Oxytocin and cardioprotection in diabetes and obesity.

Oxytocin (OT) emerges as a drug for the treatment of diabetes and obesity. The entire OT system is synthesized in the rat and human heart. The direct myocardial infusion with OT into an ischemic or failing heart has the potential to elicit a variety of cardioprotective effects. OT treatment attenuates cardiomyocyte (CMs) death induced by ischemia-reperfusion by activating pro-survival pathways within injured CMs in vivo and in isolated cells. OT treatment reduces cardiac apoptosis, fibrosis, and hypertrophy. The OT/OT receptor (OTR) system is downregulated in the db/db mouse model of type 2 diabetes which develops genetic diabetic cardiomyopathy (DC) similar to human disease. We have shown that chronic OT treatment prevents the development of DC in the db/db mouse. In addition, OT stimulates glucose uptake in both cardiac stem cells and CMs, and increases cell resistance to diabetic conditions. OT may help replace lost CMs by stimulating the in situ differentiation of cardiac stem cells into functional mature CMs. Lastly, adult stem cells amenable for transplantation such as MSCs could be preconditioned with OT ex vivo and implanted into the injured heart to aid in tissue regeneration through direct differentiation, secretion of protective and cardiomyogenic factors and/or their fusion with injured CMs.

Oxytocin decreases diurnal and nocturnal arterial blood pressure in the conscious unrestrained spontaneously hypertensive rat.

In this study, we assessed the effects of oxytocin (OT) on mean arterial blood pressure (MAP), heart rate (HR), and locomotor activity (LA) in male spontaneous hypertensive rats (SHR) and Sprague-Dawley (SDR) controls using telemetry. OT was given by intravenous injections of 0.1, 0.2 or 0.4mg/kg to assess short term acute effects or by daily subcutaneous injections of 0.5 or 1.0mg/kg for 5 days. Compared to the saline infusion, (i) intravenous OT, regardless of concentration, increased MAP in SHR and SDR, (ii) HR increased, but was periodically lower in both strains with 0.2 or 0.4mg/kg, and (iii) no effects of OT on LA were observed. Subcutaneous injections demonstrated that (i) 1.0mg/kg for 5days lowered diurnal MAP and HR in SDR and SHR, persisting for 6 days, (ii) 1.0mg/kg decreased nocturnal HR in SDR, (iii) 0.5 and 1.0mg/kg decreased MAP with minor effects on HR in the SHR, and lastly (iv) OT decreased LA mainly during the diurnal cycle in both strains. Our main results show that OT induces significant beneficial effects on cardiovascular function over several diurnal and nocturnal cycles in the SHR, with the most prominent effect being a robust decrease in MAP.

Treatment with brain natriuretic peptide prevents the development of cardiac dysfunction in obese diabetic db/db mice.

AIMS/HYPOTHESIS: Obesity and diabetes increase the risk of developing cardiovascular diseases and heart failure. These metabolic disorders are generally reflected by natriuretic peptide system deficiency. Since brain natriuretic peptide (BNP) is known to influence metabolism and cardioprotection, we investigated the effect of chronic exogenous BNP treatment on adverse myocardial consequences related to obesity and diabetes. METHODS: Ten-week-old C57BL/KsJ-db/db obese diabetic mice (db/db) and their lean control littermates (db/+) were treated with BNP (0.6 µg kg(-1) h(-1)) or saline for 12 weeks (n = 10/group). Serial blood and tomography analysis were performed. Cardiac function was determined by echocardiography, and biochemical and histological heart and fat analyses were also performed. RESULTS: BNP treatment resulted in an average increase in plasma BNP levels of 70 pg/ml. An improvement in the metabolic profile of db/db mice was observed, including a reduction in fat content, increased insulin sensitivity, improved glucose tolerance and lower blood glucose, despite increased food intake. db/db mice receiving saline displayed both early systolic and diastolic dysfunction, whereas these functional changes were prevented by BNP treatment. The cardioprotective effects of BNP were attributed to the inhibition of cardiomyocyte apoptosis, myocardial fibrosis, cardiac hypertrophy and the AGE-receptor for AGE (RAGE) system as well as normalisation of cardiac AMP-activated protein kinase and endothelial nitric oxide synthase activities. CONCLUSIONS/INTERPRETATION: Our results indicate that chronic BNP treatment at low dose improves the metabolic profile and prevents the development of myocardial dysfunction in db/db mice.

Cardiac oxytocin receptor blockade stimulates adverse cardiac remodeling in ovariectomized spontaneously hypertensive rats.

An increasing amount of evidence demonstrates the beneficial role of oxytocin (OT) in the cardiovascular system. Similar actions are attributed to genistein, an isoflavonic phytoestrogen. The treatment with genistein activates the OT system in the aorta of ovariectomized (OVX) Sprague-Dawley (SD) rats. The objective of this study was to determine the effects of low doses of genistein on the OT-induced effects in rat hypertension. The hypothesis tested was that treatment of OVX spontaneously hypertensive rats (SHRs) with genistein improves heart structure and heart work through a mechanism involving the specific OT receptor (OTR). OVX SHRs or SD rats were treated with genistein (in microg/g body wt sc, 10 days) in the presence or absence of an OT antagonist (OTA) [d(CH(2))(5), Tyr(Me)(2), Orn(8)]-vasotocin or a nonspecific estrogen receptor antagonist (ICI-182780). Vehicle-treated OVX rats served as controls. RT-PCR and Western blot analysis demonstrated that left ventricular (LV) OTR, downregulated by ovariectomy, increased in response to genistein. In SHRs or SD rats, this effect was blocked by OTA or ICI-182780 administration. The OTR was mainly localized in microvessels expressing the CD31 marker and colocalized with endothelial nitric oxide synthase. In SHRs, the genistein-stimulated OTR increases were associated with improved fractional shortening, decreased blood pressure (12 mmHg), decreased heart weight-to-body weight ratio, decreased fibrosis, and lowered brain natriuretic peptide in the LV. The prominent finding of the study is the detrimental effect of OTA treatment on the LV of SHRs. OTA treatment of OVX SHRs resulted in a dramatic worsening of ejection fractions and an augmented fibrosis. In conclusion, these results demonstrate that cardiac OTRs are involved in the regulation of cardiac function of OVX SHRs. The decreases of OTRs may contribute to cardiac pathology following menopause.

Anti-inflammatory effect of oxytocin in rat myocardial infarction.

While an increasing amount of evidence demonstrates the homeostatic functions of the cardiac oxytocin (OT) system, less is known about the role of this hormone in the injured heart. The current study examined the effect of OT infusion on cell apoptosis, expression of proliferating cell nuclear antigen (PCNA) and inflammation in the acute and subacute phases of myocardial infarction (MI). Prior MI male Sprague-Dawley rats were infused subcutaneously with OT 25 or 125 ng/(kg h) for 3 or 7 days. Saline-treated MI and sham-operated rats served as controls. Echocardiography and analysis of cardiac sections were used to disclose OT actions. Left ventricular fractional shortening, estimated to be 46.0 +/- 1.8% in sham controls, declined to 21.1 +/- 3.3% in vehicle-treated MI rats and was improved to 34.2 +/- 2.1 and to 30.9 +/- 2.5% after treatment with OT 25 and 125 ng/(kg h), respectively. OT infusion resulted in: (1) increase of cells expressing PCNA in the infarct zone, diminished cell apoptosis and fibrotic deposits in the remote myocardium; (2) suppression of inflammation by reduction of neutrophils, macrophages and T lymphocytes; (3) depression of the expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 with promotion of transforming growth factor-beta. OT treatment reduced expression of atrial and brain natriuretic peptides in the infarcted ventricle, as well as the concentration of both peptides in the circulation. These results indicate that continuous OT delivery reduces inflammation and apoptosis in infarcted and remote myocardium, thus improving function in the injured heart.

Exercise training decreases hepatic SCD-1 gene expression and protein content in rats.

Stearoyl-CoA desaturase-1 (SCD-1) is the rate limiting enzyme in the biosynthesis of saturated-derived monounsaturated fats that are the major constituents of very-low-density-lipoproteins-triacylglycerol (VLDL-TAG) and are involved in regulating cellular metabolism. The purpose of this study was to evaluate the effects of an 8-week exercise training program on the hepatic gene expression of this crucial enzyme. Female rats either trained (TR) or kept sedentary (Sed) for 8 weeks were submitted either to standard (SD) diet for 8 or for 6 weeks followed by high-fat (HF; 42% kcal) diet for 2 weeks. The 2-week-high fat feeding resulted in an increase in liver triacylgycerol (TAG), plasma free-fatty-acids (FFA), abdominal fat mass, sterol-regulatory-element-binding protein-1c (SREBP-1c), and carbohydrate-response-element-binding protein (ChREBP) gene expression in liver along with a decrease in SCD-1 gene expression and plasma and liver SCD-1 desaturation index (C16:1/C16:0). Liver TAG, plasma FFA, SREBP-1c mRNA, and SCD-1 desaturation indexes (C16:1/C16:0; C18:1/C18:0) were not changed in liver or in plasma by the training program. Nevertheless, training resulted in an important decrease in fat mass (P < 0.01), hepatic SCD-1 mRNA levels (P < 0.01), and protein content (P < 0.05) in both SD and HF fed rats. It is concluded that despite an absence of decreased liver TAG, exercise training contributes to the proper regulation of fat metabolism by down-regulating hepatic SCD-1 gene expression and protein content.

The Role of Oxytocin in Cardiovascular Protection.

The beneficial effects of oxytocin on infarct size and functional recovery of the ischemic reperfused heart are well documented. The mechanisms for this cardioprotection are not well defined. Evidence indicates that oxytocin treatment improves cardiac work, reduces apoptosis and inflammation, and increases scar vascularization. Oxytocin-mediated cytoprotection involves the production of cGMP stimulated by local release of atrial natriuretic peptide and synthesis of nitric oxide. Treatment with oxytocin reduces the expression of proinflammatory cytokines and reduces immune cell infiltration. Oxytocin also stimulates differentiation stem cells to cardiomyocyte lineages as well as generation of endothelial and smooth muscle cells, promoting angiogenesis. The beneficial actions of oxytocin may include the increase in glucose uptake by cardiomyocytes, reduction in cardiomyocyte hypertrophy, decrease in oxidative stress, and mitochondrial protection of several cell types. In cardiac and cellular models of ischemia and reperfusion, acute administration of oxytocin at the onset of reperfusion enhances cardiomyocyte viability and function by activating Pi3K and Akt phosphorylation and downstream cellular signaling. Reperfusion injury salvage kinase and signal transducer and activator of transcription proteins cardioprotective pathways are involved. Oxytocin is cardioprotective by reducing the inflammatory response and improving cardiovascular and metabolic function. Because of its pleiotropic nature, this peptide demonstrates a clear potential for the treatment of cardiovascular pathologies. In this review, we discuss the possible cellular mechanisms of action of oxytocin involved in cardioprotection.

Downregulation of oxytocin and natriuretic peptides in diabetes: possible implications in cardiomyopathy.

Regular physical activity is beneficial in preventing the risk of cardiovascular complications of diabetes. Recent studies showed a cardioprotective role of oxytocin (OT) to induce natriuretic peptides (NPs) and nitric oxide (NO) release. It is not known if the diabetic state is associated with a reduced OT-NPs-NO system and if exercise training improves this system. To address this, we investigated the effects of treadmill running using the db/db mouse model of type 2 diabetes. Eight-week-old db/db mice were subjected to running 5 days per week for a period of 8 weeks. The lean db/+ littermates were used as controls. Sedentary db/db mice were obese and hyperglycaemic, and exercise training was not effective in reducing body weight and the hyperglycaemic state. Compared to control mice, db/db mice had lower heart weight and heart-to-body weight ratios. In these mice, this was associated with augmented cardiac apoptosis, cardiomyocyte enlargement and collagen deposits. In addition, db/db mice displayed significant downregulation in gene expression of OT (76%), OT receptors (65%), atrial NP (ANP; 43%), brain NP (BNP; 87%) and endothelial nitric oxide synthase (eNOS) (54%) in the heart (P < 0.05). Exercise training had no effect on expression of these genes which were stimulated in control mice. In response to exercise training, the significant increment of anti-apoptotic Bcl-2 gene expression was observed only in control mice (P < 0.05). In conclusion, downregulation of the OT-NPs-NO system occurs in the heart of the young db/db mouse. Exercise training was not effective in reversing the defect, suggesting impairment of this cardiac protective system in diabetes.

Functional activity of the carboxyl-terminally extended oxytocin precursor Peptide during cardiac differentiation of embryonic stem cells.

The hypothalamic post-translational processing of oxytocin (OT)-neurophysin precursor involves the formation of C-terminally extended OT forms (OT-X) that serve as intermediate prohormones. Despite abundant expression of the entire functional OT system in the developing heart, the biosynthesis and implication of OT prohormones in cardiomyogenesis remain unknown. In the present work, we investigated the involvement of OT-X in cardiac differentiation of embryonic stem (ES) cells. Functional studies revealed the OT receptor-mediated cardiomyogenic action of OT-Gly-Lys-Arg (OT-GKR). To obtain further insight into the mechanisms of OT-GKR-induced cardiac effects, we generated ES cell lines overexpressing the OT-GKR gene and enhanced green fluorescent protein (EGFP). The functionality of the OT-GKR/EGFP construct was assessed by fluorescence microscopy and flow cytometry, with further confirmation by radioimmunoassay and immunostaining. Increased spontaneously beating activity of OT-GKR/EGFP-expressing embryoid bodies and elevated expression of GATA-4 and myosin light chain 2v cardiac genes indicated an inductive effect of endogenous OT-GKR on ES cell-derived cardiomyogenesis. Furthermore, patch-clamp experiments demonstrated induction of ventricular phenotypes in OT-GKR/EGFP-transfected and in OT-GKR-treated cardiomyocytes. Increased connexin 43 protein in OT-GKR/EGFP-expressing cells further substantiated the evidence that OT-GKR modifies cardiac differentiation toward the ventricular sublineage. In conclusion, this report provides new evidence of the biological activity of OT-X, notably OT-GKR, during cardiomyogenic differentiation.

Exercise training decreases plasma leptin levels and the expression of hepatic leptin receptor-a, -b, and, -e in rats.

In addition to acting in the central nervous system, leptin also acts on peripheral tissues such as liver to provide a protection against lipid accretion. Previous evidence from human and animal model indicates that exercise training reduces circulating leptin levels beyond the changes in adiposity levels. Because liver is one of the main peripheral organs for leptin action, this present study was designed to determine whether leptin receptors expression in liver is changed by exercise training. Female rats trained (TR) or kept sedentary (Sed) for 8 weeks were submitted either to a standard (SD) diet for 8 weeks or for 6 weeks followed by 2 weeks of high-fat (HF) or high-carbohydrate (HC) feeding. Food intake, adiposity levels, circulating plasma leptin and insulin concentrations along with the hepatic expression of leptin receptors (ObR-a, -b, and -e) and peroxisome proliferator-activated receptor alpha (PPARalpha) and peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha), were measured in all the animals. Intra-abdominal fat depots were increased under the HF but not under the HC diet. As expected, exercise training decreases intra-abdominal adiposity in animals fed with the SD and the HF diet, and to a lesser extent in HC-fed rats. Plasma leptin levels either expressed in absolute values or in values relative to adiposity levels were significantly (P < 0.05) increased with the HF diet and significantly decreased in TR animals, independently of the diet. Moreover, a significant (P < 0.01) reduction in hepatic gene expression of ObR-a, -b and -e was found in TR animals in all the three diet conditions. PPARalpha and PGC-1alpha mRNAs were also decreased (P < 0.05) in TR animals in two out of three diet conditions. The present findings indicate that exercise training-induced decrease in plasma leptin levels is accompanied by a reduction in gene expression of three different isoforms of leptin receptors in liver.

Hyperhomocysteinemia is not sufficient to cause preeclampsia in an animal model: the importance of folate intake.

OBJECTIVE: The objective of our study was to determine whether methylenetetrahydrofolate reductase (Mthfr)-deficient mice develop preeclampsia (PE). STUDY DESIGN: Mice were placed on a normal or low-folate/high-methionine (LF/HM) diet to assess the impact of mild and severe homocysteinemia. Blood pressure and proteinuria were measured throughout gestation in Mthfr-deficient and control mice on both diets, by radiotelemetry and by determining the urinary albumin/creatinine ratio by enzyme-linked immunosorbent assay, respectively. RESULTS: Although Mthfr-deficient mice have endothelial dysfunction, they do not develop hypertension or proteinuria during gestation. The LF/HM diet induced proteinuria, growth restriction, and a decrease in the number of pups per litter in all mice without any effect on the placenta. CONCLUSION: Our study clearly demonstrates that hyperhomocysteinemia is not sufficient to cause PE in this animal model. Furthermore, it confirms the importance of folate intake on pregnancy outcomes.

Retinoid acid-induced effects on atrial and pacemaker cell differentiation and expression of cardiac ion channels.

Whereas retinoid acid (RA) signaling has been implicated in embryonic heart development, its significance in differentiation of specific cardiac subtypes remains largely unknown. In the present study, we took advantage of lineage-specific expression of atrial natriuretic peptide (ANP) in embryonic stem (ES) cells to study RA-induced effects on differentiation of atrial- and pacemaker-like phenotypes. Embryoid bodies (EB) were exposed to 10(-5), 10(-7), and 10(-9) M RA at early (days 1-5 [d1-5]) and late (d6-10) developmental stages, and RA effects on expression of lineage-specific cardiac markers and ion channels were examined. Our initial experiments revealed a detrimental effect of 10(-5) M RA on EB development by inducing marked apoptosis. Morphologic and expression analysis demonstrated that 10(-7) M RA applied at d1-5 was most effective to induce the atrial sublineage. RA did not affect differentiation of pacemaker-like cells, independent of RA concentration and application time. Conversely, RA exposure at an early developmental stage inhibited ventricular-specific MLC-2v gene expression. Late-stage RA administration exhibited no significant alterations in cardiomyogenic differentiation. Terminally differentiated cardiomyocytes exposed to RA at d1-5 or d6-10 displayed unchanged I(Ca,L) and I(to) channel expression compared with untreated cells. However, patch clamp studies revealed a significant increase of I(Ca,L) and I(to) current densities associated with increased levels of the underlying channel subunits in 6-7-day-old cardiomyocytes upon early RA exposure. In contrast, I(f) current density and HCN4 expression remained largely unaffected by RA. Our results imply that RA induces differentiation of ANP-expressing EBs toward an atrial phenotype in a time- and concentration-dependent manner and accelerates expression of I(Ca,L) and I(to) ion channels without affecting differentiation of pacemaker cells.

Anti-inflammatory and angiogenic effects of exercise training in cardiac muscle of diabetic mice.

Background: Improved glycemic control and cardiovascular function are major benefits of regular exercise training (ET) in type 2 diabetes. Recent work has demonstrated that ET improves cardiac and vascular functions independent of obesity, inflammation, and glucose control in the diabetic db/db mouse. In this study, we determined whether ET can overcome the effects of elevated inflammatory cytokines and hyperglycemia on markers of cardiac angiogenesis and inflammation in the diabetic mouse. Methods: Male diabetic db/db mice were assigned to a sedentary and exercise-trained group. Sedentary lean control littermates were used as controls. ET was performed at moderate intensity on a treadmill 5 days a week for a period of 8 weeks. After ET, blood was collected for assay of glucose, hemoglobin (HB and HB1AC), C-reactive protein (CRP), and IL-6. Markers of inflammation and insulin resistance (IL-6, IL-1ß, and tumor necrosis factor-alpha [TNF-α]) and angiogenesis (endothelial nitric oxide synthase [eNOS], vascular endothelial growth factor-A [VEGF-A], and hypoxia-inducible factor-1α [HIF-1α]) were measured in hearts. Results: Diabetic db/db mice remained obese and hyperglycemic after ET. Percent total HB and HB1AC were significantly higher in ET db/db mice compared to sedentary db/db mice, indicating further deterioration of glucose control with ET. Plasma levels of CRP and IL-6 were higher in sedentary db/db mice compared to control mice and were unaffected by ET. However, in the presence of hyperglycemia and elevated plasma cytokines, protein expression of eNOS, mRNA expression of VEGF-A, and HIF-1α was increased in db/db hearts after ET. On the other hand, protein expression of TNF-α and mRNA expression IL-6 and IL-1ß was significantly decreased by ET in hearts of db/db mice. Conclusion: Our results indicate that ET improves cardiac markers of angiogenesis, insulin resistance, and endothelial dysfunction in the db/db mouse. This was observed independently of obesity, hyperglycemia, and the systemic inflammatory state.

Dissociation of natriuresis and diuresis by oxytocin molecular forms in rats.

In the rat, oxytocin (OT) produces dose-dependent diuretic and natriuretic responses. Post-translational enzymatic conversion of the OT biosynthetic precursor forms both mature and C-terminally extended peptides. The plasma concentrations of these C-terminally extended peptides (OT-G; OT-GK and OT-GKR) are elevated in newborns and pregnant rats. Intravenous injection of OT-GKR to rats inhibits diuresis, whereas injection of amidated OT stimulates diuresis. Since OT and OT-GKR show different effects on the urine flow, we investigated whether OT-GKR modulates renal action by inhibition of the arginine-vasopressin (AVP) receptor V2 (V2R), the receptor involved in renal water reabsorption. Experiments were carried out in the 8-week-old Wistar rats receiving intravenous (iv) injections of vehicle, OT, OT-GKR or OT+OT-GKR combination. OT (10 µmol/kg) increased urine outflow by 40% (P<0.01) and sodium excretion by 47% (P<0.01). Treatment with OT-GKR (10 µmol/kg) decreased diuresis by 50% (P<0.001), decreased sodium excretion by 50% (P<0.05) and lowered potassium by 42% (P<0.05). OT antagonist (OTA) reduced diuresis and natriuresis exerted by OT, whereas the anti-diuretic effect of OT-GKR was unaffected by OTA. The treatment with V2R antagonist (V2A) in the presence and absence of OT induced diuresis, sodium and potassium outflow. V2A in the presence of OT-GKR only partially increased diuresis and natriuresis. Autoradiography and molecular docking analysis showed potent binding of OT-GKR to V2R. Finally, the release of cAMP from CHO cells overexpressing V2 receptor was induced by low concentration of AVP (EC50:4.2e-011), at higher concentrations of OT (EC50:3.2e-010) and by the highest concentrations of OT-GKR (EC50:1.1e-006). OT-GKR potentiated cAMP release when combined with AVP, but blocked cAMP release when combined with OT. These results suggest that OT-GKR by competing for the OT renal receptor (OTR) and binding to V2R in the kidney, induces anti-diuretic, anti-natriuretic, and anti-kaliuretic effects.

Evidence for non-linear pharmacokinetics of oxytocin in anesthetizetized rat.

PURPOSE: Because oxytocin (OT) is potentially useful in cardiovascular therapy but has hormonal roles on the cardiovascular and renal systems, we characterized its pharmacokinetic (PK) properties as a function of dose. METHODS: A single intravenous bolus of OT was given at doses of 200, 300, 500, 1000, 3000, 5000 and 10000 ng/kg to anesthetized male rats (n >= 4 per dose). Blood samples (6) were taken over 72 min to 150 min, depending on dose. The individual time-courses of plasma OT concentrations were analyzed with a one- or an open two-compartment PK model. Kruskal-Wallis tests (alpha=0.05) were used to compare the PK parameters among groups. RESULTS: At doses up to 500 ng/kg, OT showed a higher median systemic clearance (CLT = 0.0624 L/(min*kg); 0.0622 +/- 0.0228 as mean +/- SD value), a higher median central compartment volume of distribution (VC = 0.7906 L/kg; 0.6961 +/- 0.1754), and a lower median elimination half life (t(1/2)(lambdaz) 7.94 min; 9.08 +/- 4.3) with respect to the higher doses (CLT = 0.0266 L/(min*kg); 0.0284 +/- 0.0098, VC = 0.2213 L/kg; 0.2227 +/- 0.1142, and t(1/2)(lambdaz) 21.09 min; 28.36 +/- 21.8), all differences being significant (p 0.0008). Minimal differences were found for the estimates of these PK parameters among the 4 higher OT doses. CONCLUSION: The PK properties and persistence of exogenous OT are not proportional to dose, therefore this must be accounted for in dosing regimen design for potential cardiovascular therapy.

The effects of exercise training and caloric restriction on the cardiac oxytocin natriuretic peptide system in the diabetic mouse.

BACKGROUND: Regular exercise training (ET) and caloric restriction (CR) are the frontline strategies in the treatment of type 2 diabetes mellitus with the aim at reducing cardiometabolic risk. ET and CR improve body weight and glycemic control, and experimental studies indicate that these paradigms afford cardioprotection. In this study, the effects of combined ET and CR on the cardioprotective oxytocin (OT)-natriuretic peptide (NP) system were determined in the db/db mouse, a model of type 2 diabetes associated with insulin resistance, hyperglycemia, and obesity. METHODS: Five-week-old male db/db mice were assigned to the following groups: sedentary, ET, and ET + CR. Nonobese heterozygote littermates served as controls. ET was performed on a treadmill at moderate intensity, and CR was induced by reducing food intake by 30% of that consumed by sedentary db/db mice for a period of 8 weeks. RESULTS: After 8 weeks, only ET + CR, but not ET, slightly improved body weight compared to sedentary db/db mice. Regardless of the treatment, db/db mice remained hyperglycemic. Hearts from db/db mice demonstrated reduced expression of genes linked to the cardiac OT-NP system. In fact, compared to control mice, mRNA expression of GATA binding protein 4 (GATA4), OT receptor, OT, brain NP, NP receptor type C, and endothelial nitric oxide synthase (eNOS) was decreased in hearts from sedentary db/db mice. Both ET alone and ET + CR increased the mRNA expression of GATA4 compared to sedentary db/db mice. Only ET combined with CR produced increased eNOS mRNA and protein expression. CONCLUSION: Our data indicate that enhancement of eNOS by combined ET and CR may improve coronary endothelial vasodilator dysfunction in type 2 diabetes but did not prevent the downregulation of cardiac expression in the OT-NP system, possibly resulting from the sustained hyperglycemia and obesity in diabetic mice.

Receptors involved in moxonidine-stimulated atrial natriuretic peptide release from isolated normotensive rat hearts.

Imidazoline I1-receptors are present in the heart and may be involved in atrial natriuretic peptide (ANP) release. The following studies investigated whether moxonidine (an antihypertensive imidazoline I1-receptor and alpha2-adrenoceptor agonist) acts directly on the heart to stimulate ANP release, and to characterize the receptor type involved in this action. Perfusion of rat (200-225 g) isolated hearts with moxonidine (10(-6) and 10(-5) M), for 30 min, resulted in ANP release (83+/-29 and 277+/-70 ng/30 min, above basal, respectively), significantly (P<0.01) different from perfusion with buffer (-6+/-31 ng/30 min). ANP release stimulated by moxonidine (10(-6) M) was inhibited by co-perfusion with the antagonists, AGN192403 (imidazoline I1-receptor), phenoxybenzamine (alpha2>alpha1-adrenoceptors), and prazosin (alpha1>alpha2-adrenoceptors), but increased by rauwolscine (alpha2-adrenoceptors). Perfusion with 10(-5) M brimonidine (full alpha2-adrenoceptor agonist) inhibited moxonidine-stimulated ANP release. Similarly, moxonidine (10(-6) M) tended to reduce coronary flow, but significantly increased coronary flow in the presence of brimonidine, which was vasoconstrictive when perfused alone. Coronary flow was reduced by 10(-5) M each, brimonidine>clonidine>moxonidine; while similar bradycardia was observed with clonidine and moxonidine, but not with brimonidine. In conclusion, these results argue in favor of moxonidine acting primarily on imidazoline I1-receptors to release ANP, with both alpha2-adrenoceptor and imidazoline I1-receptors exerting inhibitory inter-relation. In contrast, the coronary vasodilatory effect of moxonidine requires full activation of alpha2-adrenoceptor. The sympatholytic and ANP-releasing effects of moxonidine appear to be mediated by cardiac imidazoline receptors that may be differentially localized. Most importantly, moxonidine can stimulate ANP release from the heart without contribution of the central nervous system.

Prorenin/renin receptor blockade promotes a healthy fat distribution in obese mice.

OBJECTIVE: Administration of the handle region peptide (HRP), a (pro)renin receptor blocker, decreases body weight gain and visceral adipose tissue (VAT) in high-fat/high-carbohydrate (HF/HC) diet-fed mice. The objective of this study was to elucidate potential mechanisms implicated in these observations. METHODS: Mice were given a normal or a HF/HC diet along with saline or HRP for 10 weeks. RESULTS: In HF/HC-fed mice, HRP increased the expression of several enzymes implicated in lipogenesis and lipolysis in subcutaneous fat (SCF) while the expression of the enzyme implicated in the last step of lipogenesis decreased in VAT. A reduction was also observed in circulating free fatty acids in these animals which was accompanied by normalized adipocyte size in VAT and increased adipocyte size in SCF. ''Beiging'' is the evolution of a white adipose tissue toward a brown-like phenotype characterized by an increased mitochondrial density and small lipid droplets. HRP increased the expression of' "beiging" markers in SCF of HF/HC diet-fed mice. CONCLUSIONS: HRP treatment may favor healthy fat storage in SCF by activating a triglyceride/free fatty acid cycling and "beiging," which could explain the body weight and fat mass reduction.

Urinary responses to acute moxonidine are inhibited by natriuretic peptide receptor antagonist.

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P<0.01) diuresis in SHR (0.21+/-0.04 vs 1.16+/-0.06 ml h(-1) 100 g(-1)), SD (0.42+/-0.06 vs 1.56+/-0.19 ml h(-1) 100 g(-1)) and WKY (0.12+/-0.04 vs 1.44+/-0.21 ml h(-1) 100 g(-1)). Moxonidine-stimulated urine output was lower in SHR than in SD and WKY. Moxonidine-stimulated sodium and potassium excretions were lower in SHR than in SD, but not WKY, demonstrating an influence of strain but not of pressure. Pretreatment with the natriuretic peptide antagonist anantin (5 or 10 microg) resulted in dose-dependent inhibition of moxonidine-stimulated urinary actions. Anantin (10 microg) inhibited (P<0.01) urine output to 0.38+/-0.06, 0.12+/-0.01, and 0.16+/-0.04 ml h(-1) 100 g(-1) in SD, WKY, and SHR, respectively. Moxonidine increased (P<0.01) plasma ANP in SD (417+/-58 vs 1021+/-112 pg ml(-1)) and WKY (309+/-59 vs 1433+/-187 pg ml(-1)), and in SHR (853+/-96 vs 1879+/-229 pg ml(-1)). These results demonstrate that natriuretic peptides mediate the urinary actions of moxonidine through natriuretic peptide receptors.

Genistein supplementation stimulates the oxytocin system in the aorta of ovariectomized rats.

OBJECTIVE: In the present study, we localized oxytocin (OT) and its receptor (OTR) in the rat aorta, and investigated whether genistein, an isoflavonic phytoestrogen, influences their expression in ovariectomized (OVX) rats deficient in estrogen. METHODS AND RESULTS: OVX Sprague-Dawley rats were randomized to the following groups: genistein (from 0.02 to 5 microg/g/day, s.c. for 10 days), estradiol (E(2,) 0.1 microg/g/day, s.c. for 10 days) or their respective vehicles. OT and OTR immunostaining was concentrated in the aortic tunica intima, suggesting their paracrine/autocrine action within endothelial cells. Reverse transcription-polymerase chain reaction analysis showed that 1 and 5 microg/g but not 0.1 microg/g genistein elevated OT mRNA (2-fold P<0.05), OTR mRNA (2.5-fold, P<0.05) and endothelial nitric oxide synthase (eNOS) mRNA (2-fold, P<0.05) in the aorta of OVX rats. In addition, genistein treatment increased estrogen receptor alpha (ERalpha) (2- to 3-fold, P<0.05) but resulted in a 50% decrease of ERbeta (P<0.05). These genistein effects were neutralized by treatment of OVX rats with the ER antagonist ICI 182,780 (1.5 microg/g/day, s.c. for 10 days). Similarly, Western blot analysis revealed an increase of 67-kDa OTR, 140-kDa eNOS, 62-kDa ERalpha and a decrease of 55-kDa ERbeta (P<0.05) in the aorta of OVX rats treated with genistein. In contrast, the treatment of OVX rats with E(2) elevated ERbeta mRNA (1.5 fold, P<0.05) but similarly to genistein increased OT, OTR, eNOS and ERalpha mRNA. CONCLUSION: These results provide the first evidence of OT and OTR co-localization in endothelial cells. The response to genistein via ER activation can be regarded as a recovery from endothelial dysfunction induced by ovariectomy.