| 1. |
- Fredriksson, Katarina, et al.
(författare)
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Dysregulation of Mitochondrial Dynamics and the Muscle Transcriptome in ICU Patients Suffering from Sepsis Induced Multiple Organ Failure
- 2008
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 3:11, s. e3686-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Septic patients treated in the intensive care unit (ICU) often develop multiple organ failure including persistent skeletal muscle dysfunction which results in the patient's protracted recovery process. We have demonstrated that muscle mitochondrial enzyme activities are impaired in septic ICU patients impairing cellular energy balance, which will interfere with muscle function and metabolism. Here we use detailed phenotyping and genomics to elucidate mechanisms leading to these impairments and the molecular consequences. Methodology/Principal Findings: Utilising biopsy material from seventeen patients and ten age-matched controls we demonstrate that neither mitochondrial in vivo protein synthesis nor expression of mitochondrial genes are compromised. Indeed, there was partial activation of the mitochondrial biogenesis pathway involving NRF2a/GABP and its target genes TFAM, TFB1M and TFB2M yet clearly this failed to maintain mitochondrial function. We therefore utilised transcript profiling and pathway analysis of ICU patient skeletal muscle to generate insight into the molecular defects driving loss of muscle function and metabolic homeostasis. Gene ontology analysis of Affymetrix analysis demonstrated substantial loss of muscle specific genes, a global oxidative stress response related to most probably cytokine signalling, altered insulin related signalling and a substantial overlap between patients and muscle wasting/inflammatory animal models. MicroRNA 21 processing appeared defective suggesting that post-transcriptional protein synthesis regulation is altered by disruption of tissue microRNA expression. Finally, we were able to demonstrate that the phenotype of skeletal muscle in ICU patients is not merely one of inactivity, it appears to be an actively remodelling tissue, influenced by several mediators, all of which may be open to manipulation with the aim to improve clinical outcome. Conclusions/Significance: This first combined protein and transcriptome based analysis of human skeletal muscle obtained from septic patients demonstrated that losses of mitochondria and muscle mass are accompanied by sustained protein synthesis (anabolic process) while dysregulation of transcription programmes appears to fail to compensate for increased damage and proteolysis. Our analysis identified both validated and novel clinically tractable targets to manipulate these failing processes and pursuit of these could lead to new potential treatments.
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| 2. |
- Scheele, Camilla, et al.
(författare)
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Altered regulation of the PINK1 locus: a link between Type 2 diabetes and neurodegeneration?
- 2007
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 21:13, s. 3653-3665
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in PINK1 cause the mitochondrial-related neurodegenerative disease Parkinson’s. Here we investigate whether obesity, type 2 diabetes, or inactivity alters transcription from the PINK1 locus. We utilized a cDNA-array and quantitative real-time PCR for gene expression analysis of muscle from healthy volunteers following physical inactivity, and muscle and adipose tissue from nonobese or obese subjects with normal glucose tolerance or type 2 diabetes. Functional studies of PINK1 were performed utilizing RNA interference in cell culture models. Following inactivity, the PINK1 locus had an opposing regulation pattern (PINK1 was down-regulated while natural antisense PINK1 was up-regulated). In type 2 diabetes skeletal muscle, all transcripts from the PINK1 locus were suppressed and gene expression correlated with diabetes status. RNA interference of PINK1 in human neuronal cell lines impaired basal glucose uptake. In adipose tissue, mitochondrial gene expression correlated with PINK1 expression although remained unaltered following siRNA knockdown of Pink1 in primary cultures of brown preadipocytes. In conclusion, regulation of the PINK1 locus, previously linked to neurodegenerative disease, is altered in obesity, type 2 diabetes and inactivity, while the combination of RNAi experiments and clinical data suggests a role for PINK1 in cell energetics rather than in mitochondrial biogenesis.
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| 3. |
- Scheele, Camilla
(författare)
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Functional genomics studies of PINK1
- 2007
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Functional genomics has become an important and established research discipline during the last 10 years, mainly as a consequence of the completion of large-scale genome sequencing projects. The human genome is now predicted to transcribe 20,000-25,000 protein coding genes, which is only a quarter of the number suggested a few years ago. Instead, a dynamic RNA universe seems to provide diversity to mammalian cells. Around 60-70% of protein coding genes are predicted to generate two or more transcripts by alternative splicing, while non-protein coding RNA are also directly transcribed from the genome. One category of such non-protein coding RNA is cistranscribed natural antisense (NAT). Cis-NATs are transcribed from a gene s antisense DNA strand and are suggested to have regulatory functions through direct interaction. The work described in this thesis includes functional genomics studies of the PINK1 locus. We undertook a study to discover novel candidate genes associated with physical inactivity, a known risk factor for type 2 diabetes. Gene expression profiling of skeletal muscle from subjects before and after 5 weeks of inactivity by quantitative real-time PCR demonstrated a co-ordinated reduction in mitochondrial gene expression. We thus established a human in vivo model for mitochondrial dysfunction. Microarray analyses of the same sample-set suggested that PINK1, a novel mitochondrial kinase, was down regulated during inactivity. PINK1 is transcribed from a complex locus, alternatively spliced and with an annotated cis-NAT. Mutations at this locus had also been linked to Parkinson s disease and we thus selected this locus for subsequent functional genomics studies. We utilized human in vivo models, gene expression and genomic association analysis and RNA interference (RNAi) to study the regulation of PINK1. We demonstrated dynamic expression from the PINK1 locus during modulation of mitochondria in vivo in human skeletal muscle. PINK1 was down regulated in our mitochondrial dysfunction model, while a shorter splice variant of PINK1 (svPINK1) and the NAT (naPINK1) were concordantly up regulated. The opposite expression pattern was obtained in a human in vivo model for increased mitochondrial activity, suggesting a direct association between svPINK1 and naPINK1. Knockdown of naPINK1 utilizing siRNAs targeting two different sites of naPINK1 reduced the level of svPINK1. This directly supports a role for naPINK1 in promoting the abundance of svPINK1, a novel mechanism for regulation by natural antisense. In contrast, all transcripts from the PINK1 locus were less abundant in muscle tissue from diabetics, compared to healthy controls. To investigate whether PINK1 transcript levels could affect metabolic fitness or if the lower expression rather was a secondary effect of diabetes, we measured PINK1 tagging single nucleotide polymorphisms (SNPs). Several SNPs associated with PINK1 transcripts levels. The genotypes associating with higher expression of PINK1 also associated with lower plasma levels of non-esterified fatty acid levels (NEFA) and glucose. Two sets of RNA interference studies provided support for these clinical associations. Firstly, knockdown of PINK1 in human neuroblastoma cells resulted in impaired basal glucose uptake. Secondly, FABP4, a lipid transport protein, was selectively down regulated following PINK1 knockdown in adipocytes. However, mitochondrial genes were not altered when PINK1 expression was ablated, despite the in vivo association between such genes. Taken together, our data suggest a role of PINK1 in lipid and glucose metabolism while PINK1 does not appear to be essential for mitochondrial biogenesis in mammalian cells.
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| 4. |
- Timmons, James A, et al.
(författare)
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Expression profiling following local muscle inactivity in humans provides new perspective on diabetes-related genes
- 2006
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Ingår i: Genomics. - : Elsevier BV. - 0888-7543 .- 1089-8646. ; 87:1, s. 165-172
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Tidskriftsartikel (refereegranskat)abstract
- Physical activity enhances muscle mitochondrial gene expression, while inactivity and mitochondrial dysfunction are both risk factors for developing diabetes. Defective activation of the transcriptional coactivator PGC-1 may contribute to the gene expression pattern observed in diabetic and insulin-resistant skeletal muscle. We proposed that greater insight into the mitochondrial component of skeletal muscle “diabetes” would be possible if the clinical transcriptome data were contrasted with local muscle inactivity-induced modulation of mitochondrial genes in otherwise healthy subjects. We studied PPARGC1A (PGC-1), PPARGC1B (PGC-1β), NRF1, and a variety of mitochondrial DNA (mtDNA) and nuclear-encoded mitochondrial genes critical for oxidative phosphorylation in soleus muscle biopsies obtained from six healthy men and women before and after 5 weeks of local muscle inactivity. Muscle inactivity resulted in a coordinated down-regulation of PGC-1 and genes involved with mitochondrial metabolism, including muscle substrate delivery genes. Decreased expression of the mtDNA helicase Twinkle was related to the decline in mitochondrial RNA polymerase (r = 0.83, p < 0.04), suggesting that mtDNA transcription and replication are coregulated in human muscle tissue. In contrast to the situation in diabetes, PGC-1β expression was not significantly altered, while NRF1 expression was actually up-regulated following muscle inactivity. We can conclude that reduced PGC-1 expression described in Type 2 diabetes may be partly explained by muscle inactivity. Further, although diabetes patients are typically inactive, our analysis indicates that local muscle inactivity may not be expected to contribute to the decreased NRF1 and PGC-1β expression noted in insulin-resistant and Type 2 diabetes patients, suggesting these changes may be more disease specific.
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| 5. |
- Timmons, James A., et al.
(författare)
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Using molecular classification to predict gains in maximal aerobic capacity following endurance exercise training in humans
- 2010
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Ingår i: Journal of applied physiology. - : American Physiological Society. - 8750-7587 .- 1522-1601. ; 108:6, s. 1487-1496
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Tidskriftsartikel (refereegranskat)abstract
- Timmons JA, Knudsen S, Rankinen T, Koch LG, Sarzynski M, Jensen T, Keller P, Scheele C, Vollaard NB, Nielsen S, Akerstrom T, MacDougald OA, Jansson E, Greenhaff PL, Tarnopolsky MA, van Loon LJ, Pedersen BK, Sundberg CJ, Wahlestedt C, Britton SL, Bouchard C. Using molecular classification to predict gains in maximal aerobic capacity following endurance exercise training in humans. J Appl Physiol 108: 1487-1496, 2010. First published February 4, 2010; doi:10.1152/japplphysiol.01295.2009.-A low maximal oxygen consumption ((V) over dotO(2max)) is a strong risk factor for premature mortality. Supervised endurance exercise training increases (V) over dotO(2max) with a very wide range of effectiveness in humans. Discovering the DNA variants that contribute to this heterogeneity typically requires substantial sample sizes. In the present study, we first use RNA expression profiling to produce a molecular classifier that predicts (V) over dotO(2max) training response. We then hypothesized that the classifier genes would harbor DNA variants that contributed to the heterogeneous (V) over dotO(2max) response. Two independent preintervention RNA expression data sets were generated (n = 41 gene chips) from subjects that underwent supervised endurance training: one identified and the second blindly validated an RNA expression signature that predicted change in (V) over dotO(2max) (""predictor"" genes). The HERITAGE Family Study (n = 473) was used for genotyping. We discovered a 29-RNA signature that predicted (V) over dotO(2max) training response on a continuous scale; these genes contained similar to 6 new single-nucleotide polymorphisms associated with gains in (V) over dotO(2max) in the HERITAGE Family Study. Three of four novel candidate genes from the HERITAGE Family Study were confirmed as RNA predictor genes (i.e., ""reciprocal"" RNA validation of a quantitative trait locus genotype), enhancing the performance of the 29-RNA-based predictor. Notably, RNA abundance for the predictor genes was unchanged by exercise training, supporting the idea that expression was preset by genetic variation. Regression analysis yielded a model where 11 single-nucleotide polymorphisms explained 23% of the variance in gains in (V) over dotO(2max), corresponding to similar to 50% of the estimated genetic variance for (V) over dotO(2max). In conclusion, combining RNA profiling with single-gene DNA marker association analysis yields a strongly validated molecular predictor with meaningful explanatory power. (V) over dotO(2max) responses to endurance training can be predicted by measuring a similar to 30-gene RNA expression signature in muscle prior to training. The general approach taken could accelerate the discovery of genetic biomarkers, sufficiently discrete for diagnostic purposes, for a range of physiological and pharmacological phenotypes in humans.
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