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Biochemical and molecular targets of dysfunction on fatty acid transport and mitochondrial oxidation induced by steatogenic drugs
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Resumo(s)
Mitochondrial dysfunction induced by steatogenic drugs may result from several biochemical
mechanisms, of which some have already been described whereas many others remain far
to be elucidated. The studies presented in this thesis were planned to bring new insights on
the interaction of some of these drugs with mitochondrial fatty acid oxidation (FAO) and to
explore other potential targets, not only inside mitochondria but also in the extramitochondrial
compartment. Previous work from our group provided important mechanistic
explanations underlying the toxicity of one specific drug, i.e. valproic acid (VPA), used as a
model of a steatogenic drug. A number of unanswered queries have also been raised along
this research work, and thus, the studies presented in this thesis using VPA represent new
advances in this field, focused on the impairment of mitochondrial functions. Additionally, the
non-steroidal anti-inflammatory drug Ibuprofen was also studied in order to test whether the
mechanisms found to explain specific aspects of mitochondrial toxicity of VPA could also be
observed in Ibuprofen-induced steatosis.
The first part of this thesis comprises a general introduction to the experimental work. This
includes the objectives of the present thesis (chapter 1) and a review of the literature
(chapter 2) covering the different pathways which may be affected by VPA and other
steatogenic drugs, as well as the consequences of a general mitochondrial dysfunction.
The interplay of mitochondrial FAO with VPA metabolism was evaluated in several
perspectives. First, an additional novel step on VPA biotransformation in the extramitochondrial
compartment of the cell was proposed and clarified (chapter 3). For this
purpose, purified mitochondrial and cytosolic fractions obtained from livers of VPA-treated
rats were analysed in search for the activated metabolite of VPA, valproyl-CoA (VP-CoA).
The result provided evidence that VPA is not only activated in the mitochondrial matrix,
where it can be further oxidized, but can also be activated in the extra-mitochondrial space.
The characterization of the extra-mitochondrial synthesis of VP-CoA was performed in vitro
using the specific acyl-CoA synthetase substrates. It was concluded that in the cell there are
two possible enzymes that can catalyse the formation of VP-CoA, one in the mitochondria
and another in the cytosol. A similar bi-compartmental activation of ibuprofen was also
observed since the cytosolic formation of ibuprofenoyl-CoA was demonstrated in parallel
with the drug’s activation in the mitochondria (chapter 5). The activation to CoA-esters of
carboxylic steatogenic drugs may deplete the correspondent concentrations of CoA in the respective cellular compartments, since CoA does not cross mitochondrial membranes. This
hypothesis might be on the basis of a major cause for mitochondrial FAO impairment. The
accumulation of long-chain fatty acids in the cytosol can thus result from cytosolic depletion
of CoA.
The formation of extra-mitochondrial VP-CoA opened up new perspectives and generated
novel hypotheses concerning the interference of this drug with cytosolic reactions that could
also contribute to mitochondrial dysfunction in particular, and to cellular dysregulation in
general. The interaction of extra-mitochondrial CoA esters of VPA with the transport of longchain
fatty acids (LCFA) to the mitochondrial matrix by the carnitine shuttle was evaluated. A
competition between VP-CoA as formed in the cytosol and endogenous long-chain fatty
acyl-CoA’s for CPT I activity (chapter 4) was observed in vitro, using human control
fibroblasts. Additionally, the mechanism of interaction of VP-CoA with CPT I was
characterized using three plasmids of rat CPT IA with different sensitivities towards malonyl-
CoA. The results obtained with these proteins suggested that both VP-CoA and malonyl-
CoA inhibit CPT I activity but through different mechanisms. Also, evidence is provided that
VP-CoA interferes at the catalytic domain of CPT I affecting the sensitivity of this enzyme for
malonyl-CoA. The interference of VP-CoA with this pivotal enzyme in mitochondrial FAO
may be a crucial mechanism in the drug-induced hepatotoxicity and the weight gain
frequently observed in patients under VPA therapy.
The in vivo urinary excretion of carnitine esters derived from VPA metabolism, namely
valproylcarnitine, prompted us to identify the drug metabolizing enzyme responsible for the
formation of this drug conjugate and led us to investigate the possibility that extramitochondrial
VP-CoA might be a substrate for carnitine acyltransferases (chapter 6).
Several yeast expressed enzymes, such as CPT I, CPT II, COT, from different species (rat,
mouse and human), were incubated with the acyl-CoA substrates and carnitine, plus VPCoA.
Surprisingly, none of the studied carnitine acyltransferases was able to synthesize
valproylcarnitine from VP-CoA. Also, the fact that CPT I could not transfer the acyl moiety of
VP-CoA to carnitine, suggests that extra-mitochondrial VP-CoA is not transported into the
mitochondrial matrix, but rather may accumulate in the cytosol aggravating the toxicity
induced by this drug. Valproylcarnitine was also not formed in mouse intestinal bacteria after
incubation with VP-CoA but it is suggested that it can be formed by direct esterification of
VPA with carnitine. It is still not clear if urinary valproylcarnitine excretion can result from
bacterial metabolism of VPA and which enzyme is playing this role. It was previously demonstrated by our group that the VPA-induced impairment of
mitochondrial FAO was also associated with an indirect mechanism, the inhibition of
pyruvate-driven oxidative phosphorylation, and subsequent reduction on the energetic
control of the cell. This effect was further investigated based on the possible unavailability of
mitochondrial pyruvate (chapter 7) triggered by the interference of VPA or its cytosolic
products (VP-CoA and D
4-VPA) with the transport of this substrate across mitochondrial
membranes. Using a controlled inner-mitochondrial membrane system, the proton-driven
uptake of [14C]-pyruvate was quantified and the effect of VPA as well as some important
metabolic intermediates evaluated. The obtained results demonstrate that the activated CoA
metabolites of VPA and D
4-VPA strongly inhibit the uptake of pyruvate in mitochondrial
membranes, respectively by 40% and 60%.
A major consequence of FAO inhibition, either induced by CoA depletion or by other abovementioned
mechanisms, involves the decreased formation of acetyl-CoA, the end product of
FAO. Considering the crucial role of acetyl-CoA as a central point of convergence in many
other major metabolic pathways, their functions will be ultimately affected if acetyl-CoA
production decreases. This is true for the urea cycle, responsible for the elimination of
excess of nitrogen resulting from amino acid degradation. The relation between VPA therapy
and urea cycle is undisputed, based on the clinical hyperammonemia frequently observed in
VPA-treated patients. The biochemical mechanisms underlying this adverse effect were also
studied (chapter 8), giving special attention to the critical role of N-acetylglutamate (NAG) in
the regulation of the flux through the urea cycle. The synthesis of NAG was found to be
inhibited by VP-CoA, and the characterization of this inhibition was performed. In addition to
the reduced levels of acetyl-CoA observed as result of FAO inhibition, the depletion of NAG
may strongly aggravate the accumulation of ammonia induced by VPA. The potential
combination of other therapeutics with VPA was also evaluated in order to reduce this
undesired side effect.
The final part of this thesis includes a global discussion of the major results obtained and
conclusions. A comprehensive analysis of the different studied perspectives and the impact
of the results presented in this thesis on the different mitochondrial and extra-mitochondrial
reactions that are linked to fatty acid transport and mitochondrial oxidation, hepatic steatosis
and drug-induced dysfunction will be presented (chapter 9).
Descrição
Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2010
Palavras-chave
Bioquímica Teses de doutoramento - 2010