Orientador: Adelar Bracht

Data da Defesa: 11/12/2013



INTRODUCTION p-Synephrine and octopamine have been identified as important active components in preparations of Citrus aurantium and other citrus species. The fruit of C. aurantium, commonly known as bitter orange, is sometimes used as a food, but it is more widely ingested as a medicinal or dietary supplement. Extracts of C. aurantium have been claimed to promote weight loss. The active ingredients of C. aurantium include various alkaloids with adrenergic activity, including p-synephrine and octopamine. Structurally, these active components in C. aurantium are closely related to endogenous neurotransmitters and ephedrine. p-Synephrine is similar in structure to epinephrine and octopamine is similar to norepinephrine. Recently it was shown that octopamine and psynephrine activate lipolysis both in rat and human adipocytes. These amines, however, are much less efficient in human than in rat adipocytes. Evidence has been presented that octopamine and p-synephrine are 3-agonists in mammalian fat cells. Up to now little attention has been given to the possible effects of octopamine and p-synephrine on the liver cells. The ingestion of C. aurantium preparations for weight loss purposes is obviously directed toward the fat cells, but one cannot avoid metabolic actions on other tissues, especially on the liver which is the metabolic organ par excellence and the first that receives orally ingested compounds via the portal vein. A recent first approach to the effects of the C. aurantium amines in the rat liver has shown that commercial extracts are able to affect several hepatic metabolic variables. The effects include stimulations of glycogen catabolism (glycogenolysis) and oxygen uptake and inhibition of gluconeogenesis. Hemodynamic effects were observed as well, more precisely a pronounced increase in the portal perfusion pressure. The effects on glycogenolysis, oxygen uptake and hemodynamics are sensitive to 1- and 1-2- adrenergic antagonists, but are insensitive to the 3-antagonist SR59230A. Also, most of the effects of the C. aurantium extract were reproduced by 200 µM psynephrine. The main exception was gluconeogenesis inhibition, which was not found with 200 µM p-synephrine and is thus exerted by other compounds in the C. aurantium extract. On the other hand, the action of octopamine on liver metabolism is unknown, as no experiments on the subject have so far been reported. AIMS Taking into account what was exposed above, the aims of the present work were: (1) to establish the concentration dependences of the effects of psynephrine on carbohydrate metabolism and hemodynamics in the liver, their sensitivity to various hormone antagonists and their possible dependence on cofactors such as calcium ions and cAMP. The latter experiments should inform about the participation of Ca2+ and cAMP in the signalling pathways leading to the hemodynamic and metabolic effects of p-synephrine; (2) to characterize the metabolic and hemodynamic effects of octopamine in the liver by measuring variables related to both carbohydrate and fatty acid metabolism, by investigating their concentration dependences and their sensitivity to adrenergic antagonists; 9 MATERIAL AND METHODS Male Wistar rats weighing 200-280 g were used in all experiments. Animals were fed ad libitum with a standard laboratory diet (Nuvilab®, Colombo, Brazil) and maintained on a regulated light-dark cycle. In accordance with the requirements of the experimental protocols, fed rats as well as 18 h fasted rats were used. Hemoglobin-free, non-recirculating liver perfusion was performed. After cannulation of the portal and cava veins the liver was positioned in a plexiglass chamber. The constant perfusate flow was provided by a peristaltic pump and adjusted between 30 and 33 ml per minute, depending on the liver weight. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4) containing 25 mg% bovine-serum albumin, saturated with a mixture of oxygen and carbon dioxide (95:5) by means of a membrane oxygenator with simultaneous temperature adjustment (37 °C). In the effluent perfusion fluid the following compounds were assayed by means of standard enzymatic procedures: glucose, lactate and pyruvate. The oxygen concentration in the outflowing perfusate was monitored continuously, employing a Teflon-shielded platinum electrode adequately positioned in a plexiglass chamber at the exit of the perfusate. Metabolic rates were calculated from input-output differences and the total flow rates and were referred to the wet weight of the liver. The 14CO2 resulting from [1- 14C]octanoate or [1- 14C]oleate transformation was trapped in phenylethylamine and the radioactivity was measured by liquid scintillation spectroscopy. 45Ca was also quantified by liquid scintillation counting. Measurement of adenosine 2,5 cyclic monophosphate (cAMP) was done by an ELISA commercial kit (cAMP EIA kit from Enzo Life Sciences). The portal perfusion pressure was monitored by means of a pressure transducer (Hugo Sachs Elektronic-Harvard Apparatus GmbH, March-Hugstetten, Germany). The sensor was positioned near the entry of the portal vein, and the transducer was connected to a recorder. RESULTS AND DISCUSSION Octopamine and p-synephrine increased glycogenolysis, glycolysis from endogenous glycogen, oxygen uptake driven mainly by endogenous fatty acids, and lactate gluconeogenesis in a concentration-dependent manner in the range up to 500 µM and presented saturation at the highest concentrations or even diminution. The latter phenomenon occurred with both octopamine and psynephrine especially in the case of the lactate driven gluconeogenesis and the associated increment in oxygen uptake. Table 1 (next page) allows to compare the effectiveness of both compounds in terms of their concentrations for halfmaximal stimulation. The data in the table above reveal that in most cases psynephrine is more effective than octopamine with the exception of the oxygen uptake driven by endogenous fatty acids. The portal perfusion pressure was considerably increased by both psynephrine and octopamine, their actions being close to the maximal at the concentration of 10 µM. Octopamine also accelerated the oxidation of exogenous fatty acids (octanoate and oleate), as revealed by the increase in 14CO2 production derived from the 14C-labeled precursors and the increase in ketogenesis. 10 Table 1 Variable Concentrations for half-maximal stimulation p-Synephrine (µM) Octopamine (µM) Glucose output (glycogenolysis) 26.1 70.7 Glucose output (gluconeogenesis) 4.1 ~40 Oxygen uptake (endogenous fatty acids oxidation) 8.95


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