Pohl, Elena E.

Rupprecht, Anne

Sokolenko, Elena A.

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10P.15 Identification and characterization of uncoupling protein
in fat body and muscle mitochondria isolated from cockroach
Gromphadorhina coquereliana
Malgorzata Slocinska1, Nina Antos-Krzeminska2,
Grzegorz Rosinski1, Wieslawa Jarmuszkiewicz2
1Departament of Animal Physiology and Development,
Adam Mickiewicz University, Poznan, Poland
2Departament of Bioenergetics, Adam Mickiewicz University, Poznan,
Poland
E-mail: slocina@amu.edu.pl
Uncoupling proteins (UCPs) are members of the mitochondrial
anion carrier family dissipating the electrochemical proton gradient
across the inner mitochondrial membrane. Homologues including
closely related UCP1–UCP3 and more diverged UCP4 and UCP5 were
found in animals, plants, fungi and protists. The first insect
uncoupling protein, dmUCP was identified in Drosophila melanogaster
and characterized in the heterologous yeast system. The aim of our
study was to characterize UCP in mitochondria of cockroach
Grompadorhina coquereliana isolated from a trophic tissue of fat body
(insect analogue of mammalian liver) and skeletal muscle (a high
metabolic energy tissue). Bioenergetic studies support evidence for
the functioning of UCP in both tissues. UCP activity was subsequently
stimulated by addition of micromolar concentrations of fatty acids
(palmitic acid and linoleic acid) and inhibited by the purine
nucleotide GTP. Cockroach UCP was immunodetected by polyclonal
antibodies raised against mouse UCP2, human UCP3, UCP4 and UCP5.
All detections showed a single around 36 kDa band. Expression of
UCP in cockroach muscle mitochondria was approximately three
times higher than that in mitochondria isolated from fat body. LC–
MS/MS analysis revealed peptides with high sequence identity of the
purified protein to previously characterized UCP4 in human and D.
melanogaster. The discovery of UCP protein in cockroach G. coquer-
eliana might have implications for understanding the bioenergetics,
metabolism and function of fat body and muscle in insects.
This work was supported by the grant (N N 303291634) of the
Polish Ministry of Science.
doi:10.1016/j.bbabio.2010.04.271
10P.16 Mitochondrial uncoupling protein 4 (UCP4) expression
in rat inner ear during development and after oxidative
stress induction
Alina Smorodchenko1, Anne Rupprecht1, Julia Fuchs2,
Johann Gross2, Elena E. Pohl1
1Institute of Molecular Physiology and Biophysics,
University of Veterinary Medicine, Vienna, Austria
2Department of Othorhinolaryngology, Charité–Universitätsmedizin Berlin,
Berlin, Germany
E-mail: Alina.Smorodchenko@vetmeduni.ac.at
UCP4 belongs to the mitochondrial anion transporter family and is
proposed to participate in the regulation of free radical production,
calcium homeostasis, thermogenesis and apoptosis in mammalian
brain. Using a new designed antibody we have previously shown that
UCP4 appears in fetal murine brain tissue as early as on embryonic
days 12–14 and suggested that it may be involved in neuronal cell
differentiation (Smorodchenko A et al., 2009). Here we proved the
hypothesis that UCP4 contributes to the regulation of oxidative stress,
using the model from our previous work (Gross J et al., 2007). For this
we compared UCP4 expression level in freshly isolated explants of
organ of Corti (OC), modiolus (MOD) and stria vascularis (SV) from
neonatal rats with explants cultured under normoxic or hypoxic
conditions. Western Blot analysis showed that protein level is
decreased in MOD both at normoxic and hypoxic conditions but
was not changed in OC and spiral limbus. UCP4 down-regulation
coincided with the decrease of VDAC content and possibly reflected
the mitochondrial damage during oxidative stress. Comparing further
the UCP4 level at different postnatal stages we found that proteinwas
developmentally regulated and its expressionwas region-specific. We
hypothesized that UCP4 may play an important role in functional
maturation of rat inner ear.
References
[1] Gross J et al. (2007) Brain Res. 1162: 56–68.
[2] Smorodchenko A et al. (2009) Biochim. Biophys. Acta 1788.10:
2309–2319.
doi:10.1016/j.bbabio.2010.04.272
10P.17 Involving of 4-hydroxy-2-nonenal in the activation of UCP1
Elena A. Sokolenko1,2, Anne Rupprecht1,2, Elena E. Pohl1,2
1Department of Biomedical Sciences, University of Veterinary Medicine,
Vienna, Austria
2Institute of Cell Biology and Neurobiology,
Charité – Universitätsmedizin, Berlin, Germany
E-mail: elena.pohl@vetmeduni.ac.at
Uncoupling proteins (UCPs) is a subfamily in a family of
mitochondrial membrane anion carriers. One of the most controver-
sially discussed UCP function is their ability to regulate mitochondrial
ROS production and/or to be regulated by ROS or ROS derivatives.
Here, we investigated whether the aldehydic product of lipid
peroxidation 4-hydroxy-2-nonenal (HNE) activates UCP1. For that
we used a well-defined model of lipid membranes reconstituted with
recombinant UCP1 and arachidonic acid, AA [1, 2]. The comparison of
protein-free and protein-containing membranes in the presence of
HNE demonstrated no direct activating effect of aldehyde on UCP1.
However, the addition of HNE led to the strong potentiation of the
AA-mediated increase of the membrane conductance, Gm, both in
UCP-containing and in UCP-free membranes. The application of high
potentials [3] additionally stimulated the Gm increase. Alteration of
lipid membrane properties due to addition of HNE is proposed to be
responsible for the observed protein-independent HNE activity.
References
[1] Beck V et al. (2006) Biochim. Biophys. Acta 1757: 474–479.
[2] Beck V et al. (2007) FASEB J. 21: 1137 1144.
[3] Rupprecht A et al. (2010) Biophys. J. 98: 1–9.
doi:10.1016/j.bbabio.2010.04.273
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Involving of 4-hydroxy-2-nonenal in the activation of UCP1 

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Involving of 4-hydroxy-2-nonenal in the activation of UCP1

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