• Publikační typ
• abstrakt z konference MeSH

We investigated hydrogen peroxide production in mitochondria with low (liver, heart, brain) and high (brown adipose tissue, BAT) content of glycerophosphate dehydrogenase (mGPDH). ROS production at state 4 due to electron backflow from mGPDH was low, but after inhibition of electron transport with antimycin A high rates of mGPDH-dependent ROS production were observed in liver, heart and brain mitochondria. When this ROS production was related to activity of mGPDH, many-fold higher ROS production was found in contrast to succinate- (39-, 28-, 3-fold) or pyruvate plus malate-dependent ROS production (32-, 96-, 5-fold). This specific rate of mGPDH-dependent ROS production was also exceedingly higher (28-, 66-, 22-fold) compared to that in BAT. mGPDH-dependent ROS production was localized to the dehydrogenase+CoQ and complex III, the latter being the highest in all mitochondria but BAT. Our results demonstrate high efficiency of mGPDH-dependent ROS production in mammalian mitochondria with a low content of mGPDH and suggest its endogenous inhibition in BAT.

• MeSH
• antimycin A farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hnědá tuková tkáň metabolismus   MeSH
• jaterní mitochondrie metabolismus   účinky léků   MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• kyselina jantarová metabolismus   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• mitochondrie metabolismus   účinky léků   MeSH
• mozek metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• potkani Wistar MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• srdeční mitochondrie metabolismus   účinky léků   MeSH
• techniky in vitro MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH

Prostate cancer is one of the most prominent cancers diagnosed in males. Contrasting with other cancer types, glucose utilization is not increased in prostate carcinoma cells as they employ different metabolic adaptations involving mitochondria as a source of energy and intermediates required for rapid cell growth. In this regard, prostate cancer cells were associated with higher activity of mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key rate limiting component of the glycerophosphate shuttle, which connects mitochondrial and cytosolic processes and plays significant role in cellular bioenergetics. Our research focused on the role of mGPDH biogenesis and regulation in prostate cancer compared to healthy cells. We show that the 42 amino acid presequence is cleaved from N-terminus during mGPDH biogenesis. Only the processed form is part of the mGPDH dimer that is the prominent functional enzyme entity. We demonstrate that mGPDH overexpression enhances the wound healing ability in prostate cancer cells. As mGPDH is at the crossroad of glycolysis, lipogenesis and oxidative metabolism, regulation of its activity by intramitochondrial processing might represent rapid means of cellular metabolic adaptations.

• MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• mitochondrie genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty genetika   metabolismus   MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial respiratory chain enzyme Complexes are present in placenta at proportion similar to other tissues with exception of glycerophosphate dehydrogenase (mGPDH) which is expressed at a very high rate. As shown by Western blot quantification and respiratory chain enzyme activity measurements, the specific content of mGPDH is similar to that of succinate dehydrogenase or NADH dehydrogenase. Using fluorometric probe dichlorodihydrofluorescein diacetate we found that placental mitochondria display high rate of glycerophosphate-dependent hydrogen peroxide production. This was confirmed by oxygraphic detection of glycerophosphate-induced, KCN- or antimycin A-insensitive oxygen uptake. Hydrogen peroxide production by mGPDH was highly activated by one-electron acceptor, potassium ferricyanide and it was depressed by inhibitors of mGPDH and by cytochrome c. Our results indicate that mGPDH should be considered as an additional source of reactive oxygen species participating in induction of oxidative stress in placenta.

• MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• kyslík metabolismus   MeSH
• lidé MeSH
• mitochondrie enzymologie   MeSH
• oxidoreduktasy metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• placenta enzymologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Overproduction of reactive oxygen species (ROS) has been implicated in a range of pathologies. Mitochondrial flavin dehydrogenases glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH) represent important ROS source, but the mechanism of electron leak is still poorly understood. To investigate the ROS production by the isolated dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements and hydrogen peroxide production studies by Amplex Red fluorescence, and luminol luminescence in combination with oxygraphy revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q as the site of ROS production in the case of mGPDH. Distinct mechanism of ROS production by the two dehydrogenases is also apparent from induction of ROS generation by ferricyanide which is unique for mGPDH. Furthermore, using native electrophoretic systems, we demonstrated that mGPDH associates into homooligomers as well as high molecular weight supercomplexes, which represent native forms of mGPDH in the membrane. By this approach, we also directly demonstrated that isolated mGPDH itself as well as its supramolecular assemblies are all capable of ROS production.

• MeSH
• ferrikyanidy metabolismus   MeSH
• glycerolfosfátdehydrogenasa chemie   metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• krysa rodu rattus MeSH
• mitochondrie enzymologie   metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• savci MeSH
• sukcinátdehydrogenasa chemie   metabolismus   MeSH
• transport elektronů * MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.

• MeSH
• alosterická regulace MeSH
• glycerolfosfátdehydrogenasa genetika   fyziologie   MeSH
• glykolýza MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondrie enzymologie   MeSH
• orgánová specificita MeSH
• oxidativní fosforylace MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• sukcinátdehydrogenasa metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

α-Tocopheryl succinate (TOS), a redox-silent analogue of vitamin E, suppresses cell growth in a number of clinical and experimental cancers, inhibits mitochondrial succinate dehydrogenase (SDH) and activates reactive oxygen species (ROS) generation. The aim of this study was to test whether TOS also inhibits glycerol-3-phosphate dehydrogenase (mGPDH), another flavoprotein-dependent enzyme of the mitochondrial respiratory chain because there are differences between electron transfer pathway from SDH and mGPDH to coenzyme Q pool. For our experiments brown adipose tissue mitochondria with high expression of mGPDH were used. Our data showed that inhibition of glycerol-3-phosphate (GP)-dependent oxygen consumption by TOS was more pronounced than the succinate (SUC)-dependent one (50% inhibition was reached at 10 μmol/l TOS vs. 80 μmol/l TOS, respectively). A comparison of the inhibitory effect of TOS on GP-oxidase, GP-cytochrome c oxidoreductase and GP-dehydrogenase activities showed that TOS directly interacts with the dehydrogenase. After TOS application the GP-dependent generation of ROS was highly depressed. It may thus be concluded that TOS-induced inhibition of mGPDH is more pronounced than TOS-induced inhibition of SDH and that the inhibitory effect of TOS for both substrates is exerted at different locations of the particular dehydrogenases. Our data indicate that the inhibition of mGPDH activity could also play a role in TOS-induced growth suppression in neoplastic cells.

• MeSH
• alfa-tokoferol aplikace a dávkování   MeSH
• glycerolfosfátdehydrogenasa antagonisté a inhibitory   biosyntéza   MeSH
• hnědá tuková tkáň enzymologie   MeSH
• karcinogeneze genetika   MeSH
• křečci praví MeSH
• lidé MeSH
• mitochondrie účinky léků   enzymologie   MeSH
• nádory farmakoterapie   enzymologie   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• spotřeba kyslíku účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

• MeSH
• antimycin A analogy a deriváty   farmakologie   MeSH
• buněčné dýchání MeSH
• elektronová paramagnetická rezonance MeSH
• ethidium analogy a deriváty   chemie   MeSH
• ferrikyanidy farmakologie   MeSH
• financování organizované MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• glycerolfosfáty metabolismus   MeSH
• hnědá tuková tkáň enzymologie   účinky léků   ultrastruktura   MeSH
• křečci praví MeSH
• mitochondrie enzymologie   metabolismus   účinky léků   MeSH
• reaktivní formy kyslíku analýza   metabolismus   MeSH
• respirační komplex III metabolismus   MeSH
• spotřeba kyslíku MeSH
• transport elektronů MeSH
• ubichinon metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• zvířata MeSH