Browsing by Subject "OXPHOS"

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  • Tomasic, Nikica; Kotarsky, Heike; Figueiredo, Rejane de Oliveira; Hansson, Eva; Mörgelin, Matthias; Tomasic, Ivan; Kallijärvi, Jukka; Elmer, Eskil; Jauhiainen, Matti; Eklund, Erik A.; Fellman, Vineta (2020)
    Mice homozygous for the human GRACILE syndrome mutation (Bcs1l (c.A232G)) display decreased respiratory chain complex III activity, liver dysfunction, hypoglycemia, rapid loss of white adipose tissue and early death. To assess the underlying mechanism of the lipodystrophy in homozygous mice (Bcs1l(p.S)(78G)), these and wild-type control mice were subjected to a short 4-hour fast. The homozygotes had low baseline blood glucose values, but a similar decrease in response to fasting as in wild-type mice, resulting in hypoglycemia in the majority. Despite the already depleted glycogen and increased triacylglycerol content in the mutant livers, the mice responded to fasting by further depletion and increase, respectively. Increased plasma free fatty acids (FAs) upon fasting suggested normal capacity for mobilization of lipids from white adipose tissue into circulation. Strikingly, however, serum glycerol concentration was not increased concomitantly with free FM, suggesting its rapid uptake into the liver and utilization for fuel or gluconeogenesis in the mutants. The mutant hepatocyte mitochondria were capable of responding to fasting by appropriate morphological changes, as analyzed by electron microscopy, and by increasing respiration. Mutants showed increased hepatic gene expression of major metabolic controllers typically associated with fasting response (Ppargc1a, Fgf21, Cd36) already in the fed state, suggesting a chronic starvation-like metabolic condition. Despite this, the mutant mice responded largely normally to fasting by increasing hepatic respiration and switching to FA utilization, indicating that the mechanisms driving these adaptations are not compromised by the CIII dysfunction. Summary statement: Bcs1l mutant mice with severe CIII deficiency, energy deprivation and post-weaning lipolysis respond to fasting similarly to wild-type mice, suggesting largely normal systemic lipid mobilization and utilization mechanisms.
  • Hulmi, Juha J.; Penna, Fabio; Pöllänen, Noora; Nissinen, Tuuli A.; Hentila, Jaakko; Euro, Liliya; Lautaoja, Juulia H.; Ballarò, Riccardo; Soliymani, Rabah; Baumann, Marc; Ritvos, Olli; Pirinen, Eija; Lalowski, Maciej (2020)
    Objective Cancer cachexia and muscle loss are associated with increased morbidity and mortality. In preclinical animal models, blocking activin receptor (ACVR) ligands has improved survival and prevented muscle wasting in cancer cachexia without an effect on tumour growth. However, the underlying mechanisms are poorly understood. The present study aimed to identify cancer cachexia and soluble ACVR (sACVR) administration-evoked changes in muscle proteome. Methods Healthy and C26 tumour-bearing (TB) mice were treated with recombinant sACVR. The sACVR or PBS control were administered either prior to the tumour formation or by continued administration before and after tumour formation. Muscles were analysed by quantitative proteomics with further examination of mitochondria and nicotinamide adenine dinucleotide (NAD+) metabolism. To complement the first prophylactic experiment, sACVR (or PBS) was injected as a treatment following tumour cell inoculation. Results Muscle proteomics in TB cachectic mice revealed downregulated signatures for mitochondrial oxidative phosphorylation (OXPHOS) and increased acute phase response (APR). These were accompanied by muscle NAD+ deficiency, alterations in NAD+ biosynthesis including downregulation of nicotinamide riboside kinase 2 (Nrk2), and decreased muscle protein synthesis. The disturbances in NAD+ metabolism and protein synthesis were rescued upontreatment with sACVR. Across the whole proteome and APR in particular, Serpina3n represented the most upregulated protein and the strongest predictor of cachexia. However, the increase in Serpina3n expression associated with increased inflammation rather than decreased muscle mass and/or protein synthesis. Conclusions We present here an evidence implicating disturbed muscle mitochondrial OXPHOS proteome and NAD+ homeostasis in experimental cancer cachexia. Treatment of tumour-bearing mice with a blocker of activin receptor ligands restores depleted muscle NAD+ and Nrk2 as well as decreased muscle protein synthesis. These results point out putative new treatment therapies for cachexia. Our results also reveal that although acute phase protein Serpina3n may serve as a predictor of cachexia, it more likely reflects a condition of elevated inflammation.
  • Hulmi, Juha; Penna, Fabio; Pöllänen, Noora; Nissinen, Tuuli; Hentilä, Jaakko; Euro, Liliya; Lautaoja, Juulia; Ballarò, Riccardo; Soliymani, Rabah; Baumann, Marc; Ritvos, Olli; Pirinen, Eija; Lalowski, Maciej (Helsingin yliopisto, 2022)
    Syöpäkakeksia on patofysiologialtaan vielä suurelta osin tuntematon, monitekijäinen aineenvaihdunnallinen sairaus, jossa paino ja lihaskudoksen määrä laskevat. Kakeksia vaikuttaa suuren osaan syöpäpotilaista lisäten heidän kuolleisuuttaan ja heikentäen elämänlaatua. Eläinmalleilla lihaskadon estäminen kokeellisella hoidolla, aktiviinireseptorin salpauksella, on parantanut selviytymistä ja vähentänyt lihasmassan katoa, vaikka syöpä itsessään ei ole parantunut. Tutkimuksemme tavoitteena oli selvittää tämän hoidon vaikutusmekanismi, sillä se on tällä hetkellä vielä epäselvää. Tutkimuksessa hiirille injektoitiin suolistosyöpäsoluja (C26) selän rasvakudokseen, mikä aiheuttaa pahanlaatuisen kasvaimen muodostumisen ja nopean lihaskadon. Hiirille annettiin kokeellista hoitoa yhdisteellä, joka estää lihasmassan kasvua rajoittavien proteiinien, aktiviinien ja myostatiinien, kiinnittymistä niiden solureseptoreihin. Havaitsimme laajoissa proteiinianalyyseissa akuutin faasin reaktion, erityisesti Serpina3n-proteiinin, lisääntymisen ja lihaksen mitokondrioiden energiaa tuottavan oksidatiivisen fosforylaation (OXPHOS) komponenttien määrän vähentyneen syöpäryhmässä. Niinpä analysoimme tarkemmin mitokondrioiden aktiivisuutta histokemiallisesti in situ ja määritimme mitokondrioiden energiantuotantoreaktioissa toimivan elintärkeän kofaktorin, nikotiiniamidiadeniinidinukleotidin (NAD+), pitoisuuksia lihaskudoksessa. Mitokondrioaktiivisuus in situ oli osittain heikentynyt syöpäryhmässä, jossa myös NAD+:n ja sen metaboliitin NADH:n määrät vähentyivät. Lisäksi havaitsimme voimakkaan laskun NAD+-biosynteesiin osallistuvan geenin, Nrk2:den ilmaantumisessa. Proteiineista Serpina3n määrä korreloi selkeimmin painonlaskuun, mutta se paljastui ennemmin hoitamattoman kakeksian kuin yleisen lihasmassan biomarkkeriksi. Serpina3n-määrä yhdistyi myös Nrk2:den ilmentymiseen, joka viittaisi yhteyteen lihaksen Nrk2-ekspressiossa ja tulehduksen säätelyssä akuutin faasin reaktion kautta. Aktiviinireseptorin salpauksella pystyimme korjaamaan NAD+-aineenvaihdunnan häiriötä ja lieventämään OXPHOS-muutoksia. Osoitimme tässä tutkimuksessa ensimmäistä kertaa, että syöpäkakeksian taustalla on NAD+-aineenvaihdunnan häiriö ja että aktiviinireseptorien salpauksella voidaan lihaskadon estämisen lisäksi parantaa NAD+-tasoja. Vielä ei tiedetä, vaikuttaako tämä kokeellinen hoito suoraan vai välillisesti NAD+-aineenvaihduntaan. Tulevaisuudessa olisi lisäksi tärkeä tutkia Nrk2:den roolia syöpäkakeksiassa ja selvittää, voidaanko NAD+-esiasteita käyttää syöpäkakeksian hoitona.
  • Tonttila, Kialiina (Helsingin yliopisto, 2021)
    Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.
  • Thompson, Kyle; Mai, Nicole; Oláhová, Monika; Scialó, Filippo; Formosa, Luke E; Stroud, David A; Garrett, Madeleine; Lax, Nichola Z; Robertson, Fiona M; Jou, Cristina; Nascimento, Andres; Ortez, Carlos; Jimenez-Mallebrera, Cecilia; Hardy, Steven A; He, Langping; Brown, Garry K; Marttinen, Paula; McFarland, Robert; Sanz, Alberto; Battersby, Brendan J; Bonnen, Penelope E; Ryan, Michael T; Chrzanowska-Lightowlers, Zofia Ma; Lightowlers, Robert N; Taylor, Robert W (2018)
    OXA1, the mitochondrial member of the YidC/Alb3/Oxa1 membrane protein insertase family, is required for the assembly of oxidative phosphorylation complexes IV and V in yeast. However, depletion of human OXA1 (OXA1L) was previously reported to impair assembly of complexes I and V only. We report a patient presenting with severe encephalopathy, hypotonia and developmental delay who died at 5 years showing complex IV deficiency in skeletal muscle. Whole exome sequencing identified biallelic OXA1L variants (c.500507dup, p.(Ser170Glnfs*18) and c.620G>T, p.(Cys207Phe)) that segregated with disease. Patient muscle and fibroblasts showed decreased OXA1L and subunits of complexes IV and V. Crucially, expression of wild-type human OXA1L in patient fibroblasts rescued the complex IV and V defects. Targeted depletion of OXA1L in human cells or Drosophila melanogaster caused defects in the assembly of complexes I, IV and V, consistent with patient data. Immunoprecipitation of OXA1L revealed the enrichment of mtDNA-encoded subunits of complexes I, IV and V. Our data verify the pathogenicity of these OXA1L variants and demonstrate that OXA1L is required for the assembly of multiple respiratory chain complexes.