CAROLINE TESSARO MOREIRA

Título da Dissertação: METABOLIC ALTERATIONS CAUSED BY USNIC ACID IN THE LIVER

Orientador: Prof. Dr. Adelar Bracht

Data da Defesa: 28/08/2012

RESUMO GERAL

Aims

 Usnic acid is a naturally occurring dibenzofuran derivative found in several lichen species. The compound has been marketed as an ingredient of food supplements for weight reduction. There is evidence that the compound acts as an uncoupler of mitochondrial oxidative phosphorylation and it is also clear that consumption of the drug can lead to severe hepatotoxicity depending on the dosis. The most obvious consequence of the uncoupling action of usnic acid is ATP depletion, as indeed demonstrated in two studies using hepatocytes. However, this phenomenon is probably linked directly or indirectly to a variety of modifications in metabolic fluxes. Although the toxic manifestations at the cellular level have been more extensively investigated, a precise quantification of individual metabolic pathways under the influence of usnic acid is still not available. A meaningful evaluation of individual metabolic pathways is best made in the isolated perfused rat liver, a system in which true metabolic steady-state conditions can be established under conditions that are much closer to the physiological ones than isolated and cultured cells. The present study, thus, takes advantage of the perfused liver in quantifying the metabolic effects of usnic acid on both catabolic and anabolic pathways under various conditions. The results should enhance understanding of the metabolic effects of usnic acid and also allow to compare the action of this compound with that of other drugs.

 Experimental approach

 Male Wistar rats (180-220 g), fed ad libitum with a standard laboratory diet were used. The main experimental system was the isolated perfused rat liver. The liver was perfused in the non-recirculation mode. The perfusion fluid was Krebs/Henseleit-bicarbonate buffer (pH 7.4), saturated with O₂ and CO₂ (95:5) by means of a membrane oxygenator and simultaneously heated to 37 °C. The oxygen concentration in the perfusate was monitored continuously by means of polarography. The tissue content of adenine mononucleotides and the perfusate contents of other metabolites (glucose, lactate, pyruvate, b-hydroxybutyrate and acetoacetate) were determined by enzymatic methods. The ¹⁴CO₂ production from [1-¹⁴C]octanoate or [1-¹⁴C]oleate was quantified by liquid scintillation counting after trapping ¹⁴CO₂ with phenylethylamine. Fluxes were calculated from the portal-venous differences and the total flow through the liver and referred to the wet liver weight.

  Results

 1) Usnic acid caused stable increases in oxygen consumption at con-centrations up to 2.5 mM during substrate-free perfusion and in the presence of gluconeogenic substrates; at higher concentrations oxygen uptake stimulation was generally smaller and transient in nature.

2) Glycolysis and glycogenolysis from endogenous glycogen were linearly stimulated by usnic acid in the range up to 10 mM. Usnic acid also increased the lactate to pyruvate ratio, which closely reflects the cytosolic NADH/NAD⁺ ratio.

3) Usnic acid also stimulated fructolysis (fructose degradation to lactate and pyruvate); in this case the effect was a saturable function of the concentration.

4) Gluconeogenesis was inhibited, the IC₅₀ value depending on the substrate: lactate, 1.33 mM; fructose, 1.72 mM; alanine 3.61 mM. For all substrates, however, inhibition of gluconeogenesis was virtually complete at 10 mM usnic acid.

5) Usnic acid was also strongly inhibitory for urea production, with an IC₅₀ value of 2.69 mM; ammonia production, on the other hand, was stimulated, with half-maximal stimulation occurring at 1.92 mM. The L-glutamate production was also diminished by usnic acid.

6) The ¹⁴CO₂ production from [1-¹⁴C]-octanoate and [1-¹⁴C]oleate was increased by usnic acid, but ketogenesis from octanoate was diminished and that from oleate was not affected. Usnic acid did not increase oxygen upake in the presence of either octanoate or oleate. It decreased, however, the b-hydroxy-butyrate/acetoacetate ratio, an indicator for the mitochondrial NADH/NAD⁺ ratio. This decrease was much more pronounced in the presence of octanoate.

7) Concerning the hepatic contents of adenine mononucleotides, the general tendency as the usnic acid concentration was raised from 2.5  to 10 mM, was an increase in the hepatic ADP and AMP contents and a decrease in the ATP contents. With 10 mM usnic acid the ATP content was dropped to only 31% of the control value. At this usnic acid concentration both the ADP and AMP contents were also significantly higher than those in the control condition.

 Conclusions

 The conclusion is allowed that the effects of usnic acid up to 2.5 mM reflect predominantly its activity as an uncoupler. The observations corroborating uncoupling, which have also been traditionally reported for other uncouplers of oxidative phosphorylation, are: a) stimulation of oxygen consumption at low concentrations; b) diminution of the ATP content combined with diminutions in the ATP/ADP and ATP/AMP ratios; c) increase in the cytosolic NADH/NAD⁺ ratio; d) inhibition of gluconeogenesis from three different substrates, namely lactate, fructose and alanine; e) stimulation of glycolysis and fructolysis as a cytosolic compensatory phenomenon for the diminished mitochondrial ATP production; f) stimulation of glycogenolysis as a means of providing glucose 6-phosphate for the increased glycolytic flux; (g) increase in ammonia production.

Additional observations suggest that usnic acid is able to exert other effects besides uncoupling. One of these effects is inhibition of electron flow in the respiratory chain. Another effect is a direct inhibitory action on ketogenesis.

The numerous metabolic effects of usnic acid tend, in principle at least, to be harmful to the organism.  It is true that there are well fundamented claims that mild uncoupling can be beneficial for health. Mild uncoupling seems to trigger molecular mechanisms that are able to increase the expression of mitochondrial genes  and it has also been claimed that it increases longevity in mice associated with the improvement of several serological markers.  In the case of usnic acid, however, it seems that the very narrow concentration range of its action makes it very difficult to adjust the injested doses in such a way as to avoid toxic and harmful manifestations. Especially noteworthy are the combined inhibitory effects on gluconeogenesis and ureagenesis, which occur both at similar low concentrations, unlike what happens with other uncouplers. 

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