Hypothalamic Renin-Angiotensin System and Lipid Metabolism: Effects of Virgin Olive Oil versus Butter in the Diet.

The brain renin-angiotensin system (RAS) has been recently involved in the homeostatic regulation of energy. Our goal was to analyse the influence of a diet rich in saturated fatty acids (butter) against one enriched in monounsaturated fatty acids (olive oil) on hypothalamic RAS, and their relationship with the metabolism of fatty acids. Increases in body weight and visceral fat, together with an increase in aminopeptidase A expression and reductions in AngII and AngIV were observed in the hypothalamus of animals fed with the butter diet. In this group, a marked reduction in the expression of genes related to lipid metabolism (LPL, CD36, and CPT-1) was observed in liver and muscle. No changes were found in terms of body weight, total visceral fat and the expression of hepatic genes related to fatty acid metabolism in the olive oil diet. The expressions of LPL and CD36 were reduced in the muscles, although the decrease was lower than in the butter diet. At the same time, the fasting levels of leptin were reduced, no changes were observed in the hypothalamic expression of aminopeptidase A and decreases were noted in the levels of AngII, AngIV and AngIII. These results support that the type of dietary fat is able to modify the hypothalamic profile of RAS and the body energy balance, related to changes in lipid metabolism.

Purification and functional characterisation of rhiminopeptidase A, a novel aminopeptidase from the venom of Bitis gabonica rhinoceros.

BACKGROUND: Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros. METHODOLOGY AND PRINCIPAL FINDINGS: The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped beta domain, a mixed alpha and beta catalytic domain, a beta-sandwich domain and a C-terminal alpha helical domain. CONCLUSIONS: This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.

[Effects of manganese exposure on liver in rats].

OBJECTIVE: To observe effects of manganese on function and pathology of liver in rats. METHODS: 36 male SD rats were randomly divited into 3 groups, each group contained 12 rats. Solutions contained MnCl2 of 5.0 g/L and 0.5 g/L were respectively administrated to the high dose and low dose Mn-exposed groups consecutively by oral drinking for 60 days, while distilled water was given to the contal group. Fluorospectrophotometry was carried out to exame the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP), as well as the protein level in liver plasmas. HE staining for the liver sections was applied to exam the pathological changes of liver in rats. RESULTS: (1) In fluorospe-ctrophotometry, activities of ALT, AST, ALP in high and low dose Mn-exposed groups were significantly higher than those in control group in liver plasmas. In high dose Mn-exposed groups, ALT, AST, ALP was respectively (60.79 +/- 9.88), (49.77 +/- 8.36) and (49.43 +/- 8.88) U/ml, P < 0.01, in low dose Mn-exposed groups, ALT,AST,ALP was respectively (47.35 +/- 8.11), (37.31 +/- 6.77) and (32.34 +/- 4.53) U/ml (P < 0.05). Significant relationships between effects and dose were obvious in the above changes. No changes in PRO levels was found among 3 groups. (2) Pathologically in high dose and low dose Mn-exposed groups the hepatic lobules were injured in different levels. Mainly the arrangements of some hepatic cords became loose or disordered and liver cells were somewhat different in shape and apparent volume. Some hepatic cells appeared lipopexic and numerous granulocytic immersion occurred around the central vein. CONCLUSION: manganese exposure resulted in the poisonous effects on liver in rats.

Effects of alpha1-adrenergic receptor blockade by doxazosin on renin-angiotensin system-regulating aminopeptidase and vasopressin-degrading activities in male and female rat thalamus.

The thalamus has connections with central autonomic centers involved in cardiovascular control and is enervated by noradrenergic fibers. The excitability of thalamic neurons is due to a reduction of ionic currents mediated by alpha(1)-adrenoceptors. The brain renin- angiotensin system (RAS) and the peptide hormone arginine-vasopressin (AVP) are also involved in the central control of blood pressure, and fluid and electrolyte homeostasis. It has been extensively reported that aminopeptidase A (APA), aminopeptidase B (APB), aminopeptidase N (APN), and vasopressin-degrading cystyl aminopeptidase activity (AVP-DA) play an important role in the regulation of the activity of angiotensins and AVP. We have analyzed the effect of alpha(1)-adrenoceptor blockade by doxazosin on RAS-regulating aminopeptidase activities and AVP-DA in soluble and membrane-bound fractions of male and female rat thalamus. Our results show that alpha(1)-adrenoceptors blockade by doxazosin does not modify the RAS through its degrading peptidases at thalamic level either in male or female rats. However, alpha(1)-adrenoceptors blockade shows gender differences in AVP-DA, increasing in males but not in females, supporting an increased capacity of males against females to degrade AVP and, therefore, to regulate cardiovascular homeostasis, under this pharmacological manipulation.

Aspartyl, arginyl and alanyl aminopeptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats.

Acid (aspartyl), basic (arginyl) and neutral (alanyl) aminopeptidases degrade angiotensins, vasopressin, oxytocin, bradykinin and enkephalins. These peptides regulate memory, energy homeostasis, water-salt balance and blood pressure, functions that are mainly exerted in the hippocampus and hypothalamus, and that can be affected by diabetes mellitus. To evaluate the relationship between the diabetes mellitus and processing and inactivation roles of these representative aminopeptidases, we measured their activities in both brain structures of control and streptozotocin-diabetic rats. Hypothalamic soluble aspartyl and arginyl aminopeptidases presented significant decreased activity levels in diabetic rats, which were mitigated by insulin therapy. In addition to membrane-bound puromycin sensitive and insensitive alanyl aminopeptidases, its soluble puromycin sensitive form did not differ between diabetic and control rats in both brain structures. Glucose and/or insulin did not seem to alter in vitro the hypothalamic activities of soluble aspartyl and arginyl aminopeptidases. The implied hypothalamic control of regulatory peptide activity by aspartyl and arginyl aminopeptidases supports the hypothesis that the hydrolytic ability of these enzyme types could be a common link for the disruptions of water-salt balance, blood pressure and energy homeostasis in diabetes mellitus.

[A study on anti-inflammatory and analgesic effects of alkaloids of Toddalia asiatica].

OBJECTIVE: To study the pharmacological activities and toxicity of the crude alkaloids of Toddalia asiatica and to provide pharmacological data for the further development of this herbal medicine. METHODS: We observed the anti-inflammatory effects of the crude alkaloids of Toddalia asiatica, using xylol and agra to induce the turgidness and sodium carboxymethyl cellulose (CMC-Na) to induce leucocyte strolling in the rats. The analgesic effects were observed by body-distortion methods. The effects of alkaloids of Toddalia asiatica on hepatic function were observed by testing the contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum and calculating the liver index. The LD 50 and 95% creditability were calculated with developed Karber Method. RESULTS: The administration of alkaloids of Toddalia asiatica had the function of inhibiting the auricle swelling caused by xylol and joint swelling caused by agar and leucocyte migration caused by CMC-Na, decreasing the body-distortion of the rats. After two weeks administration, the contents of ALT and AST showed that there was no obvious difference between administered group and control group. The LD50 of the crude alkaloids of Toddalia asiatica was 1.622 g/kg and the 95% creditability was 1.29-2.03 g/kg. CONCLUSION: Toddalia asiatica has anti-inflammatory and analgesic effects, and there is no injury to the liver after long-term administration in rats.

[Clinical observation on treatment of nonalcoholic fatty liver disease complicating hyperuricemia by Zhifang I Decoction].

OBJECTIVE: To observe the therapeutic effect of Chinese herbal recipe Zhifang I Decoction on nonalcoholic fatty liver disease (NAFLD) complicating hyperuricemia (HUA). METHODS: Forty-six patients suffering from NAFLD complicating HUA were randomly divided into treatment group (25 cases) with Zhifang I and control group (21 cases) with Xuezhikang Capsule. One course of treatment was 8 weeks. The data were processed by SPSS 11.0 statistical package after 2 courses of treatment. RESULTS: The total effective rate of the treatment group was 80.00%, which surpassed the control group (71.43%) (P<0.01); Zhifang I could improve the image of B-mode ultrasonography and was better than Xuezhikang in ameliorating the clinical symptoms (P<0.05); Zhifang I could significantly decrease the serum uric acid (UA) (P<0.01), while Xuezhikang had no obvious therapeutic effect on it (P>0.05); Zhifang I was as good as Xuezhikang in recovering alanine aminotransferase (ALT), aspartate aminotransferase (AST),gamma-glutamyltransferase (gamma-GT), total cholesterol (TC) and triglyceride (TG) (P>0.05). CONCLUSION: Zhifang I has good effect in treating NAFLD complicating HUA.

Brain renin-angiotensin system blockade by systemically active aminopeptidase A inhibitors: a potential treatment of salt-dependent hypertension.

The hyperactivity of the brain renin-angiotensin system (RAS) has been implicated in the development and maintenance of hypertension in several types of experimental and genetic hypertension animal models. We previously reported that in the murine brain, aminopeptidase A (APA) is involved in the conversion of angiotensin II (AngII) to AngIII and that AngIII is one of the main effector peptides of the brain RAS in the control of vasopressin release. Here we report that brain AngIII exerts a tonic stimulatory effect on blood pressure in a model of salt-dependent hypertension, the DOCA-salt rat, characterized by a depressed systemic but a hyperactive brain RAS. Similar high blood pressure accompanied by a low systemic renin state was described in some patients, especially in hypertensive African Americans who are resistant to treatment by blockers of the systemic RAS. We developed RB150, a prodrug of the specific and selective APA inhibitor, EC33. RB150 given i.v. is able to cross the blood-brain barrier, to inhibit brain APA, and to block the formation of central AngIII. A single dose of systemic RB150 (15 mg/kg, i.v.) in conscious DOCA-salt rats inhibited brain APA activity and markedly reduced blood pressure for up to 24 h. These results demonstrate the crucial role of brain APA as a candidate target for the treatment of hypertension and suggest that RB150, a potent systemically active APA inhibitor, could be the prototype of a new class of antihypertensive agents for the treatment of certain forms of hypertension.