Resveratrol directly suppresses proteolysis possibly via PKA/CREB signaling in denervated rat skeletal muscle.

Although there are reports that polyphenol resveratrol (Rsv) may cause muscle hypertrophy in basal conditions and attenuate muscle wasting in catabolic situations, its mechanism of action is still unclear. Our study evaluated the ex vivo effects of Rsv on protein metabolism and intracellular signaling in innervated (sham-operated; Sham) and 3-day sciatic denervated (Den) rat skeletal muscles. Rsv (10-4 M) reduced total proteolysis (40%) in sham muscles. Den increased total proteolysis (~40%) in muscle, which was accompanied by an increase in the activities of ubiquitin-proteasome (~3-fold) and lysosomal (100%) proteolytic systems. Rsv reduced total proteolysis (59%) in Den muscles by inhibiting the hyperactivation of ubiquitin-proteasome (50%) and lysosomal (~70%) systems. Neither Rsv nor Den altered calcium-dependent proteolysis in muscles. Mechanistically, Rsv stimulated PKA/CREB signaling in Den muscles, and PKA blockage by H89 (50µM) abolished the antiproteolytic action of the polyphenol. Rsv reduced FoxO4 phosphorylation (~60%) in both Sham and Den muscles and Akt phosphorylation (36%) in Den muscles. Rsv also caused a homeostatic effect in Den muscles by returning their protein synthesis rates to levels similar to Sham muscles. These data indicate that Rsv directly inhibits the proteolytic activity of lysosomal and ubiquitin-proteasome systems, mainly in Den muscles through, at least in part, the activation of PKA/CREB signaling.

In Vitro, Oral Acute, and Repeated 28-Day Oral Dose Toxicity of a Mixed-Valence Polyoxovanadate Cluster.

Polyoxovanadates (POV) are a subgroup of polyoxometalates (POM), which are nanosized clusters with reported biological activities. This manuscript describes the first toxicity evaluation of a mixed-valence polyoxovanadate, pentadecavanadate, (Me4N)6[V15O36Cl], abbreviated as V15. Cytotoxicity experiments using peripheral blood mononuclear cells (PBMC), larvae of Artemia salina Leach, and in vivo oral acute and repeated 28-day doses in mice was carried out. The LC50 values in PBMC cells and A. salina were 17.5 ± 5.8 µmol L-1, and 17.9 µg L-1, respectively, which indicates high cytotoxic activity. The toxicity in mice was not observed upon acute exposure in a single dose, however, the V15 repeated 28-day oral administration demonstrated high toxicity using 25 mg/kg, 50 mg/kg and, 300 mg/kg doses. The biochemical and hematological analyses during the 28-day administration of V15 showed significant alteration of the metabolic parameters related to the kidney and liver, suggesting moderate toxicity. The V15 toxicity was attributed to the oxidative stress and lipid peroxidation, once thiobarbituric acid (TBAR) levels significantly increased in both males and females treated with high doses of the POV and also in males treated with a lower dose of the POV. This is the first study reporting a treatment-related mortality in animals acutely administrated with a mixed-valence POV, contrasting with the well-known, less toxic decavanadate. These results document the toxicity of this mixed-valence POV, which may not be suitable for biomedical applications.

Antidiabetic properties of oral treatment of hexane and chloroform fractions of Morus nigra leaves in streptozotocin-induced rats.

Morus nigra L. has been widely used in Brazilian folk medicine for the treatment of diabetes. We evaluate the chemical composition and antidiabetic properties of the hexane (Hex-Mn) and chloroform (Chlo-Mn) fractions obtained by partition of the crude ethanolic extract from the leaves in rats. Chemical composition analysis of Hex-Mn and Chlor-Mn was performed by gas chromatography-mass spectrometry (CG-MS). In vivo and in vitro studies were carried out to compare the antidiabetic activities of the Hex-Mn and Chlor-Mn fractions. Most of the compounds identified in Hex-Mn were α-linolenic acid, stigmast-5-en-3-ol and linolenic acid ethyl ester, while in Chlor-Mn, stigmast-5-en-3-ol, palmitic acid and α-linolenic acid were mainly identified. Only Hex-Mn treatment reduced both fasting and postprandial hyperglycemia. Additionally, Hex-Mn preserved body weight gain, preserved the hepatic glycogen content, and also reduced the thiobarbituric acid reactive substances and nitrite levels, as well as restored the superoxide dismutase. Furthermore, digestion of complex carbohydrates and intestinal glucose absorption was prevented by Hex-Mn treatment. Our results suggest that the antidiabetic activity of Hex-Mn may be explained, at least in part, by the insulin sensitivity increase, antioxidant properties and reduction in carbohydrate absorption in the small intestine.

Moderate offspring exercise offsets the harmful effects of maternal protein deprivation on mitochondrial function and oxidative balance by modulating sirtuins.

BACKGROUND AND AIMS: It has been demonstrated that maternal low protein during development induces mitochondrial dysfunction and oxidative stress in the heart. Moderate-intensity exercise in early life, conversely, increases the overall cardiac health. Thus, we hypothesize that moderate-intensity exercise performed during young age could ameliorate the deleterious effect of maternal protein deprivation on cardiac bioenergetics. METHODS AND RESULTS: We used a rat model of maternal protein restriction during gestational and lactation period followed by an offspring treadmill moderate physical training. Pregnant rats were divided into two groups: normal nutrition receiving 17% of casein in the diet and undernutrition receiving a low-protein diet (8% casein). At 30 days of age, the male offspring were further subdivided into sedentary (NS and LS) or exercised (NT and LT) groups. Treadmill exercise was performed as follows: 4 weeks, 5 days/week, 60 min/day at 50% of maximal running capacity. Our results showed that a low-protein diet decreases oxidative metabolism and mitochondrial function associated with higher oxidative stress. In contrast, exercise rescues mitochondrial capacity and promotes a cellular resilience to oxidative stress. Up-regulation of cardiac sirtuin 1 and 3 decreased acetylation levels, redeeming from the deleterious effect of protein restriction. CONCLUSION: Our findings show that moderate daily exercise during a young age acts as a therapeutical intervention opposing the harmful effects of a maternal diet restricted in protein.

Brazilian Morus nigra Attenuated Hyperglycemia, Dyslipidemia, and Prooxidant Status in Alloxan-Induced Diabetic Rats.

Morus nigra has been used popularly for several proposes, including diabetic. In an attempt to support medicinal value, the acute hypoglycemic, hypolipidemic, and antioxidant effects of the ethanolic extract of Morus nigra (EEMn 200 or 400 mg/kg b.w.) were evaluated in normal and alloxan-induced diabetic treated for 14 days. Serum biochemical and antioxidant analysis were performed at the end of experiment. Oral glucose tolerance test was performed at 10th and 15th days. Chromatographic analysis by HPLC-DAD of EEMn was performed. Insulin was used as positive control to glycemic metabolism as well as fenofibrate to lipid metabolism. EEMn (400 mg/kg/day) reduced fasting and postprandial glycaemia, improved oral glucose tolerance, and reduced lipolysis and proteolysis in diabetic rats. EEMn decreased the blood levels of total cholesterol and increased HDL level when compared to the diabetic control rats. At higher levels, EEMn reduced triglycerides and VLDL levels in diabetic rats. Also, EEMn reduced malondialdehyde and increased the reduced glutathione levels in liver of diabetic rats. Chromatographic analysis identified the presence of the flavonoids rutin, isoquercetin, and kaempferitrin. Acute EEMn treatment reduced hyperglycemia, improved oral glucose tolerance, and minimized dyslipidemia and oxidative stress leading to a reduction in atherogenic index in alloxan-induced diabetic rats.

Acute Toxicity and Cytotoxicity Effect of Ethanolic Extract of Spondias tuberosa Arruda Bark: Hematological, Biochemical and Histopathological Evaluation.

Spondias tuberosa Arruda, popularly named as umbu, is native from savanna-like vegetation and widely used for medicinal purposes, however, the toxicological profile is not available yet. This study evaluated the phytochemical profile and acute toxicity and citoxicity of Ethanolic Extract of Spondias tuberosa Arruda Bark (EEStb) in hematological, biochemical and histopathological parameters. Female Wistar rats were divided into: control (C) and animal treated single doses of 300mg/Kg (EEStb300) or 2.000mg/kg body weight (ESStb2.000) of the EEStb. After 24 hours and 14 days from gavage, the behavior, hematological, biochemical and histopathological parameters were assayed. Cytotoxicity effect was evaluated on HEp-2 cell lines. Neither EEStb300 nor EEStb2.000 produced mortality nor changes in body weight during the 14-days of observation, but EEStb2.000 reduced quietly the food and water intake as well as locomotor activity at first day. There were no changes in macroscopic, histopathological, biochemical and hematological parameters. EEStb in concentrations of 6.25- 50µg ml-1 on HEp-2 cell did not produce cytotoxic effect. These results suggest that EEStb did not cause acute toxicity and cytotoxic, suggesting a good safety rate for Spondias tuberosa Arruda.

Expression of circadian clock and melatonin receptors within cultured rat cardiomyocytes.

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demonstrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor RORα have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and RORα and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORα were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erbα expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erbα mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erbα mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity.

Multiple alpha1-adrenergic receptor subtypes support synergistic stimulation of vasopressin and oxytocin release by ATP and phenylephrine.

Simultaneous exposure of explants of the hypothalamo-neurohypophyseal system (HNS) to ATP and the α(1)-adrenergic receptor (α(1)-R) agonist, phenylephrine (ATP+PE) induces a synergistic stimulation of vasopressin and oxytocin (VP/OT) release that is sustained for hours. The current studies confirm that the synergism is dependent upon activation of α(1)-R by demonstrating that an α(1)-R antagonist prevents the response. The role of the α(1)A, B, and D-adrenergic receptor subtypes in the synergistic effect of ATP+PE on intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons and VP/OT release from neural lobe was evaluated. The increase in [Ca(2+)](i) induced by PE in SON predominantly reflects release from intracellular stores and is mediated by activation of the α(1)A adrenergic receptor subtype. The α(1)A subtype is also required for the sustained elevation in [Ca(2+)](i) induced by ATP+PE. In contrast, although synergistic stimulation of VP/OT release was eliminated by removal of PE and was blunted by benoxathian, an α(1)-R antagonist that is not subtype selective, no single α(1)-R subtype selective antagonist prevented sustained stimulation of VP/OT release by ATP+PE. Thus, sustained activation of α(1)-R is essential for the synergistic VP and OT response to ATP+PE, but multiple α(1)-R subtypes can support the response. Redundancy amongst the α(1)-R subunits in supporting this response is consistent with the predicted importance of the response for sustaining the elevated VP release required to prevent cardiovascular collapse during hemorrhage and sepsis.

The crosstalk between thyroid hormones and the Renin-Angiotensin System.

Thyroid hormones (THs) exert multiple effects on the heart and vascular system. As a consequence, altered cardiovascular function observed in the thyroid diseases corresponds to one of the most important and clinically relevant aspects found in both hyperthyroidism and hypothyroidism. Besides THs' direct effects on the heart and vascular system, in the last three decades several studies have implicated the Renin-Angiotensin System (RAS) in some of the cardiovascular effects of THs, with this interaction suggesting that RAS may be an important mediator of THs actions. In the present review, we discuss the alterations in the circulating RAS, as well as modifications in cardiac and vascular RAS which are involved in the cardiovascular alterations found during the modulation of TH levels. In addition, considering the important role that both systems present during fetal and neonatal periods, we also review the interaction between THs and the RAS in the development of cardiovascular system. A greater understanding of the role of the RAS in hyperthyroidism and hypothyroidism, during early or adult life will presumably facilitate the evolution of newer, targeted therapies.

Sustained stimulation of vasopressin and oxytocin release by ATP and phenylephrine requires recruitment of desensitization-resistant P2X purinergic receptors.

Coexposure of hypothalamo-neurohypophyseal system explants to ATP and phenylephrine [PE; an alpha1-adrenergic receptor (alpha1-AR) agonist] induces an extended elevation in vasopressin and oxytocin (VP/OT) release. New evidence is presented that this extended response is mediated by recruitment of desensitization-resistant ionotropic purinergic receptor subtypes (P2X-Rs): 1) Antagonists of the P2X2/3 and P2X7-Rs truncated the sustained VP/OT release induced by ATP+PE but did not alter the transient response to ATP alone. 2) The P2X2/3 and P2X7-R antagonists did not alter either ATP or ATP+PE-induced increases in [Ca(2+)](i). 3) P2X2/3 and P2X7-R agonists failed to elevate [Ca(2+)](i), while ATP-gamma-S, an agonist for P2X2-Rs increased [Ca(2+)](i) and induced a transient increase in VP/OT release. 4) A P2Y1-R antagonist did not prevent initiation of the synergistic, sustained stimulation of VP/OT release by ATP+PE but did reduce its duration. Thus, the desensitization-resistant P2X2/3 and P2X7-R subtypes are required for the sustained, synergistic hormone response to ATP+PE, while P2X2-Rs are responsible for the initial activation of Ca(2+)-influx by ATP and ATP stimulation of VP/OT release. Immunohistochemistry, coimmunoprecipitation, and Western blot analysis confirmed the presence of P2X2 and P2X3, P2X2/3, and P2X7-R protein, respectively in SON. These findings support the hypothesis that concurrent activation of P2X2-R and alpha1-AR induces calcium-driven recruitment of P2X2/3 and 7-Rs, allowing sustained activation of a homeostatic circuit. Recruitment of these receptors may provide sustained release of VP during dehydration and may be important for preventing hemorrhagic and septic shock.

Role of purinergic P2Y1 receptors in regulation of vasopressin and oxytocin secretion.

Pharmacological studies demonstrated that ATP elevates intracellular calcium ([Ca(2+)](i)) in supraoptic nucleus (SON) neurons primarily by activation of P2X2 and P2Y1 purinergic receptors [P2Y1R]. The current studies provide evidence for the presence of P2Y1R protein in SON neurons, evidence that activation of these P2Y1Rs induces an increase in [Ca(2+)](i) from both intracellular stores and Ca(2+) influx, and functional evidence that activation of P2Y1Rs induces vasopressin (VP) and oxytocin (OT) hormone release. Pretreatment of Fura-2 AM-loaded explants of the hypothalamo-neurohypophyseal system (HNS) with thapsigargin (TG) significantly (approximately 80%) reduced the increase in [Ca(2+)](i) induced by the P2Y1R-specific agonist, 2-methylthio-ADP (2-MeSADP). In contrast, the increase in [Ca(2+)](i) was slightly (approximately 20%) decreased in calcium-free medium. The calcium response to 2-MeSADP was completely blocked by the P2Y1R-specific antagonist, MRS2179 or by a combination of TG pretreatment and calcium-free medium. It was absent in P2Y1R knockout mice (P2Y1R(-/-)). 2-MeSADP significantly increased VP and OT release from perifused rat and wild-type mouse HNS explants compared with control. MRS2179 prevented this response in wild-type mouse, but it did not prevent ATP-induced hormone release from rat explants. 2-MeSADP did not induce hormone release from P2Y1R(-/-) explants. These findings support a potential role for P2Y1Rs in regulation of VP and OT release. The finding that P2Y1R activation induces a small Ca(2+) influx suggests that P2Y1Rs may regulate VP release by modifying ion channels such as stretch-inactivated cation channels.