJ 생산균주인 Bacillus subtilis MORI균의 대두발효 혼합물을 신생 당뇨유도쥐에 4주간 경구투여 하여 먹이섭취량, 체중변화, 혈당량, 내당능, 혈중 지질함량, 장기무게 등을 조사하였다. 전체적으로 당뇨유도 시험군은 정상시험군(NC군)과 비교하여 사료섭취량은 많았으나 증체량이 적어 낮은 사료효율을 보였다. 그러나 뽕잎(M군) 및 뽕잎함유대두발효물(MM군)의 투여에 의해 사료효율이 개선되었으며 특히 MM-60군에서 가장 높은 사료효율을 나타내었다. 1주일 간격으로 4주간에 걸쳐 시행한 혈당량 측정 결과 뽕잎이나 뽕잎함유대두발효물의 투여는 공복혈당을 크게 감소시켰으며 특히 MM-60군과 MM-120군은 당뇨대조군(DC군)에 비해 유의적으로 낮게 나타났다. 4주간의 시료투여 후 경구당부하시험에 의한 내당능 조사 결과 뽕잎투여나 뽕잎함유대두발효물의 투여는 혈당량을 꾸준히 감소시켰으며 특히 포도당 투여 후 120분에서의 MM-60군과 MM-120군은 DC군에 비해 유의적으로 낮은 혈당량을 보여 뽕잎 단독보다는 대두발효배양물 첨가에 의해 내당능이 크게 개선된 것으로 나타났다. 혈중 총콜레스테롤 및 중성지질 역시 DC군에서 높은 함량을 나타냈으나 뽕잎이나 뽕잎함유대두발효물 투여에 의해 지질함량이 감소하였으며 그 감량 정도는 총콜레스테롤에서는 MM-60군과 MM-120군에서 그리고 중성지질의 경우에는 M군, MM-60군, MM-120군에서 DC군에 비해 유의적인 감소를 나타내었다. 당뇨 유발에 의해 쥐의 간, 신장, 비장, 심장 등 체내 주요장기의 무게는 크게 증가하였으나 뽕잎 또는 뽕잎함유대두 발효물 투여에 의해 이들 장기의 무게는 대부분 감소하였으며 특히 MM-60군, MM-120군은 DC군에 비해 유의적인 감소를 나타내었다. 이상의 실험결과 뽕잎분말과 DNJ 생산균주인 B. subtilis MORI균의 대두발효물의 혼합물은 신생 당뇨유도쥐의 제반 당뇨증세 완화에 긍정적으로 작용하는 것이 확인되었으며 따라서 뽕잎함유대두발효물은 앞으로 당뇨환자들에 대한 건강식품 소재로 그 활용이 크게 기대된다 하겠다.

Peroxisome proliferator-activated receptor α activation regulates lipid metabolism in the feto-placental unit from diabetic rats

Maternal diabetes promotes an overaccumulation of lipids in the feto-placental unit and impairs feto-placental development and growth. Here, we investigated the role played by the nuclear receptor peroxisome proliferator-activated receptor (PPAR)alpha in lipid metabolism in fetuses and placentas from control and neonatal streptozotocin-induced diabetic rats. Placentas and fetuses were studied on day 13.5 of gestation. The concentrations of PPARalpha (by Western blot) and its endogenous agonist leukotriene B(4) (LTB(4)) (by enzyme immunoassay) were analysed. Placental explants and fetuses were cultured with LTB(4) or clofibrate, and then lipid metabolism analysed (concentrations and synthesis from (14)C-acetate of triglycerides, phospholipids, cholesterol and cholesteryl esters; release of glycerol and free fatty acids (FFAs)). We found that maternal diabetes led to increases in placental concentrations of triglycerides and cholesteryl esters, and fetal concentrations of phospholipids. PPARalpha agonists downregulated fetal and placental lipid concentrations in control and diabetic rats. The synthesis of lipids was reduced in the diabetic placenta but increased in fetuses from diabetic animals. PPARalpha agonists reduced the synthesis of lipids in control placenta and in the fetuses from control and diabetic rats. Glycerol and FFA release was enhanced in the diabetic placenta and in control placenta cultured with PPARalpha agonists. Maternal diabetes led to reductions in fetal and placental LTB(4) concentrations and to increases in placental PPARalpha concentrations. Overall, these data support a novel role of PPARalpha as a regulator of lipid metabolism in the feto-placental unit, relevant in maternal diabetes where fetal and placental PPARalpha, LTB(4) and lipid concentrations are altered.

Enalapril treatment corrects the reduced response to bradykinin in diabetes increasing the B2 protein expression

Considering the growing importance of the interaction between components of kallikrein–kinin and renin–angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the effect of enalapril on the reduced response of bradykinin and on the interaction between angiotensin-(1–7) (Ang-(1–7)) and bradykinin (BK), important components of these systems, in an insulin-resistance model of diabetes. For the above purpose, the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. In n-STZ diabetic rats, enalapril treatment restored the reduced response to BK but not the potentiation of BK by Ang-(1–7) present in non-diabetic rats. The restorative effect of enalapril was observed at a dose that did not correct the altered parameters induced by diabetes such as hyperglycemia, glicosuria, insulin resistance but did reduce the high blood pressure levels of n-SZT diabetic rats. There was no difference in mRNA and protein expressions of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Enalapril treatment increased the B2 kinin receptor expression. From our data, we conclude that in diabetes enalapril corrects the impaired BK response probably by increasing the expression of B2 receptors. The lack of potentiation of BK by Ang-(1–7) is not corrected by this agent.

The influence of improved glycaemic control with chlorpropamide on microvascular reactivity and nitric oxide synthase activity in diabetic rats

Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to reduced production of nitric oxide. The aim of this study was to verify the influence of improved glycaemic control with chlorpropamide on microvascular reactivity, endothelial nitric oxide synthase (e-NOS) expression, and NOS activity in neonatal streptozotocin-induced diabetic rats (n-STZ). Diabetes was induced by STZ injection into neonates Wistar rats. n-STZ diabetic rats were treated with chlorpropamide (200 mg kg(-1), 15 days, by gavage). The changes in mesenteric arteriolar and venular diameters were determined in anaesthetized control and n-STZ diabetic rats, before and after topical application of acetylcholine, bradykinin and sodium nitroprusside (SNP). We also assessed e-NOS expression (using polymerase chain reaction after reverse transcription of mRNAs into cDNAs) and NOS activity (conversion of L-arginine to citrulline) in the mesenteric vascular bed of chlorpropamide-treated n-STZ, vehicle-treated n-STZ, and control rats. In n-STZ, chlorpropamide treatment reduced high glycaemic levels, improved glucose tolerance and homoeostatic model assessment (HOMA-beta), and restored NOS activity. Impaired vasodilator responses of arterioles and venules to acetylcholine, bradykinin and SNP were partially corrected by chlorpropamide treatment in n-STZ. We concluded that improved metabolic control and restored NOS activity might be collaborating with improved microvascular reactivity found in chlorpropamide-treated n-STZ.

Lack of potentiation of bradykinin by angiotensin-(1-7) in a type 2 diabetes model: Role of insulin

Considering the growing importance of the interaction between components of kallikrein-kinin and renin–angiotensin systems in physiological and pathological processes, particularly in diabetes mellitus, the aim of the present study was to investigate the interaction between angiotensin-(1-7) (Ang-(1-7)) and bradykinin (BK), important components of these systems in an insulin resistance model of diabetes, and the effect of insulin on it. For this the response of mesenteric arterioles of anesthetized neonatal streptozotocin-induced (n-STZ) diabetic and control rats was evaluated using intravital microscopy. Though capable of potentiating BK in non-diabetic rats, Ang-(1-7) did not potentiate BK in n-STZ rats. Chronic but not acute insulin treatment restored the potentiation. This restorative effect of insulin was abolished by a K+ channel blocker (tetraethylammonium), by nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester) and by a cyclooxygenase inhibitor (indomethacin). On the other hand, Na +-,K +-ATPase inhibition (by ouabain) did not abolish the effect of insulin. There was no difference in mRNA and protein expression of B1 and B2 kinin receptor subtypes between n-STZ diabetic and control rats. Insulin treatment did not alter the kinin receptor expression. Our data allow us to conclude that diabetes impaired the interaction between BK and Ang-(1-7) and that insulin restores it. The restoring effect of insulin depends on membrane hyperpolarization, nitric oxide release and cyclooxygenease metabolites but not Na+K+-ATPase. Alteration of kinin receptor expression might not be involved in the restoring effect of insulin on the potentiation of BK by Ang-(1-7).

Effect of N-benzoyl-d-phenylalanine and metformin on insulin receptors in neonatal streptozotocin-induced diabetic rats: Studies on insulin binding to erythrocytes

In the present study, we focused on the insulin-receptor binding in circulating erythrocytes of N-benzoyl-d-phenylalanine (NBDP) and metformin in neonatal streptozotocin (nSTZ)-induced male Wistar rats. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors in NBDP and metformin-treated diabetic rats. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (53.0 ± 3.1%) than in NBDP (62.0 ± 3.1%), metformin (66.0 ± 3.3%) and NBDP and metformin combination-treated (72.0 ± 4.2%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with NBDP and metformin-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from NBDP and metformin-treated diabetic rats having NBDP 2.0 ± 0.10×10−10 M−1 (Kd1); 12.0 ± 0.85×10−8 M−1 (Kd2), Metformin 2.1 ± 0.15×10−10 M−1 (Kd1); 15.0 ± 0.80×10−8 M−1 (Kd2), NBDP and metformin 2.7 ± 0.10×10−10 M−1 (Kd1); 20.0 ± 1.2×10−8 M−1 (Kd2) compared with 0.9 ± 0.06×10−10 M−1 (Kd1); 6.0 ± 0.30×10−8 M−1 (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in nSTZ induced diabetic control rats. Treatment with NBDP along with metformin significantly improved specific insulin binding, with receptor number and affinity binding reaching almost normal non-diabetic levels. The data presented here show that NBDP along with metformin increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin.

Metformin treatment restores the altered microvascular reactivity in neonatal streptozotocin-induced diabetic rats increasing NOS activity, but not NOS expression

Abnormalities in vascular function are well recognized in diabetes. Hyperglycemia may be central to the pathogenesis of vascular dysfunction but is not certain whether improvements in glycaemic control will improve vascular function. The effects of metformin, an antidiabetic agent that improves insulin sensitivity and glycaemic control, on the microvascular reactivity have not been reported in neonatal streptozotocin-induced (n-STZ) diabetes. Diabetes was induced by STZ injection (160 mg/kg, ip) in neonates (2-day-old) Wistar rats. n-STZ diabetic rats were treated with metformin (300 mg/kg, 15 d, by gavage). Using intravital microscopy the changes in mesenteric arteriolar and venular diameters were determined in anesthetized control and n-STZ diabetic rats, before and after topical application of endothelium-dependent vasodilator agents, mediators or not of the inflammatory response, and endothelium-independent vasodilator agent. We also determined the total nitric oxide synthase (NOS) activity (conversion of L-arginine to citrulline) and endothelial(e), inducible(i), and neuronal(n) NOS expression (using polymerase chain reaction after reverse transcription of the mRNAs into cDNAs) in the mesentery of metformin-treated n-STZ diabetic and vehicle-treated n-STZ diabetic and control rats. Although metformin treatment did not correct the high glycaemic levels and the impaired glucose tolerance, the reduced vasodilator responses and total NOS activity in n-STZ diabetic rats were corrected by the treatment. Neither diabetes nor metformin treatment altered the expression of the three NOS isoforms. We concluded that metformin restores the reduced response to vasodilator agents, independently of the correction of the metabolic alterations. Improvement of total NOS activity might be in part responsible for the correction.