Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 45
Identification of signaling pathway leading to increase of mitochondrial cytochrome-c
oxidase (COX) and mitofusin-2 (Mfn-2) proteins during insulin-mediated myogenesis in
vitro
Patrycja Pawlikowska1, Barbara Gajkowska2, Micha# M. Godlewski1, Arkadiusz
Orzechowski1*
1*Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw
Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland, e-mail:
orzechowski@alpha.sggw.waw.pl, 2Department of Cell Ultrastructure MRC, Polish
Academy of Sciences, Warsaw, Poland, *corresponding author.
Mitochondrial fusion mediated by Mfn-2 has been recently shown to play a crucial role in the
embryonic development, antiapoptosis through DNA protection against free radicals-induced
lesions and maintaining unchanged mtDNA pool. The purpose of this study was to examine
the role of mitochondria in insulin-dependent myogenesis in vitro. Two differently encoded
(nuclear and mitochondrial) subunits of cytochrome-c oxidase (COX), myogenin and Mfn-2
expressions were determined by Western blots. Our studies with LY294002 confirmed
assumption that insulin stimulates myogenesis though PI3-K-dependent signaling pathway.
Similarly, if MAPKK (MEK) was inhibited by PD98059 insulin-mediated myogenesis from
C2C12 muscle cells was accelerated. Additionally, immunoblots revealed that insulin raised
the expression of subunit I and IV of COX in L6 muscle cells. Apparently, insulin-dependent
induction of COX I was mediated by PI3-K, since blockade of insulin action with LY294002
resulted in decreased level of mitochondrial but not nuclear encoded subunit of COX.
Moreover, electron and confocal microscopy showed that insulin activates formation of a
network of elongated, tubular mitochondria. Increased mitochondrial length and
interconnectivity was not observed upon PI3-K inhibition with LY294002. Subsequently, we
decided to study transmembrane GTPase – Mfn-2 which seems to be essential for
mitochondrial fusion. We observed that insulin induces Mfn-2 protein in L6 myoblasts.
Moreover, we found that inhibition of MAPKK-dependent signaling pathway additionally
elevated both Mfn-2 and myogenin protein levels. Although correlation does not prove
causation, we speculate that Mfn-2 participates in insulin-mediated muscle differentiation. We
conclude that insulin-dependent myogenesis is associated with increased level of COX I and
Mfn-2 as well as augments fusion of mitochondria. The above-mentioned effects are inhibited
by LY294002, whereas inhibition of MAPKK with PD98059 led to additional amplification
of this phenomenon.
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