MARCOS RODRIGUES MALDONADO

Título da Dissertação: Efeitos da p-sinefrina sobre atividades enzimáticas e níveis de adenina nucleotídeos no fígado de rato.

Orientador: Prof. Dr. Adelar Bracht

Data da Defesa: 22/08/2017

RESUMO GERAL

INTRODUCTION
p-synephrine is one of the main active components of bitter orange (Citrus aurantium). The current worldwide consumption of formulations containing p-synephrine has often been justified by its properties of weight loss promotion and ability to increase sports performance. Its chemical structure, similar to that of catecholamines, resembles epinephrine. It has been reported that p-synephrine binds to serotonergic as well as to adrenergic receptors. In humans, p-synephrine alone or in combination with other substances, caused an increase in the resting metabolic rate and energy expenditure and was able to promote modest weight loss when consumed for 6 to 12 weeks. Measurements in isolated cell systems and in the perfused rat liver revealed several effects that could, at least in part, justify these claims. The liver is the first organ to receive the drug after oral ingestion. Its rapid transformation in this organ suggests that this is also the place where the drug acts more intensely after an oral intake, since the other organs receive only a fraction of the total absorbed in the digestive tract. In isolated liver perfusion, synephrine was active on metabolism and hemodynamics, since it significantly stimulated glycogenolysis, glycolysis, gluconeogenesis, and oxygen consumption and increased portal perfusion pressure. Some of these phenomena are compatible with weight loss promotion and the increased energy performance reported for p-synephrine. These effects were demonstrated only in vitro, but their in vivo occurrence has not yet been demonstrated.
AIMS
The objectives of this study were to evaluate the activities of enzymes indicative of the metabolic effects of p-synephrine and the influence of this substance on the cellular energy charge in the isolated perfused rat liver as well as in vivo.
MATERIAL AND METHODS
The activities of pyruvate kinase, pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and glycogen phosphorylase and the content of adenine mononucleotides were determined in the isolated perfused rat liver (25 and 100 μM) and in vivo following oral administration of p-synephrine (50 and 300 mg/kg) using standard techniques.
RESULTS AND DISCUSSION
Glycogen phosphorylase a activity was considerably higher in the perfused liver when compared to the in vivo situation. In both cases, however, p-synephrine increased the enzyme activity. In vivo stimulation at the dose of 50 mg/kg was equal to 30%, but increased to 115% at the dose of 300 mg/kg. In the perfused liver, the percentage of phosphorylase a formation caused by p-synephrine was clearly less pronounced.
The activity of pyruvate kinase was decreased in both the perfused liver and in vivo. Both concentrations/doses employed, however, caused similar decreases. In the perfused liver, these decreases were about 35% and in vivo equal to 22.5%.
The activity of pyruvate dehydrogenase was also decreased by p-synephrine. Contrary to what happened with pyruvate kinase, in the case of pyruvate dehydrogenase there was a clear dose/concentration dependence relationship. In vivo, doses of 50 and 300 mg/kg caused decreases of 60 and 79%, respectively. In the perfused liver the concentrations of 25 and 100 μM caused decreases of 40 and 66%, respectively.
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In isolated liver perfusion the α-ketoglutarate dehydrogenase activity was decreased by 58% by 100 μM p-synephrine. No significant modification was found at the 25 μM concentration. In vivo, on the other hand, at the lowest dose (50 mg/kg), there was an increase in the α-ketoglutarate dehydrogenase activity (105%). No modification was found, however, at the dose of 300 mg/kg.
Regarding the hepatic content of adenine mononucleotides, the infusion of 100 μM p-synephrine in the perfused liver increased the ATP and ADP levels. The total content (ATP+ADP+AMP) was increased by 27%. The ATP/ADP ratio showed a decreasing tendency (from 4.00±0.11 to 3.54±0.14). The ATP/AMP ratio, on the other hand, increased from 9.34±0.52 to 13.07±1.12. Similar modifications were found in vivo, 1 hour after administration of p-synephrine (300 mg/kg). The ATP content was increased by 55% and a tendency to increase ADP was found. The ATP/ADP ratio was not modified (2.00±0.19 and 2.19±0.27 for control and treatment, respectively). There was also a slight trend towards an increased AMP content, so the ATP/AMP ratio did not change significantly. The total adenine mononucleotide content, on the other hand, was increased by 47%.
Our experiments represent attempts of confirming in the living rat effects that were reported to occur in vitro (e.g., glycogenolysis stimulation) and effects that might suggest a mechanism able to enhance or modify metabolic pathways that could justify the claims about the weight-loss properties or sport performance enhancements.
The increase in glycogen phosphorylase activity by p-synephrine found in the present work, for example, is one of these effects. This was an expected phenomenon due to the observed stimulation of glycogenolysis in isolated perfused rat liver liver and to the increase of blood glucose concentration caused by the administration of p-synephrine. Nevertheless, observation of the in vivo phenomenon confirms that concentrations of at least 10 μM were reached in the portal vein after oral administration, as this is the minimum concentration at which increased glucose production can be expected based on the experiments with liver perfusion performed in previous studies.
The observed decrease in pyruvate dehydrogenase (PDH) activity may indicate that p-synephrine is potentially capable of inhibiting the transformation of carbohydrates into lipids. PDH is essential for the complete oxidation of carbohydrates into CO2 and H2O. On the other hand, the formation of acetyl-CoA by PDH is also essential for the transformation of carbohydrates into lipids. The PDH enzymatic complex is inactivated by phosphorylation, catalyzed by pyruvate dehydrogenase kinase. This enzyme, in turn, is activated when one or more of the following proportions in the mitochondrial space is increased: ATP/ADP, NADH/NAD+ and acetyl-CoA/CoA. The exact mechanism of how p-synephrine is acting can not be deduced from the available data. The overall proportion of ATP/ADP was not increased by p-synephrine, an observation that does not exclude, however, that the mitochondrial ATP/ADP ratio has been modified. On the other hand, considering that the increase in the oxygen consumption rate caused by p-synephrine observed in previous studies is mainly due to the increased oxidation of fatty acids, it is also reasonable to expect an increased acetyl-CoA/CoA ratio, which could be one of the factors responsible for the decrease of the pyruvate dehydrogenase activity.
The only parameter measured in this work for which different results were obtained in vivo and liver perfusion was the α-ketoglutarate dehydrogenase activity. In the perfused liver there was a decrease in the activity of this enzyme in the highest concentration (100 μM), which could reflect in a decrease of the oxygen consumption in the perfused liver. What actually occurs, however, is the increase in oxygen consumption. Therefore, this observation can be considered, at least in principle, as an indication that, in vivo, the observed fluctuations in α-ketoglutarate dehydrogenase activity do not reflect changes in oxygen consumption.
p-Synephrine increased the total content of adenine mononucleotides (increase in ATP and ADP contents, with no change in ATP/ADP ratio). Possibly, synephrine is capable of enhancing the de novo synthesis of adenine mononucleotides. The stimulation of the rescue route can not be excluded either. Synthesis of adenine mononucleotides requires glucose (as a source of ribose 5'-phosphate) and amino acids. Even in liver perfused without substrate an increase in the supply of glucosyl units was available due to increased glycogenolysis. Nucleotide synthesis is an energy intensive process that uses multiple pathways in various cell compartments. Regulation is highly complex and the elucidation of the mechanism by which p-synephrine raises the levels of adenine mononucleotides necessitates specific experimental approaches that are beyond the scope of the present work. Intense exercise usually leads to degradation of adenine mononucleotides in tissues such as muscles and liver. The effect of p-synephrine as an activator of adenine mononucleotide synthesis, however, could be an interesting phenomenon if we consider the alleged property of the compound as a sports performance enhancer. This is a point which, by its obvious implications, clearly deserves further investigation.
CONCLUSIONS
The results obtained in this work reveal two possible mechanisms by which p-synephrine can exert physiological effects and which may, at least partially, justify the claims that the compound exerts weight-loss effects and enhances sports performance. Data were obtained showing that the compound could inhibit the transformation of carbohydrates into lipids and increase the levels of adenine mononucleotides in the liver. Whether these effects occur in humans will depend on the concentrations that will be achieved in the portal vein. However, with the low doses that are normally ingested by humans (rarely over 100 mg), concentrations of p-synephrine above 10 μM in the portal vein can only happen if the absorption is rapid and even so for a short period of time. In our view, it is highly desirable to conduct clinical experiments at higher but non-toxic doses to find out if the effects described in the present work can be confirmed in humans.
Key words: liver metabolism; metabolic effectors; natural protoalkaloids; weight loss effect; sport peformance.

 Artigos Publicados Vinculados a Dissertação:

https://onlinelibrary.wiley.com/doi/10.1002/cbf.3311