Project description:Skeletal muscle mitochondrial dysfunction is secondary to T2DM and can be improved by long-term regular exercise training Mitochondrial dysfunction has long been implicated to play a causative role in development of type 2 diabetes (T2DM). However, a growing number of recent studies provide data that mitochondrial dysfunction is a consequence of T2DM development. The aim of our study is to clarify in further detail the causal role of mitochondrial dysfunction in T2DM by a comprehensive ex vivo analysis of mitochondrial function combined with global gene expression analysis in muscle of pre-diabetic newly diagnosed untreated T2DM subjects and long-standing insulin treated T2DM subjects compared with age- and BMI-matched controls. In addition, we assessed the impact of long-term interval exercise training on physical activity performance, mitochondrial function and glycemic control in long-standing insulin-treated T2DM subjects. Ex vivo mitochondrial density, quality and functioning was comparable between pre-diabetic subjects and matched controls, however, gene expression analysis showed a switch from carbohydrate toward lipids as energy source in pre-diabetes subjects. In contrast, long-term insulin treated T2DM subjects had slightly decreased mitochondrial density and ex vivo function. Expression of Krebs cycle and OXPHOS related genes were decreased, indicating a decreased capacity to use lipids as an energy source. The insulin-treated T2DM subjects had a lower physical activity level than pre-diabetic and normoglycemic subjects. A 52 weeks exercise training of these subjects increased submaximal oxidative efficiency, increased in vivo PCr recovery rate, as well as mildly increased in vitro mitochondrial function. Gene expression of β-oxidation, Krebs cycle and OXPHOS-related genes was increased. Our data demonstrate that mitochondrial dysfunction is rather a consequence than a causative factor in T2DM development as it was only detected in overt diabetes and not in early diabetes. Regular exercise training stabilized exogenous insulin requirement and improved mitochondrial functioning, fatty acid oxidation and general physical work load capacity in long-standing insulin-treated T2DM subjects. As such, the present study shows for the first time that long-term exercise interventions are beneficial in this group of complex diabetes patient and may prevent further metabolic deterioration.

Project description:We characterized the global response of plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase 9 subunit. The u-ATP9 transgene driven by A9 and Apetala3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and ATP levels, with an increase in ROS and a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by RT-PCR and enzymatic or functional tests, show dramatic changes in u-ATP9 plants. Both lines present a modification in the expression of several genes involved in carbon, lipid and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial biogenesis, protein synthesis, and degradation are affected. Moreover, several mRNA levels for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone response, suggesting that several signaling pathways overlap. Indeed, the transcriptome data reveal that the mitochondrial dysfunction dramatically alters genes involved in signaling pathways, including those involved in ethylene, absicic acid and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this rapport may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells. Arabidopsis oligonucleotide microarrays fabricated by the University of Arizona contain 26,000 oligonucleotides (http://www.ag.arizona.edu/microarray/). RNA was isolated from 6-week-old flowers from u-ATP9 and wt plants. The experimental (mutant) and reference (wild type) RNA samples were reverse-transcribed and directly labeled with either Cy5-dUTP or Cy3-dUTP fluorescent dye (GE Healthcare), using random hexamer primers (Invitrogen). Excess nucleotides and primers were removed using QIAquick PCR Purification Kit (Qiagen). Labeled samples were mixed and then hybridized to microarray for 15 h at 60°C. The slides were washed at room temperature in three wash steps: 2 x SSC, 0.5% SDS; 0.5 x SSC; and 0.05 x SSC for 5 min each with gentle shaking. The slides were scanned with a GenePix 4000B Scanner (Axon Instruments Inc., Union City, CA). Normalization between the Cy3 and Cy5 fluorescent dye emission channels was achieved by adjusting the levels of both image intensities. The experiments were repeated four times with samples from different experiments, as biological replicates. In dye swapping experiments, the RNA samples from different experiments were reciprocally labeled, both as a biological and technical repetition for comparing the reproducibility of the experiments. Hybridization intensities for each microarray element were measured using ScanAlyze 4.24 (available at http://genome-www4.stanford.edu/MicroArray/SMD/restech.html). The two channels were normalized in log space using the z-score normalization on a 95% trimmed data set. We removed unreliable spots according to the following criteria: spots flagged as having false intensity caused by dust or background on the array were removed; and spots for which intensity was less than three fold above background were also eliminated. Data from multiple experiments were normalized (Bolstad, 2003) and signals from spots from different experiments were statistically analyzed using Significance Analysis of Microarrays using the one class response (SAM, http://www-stat.stanford.edu/~tibs/SAM/), cut at a false discovery rate < 10% (Tusher, 2001).

Project description:DallePazze2014 - Cellular senescene-induced mitochondrial dysfunction This model is described in the article: Dynamic modelling of pathways to cellular senescence reveals strategies for targeted interventions. Dalle Pezze P, Nelson G, Otten EG, Korolchuk VI, Kirkwood TB, von Zglinicki T, Shanley DP. PLoS Comput. Biol. 2014 Aug; 10(8): e1003728 Abstract: Cellular senescence, a state of irreversible cell cycle arrest, is thought to help protect an organism from cancer, yet also contributes to ageing. The changes which occur in senescence are controlled by networks of multiple signalling and feedback pathways at the cellular level, and the interplay between these is difficult to predict and understand. To unravel the intrinsic challenges of understanding such a highly networked system, we have taken a systems biology approach to cellular senescence. We report a detailed analysis of senescence signalling via DNA damage, insulin-TOR, FoxO3a transcription factors, oxidative stress response, mitochondrial regulation and mitophagy. We show in silico and in vitro that inhibition of reactive oxygen species can prevent loss of mitochondrial membrane potential, whilst inhibition of mTOR shows a partial rescue of mitochondrial mass changes during establishment of senescence. Dual inhibition of ROS and mTOR in vitro confirmed computational model predictions that it was possible to further reduce senescence-induced mitochondrial dysfunction and DNA double-strand breaks. However, these interventions were unable to abrogate the senescence-induced mitochondrial dysfunction completely, and we identified decreased mitochondrial fission as the potential driving force for increased mitochondrial mass via prevention of mitophagy. Dynamic sensitivity analysis of the model showed the network stabilised at a new late state of cellular senescence. This was characterised by poor network sensitivity, high signalling noise, low cellular energy, high inflammation and permanent cell cycle arrest suggesting an unsatisfactory outcome for treatments aiming to delay or reverse cellular senescence at late time points. Combinatorial targeted interventions are therefore possible for intervening in the cellular pathway to senescence, but in the cases identified here, are only capable of delaying senescence onset. This model is hosted on BioModels Database and identified by: BIOMD0000000582. To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals

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