Papers by Umberto De Marchi
Nature Communications
Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as h... more Sustained ryanodine receptor (RyR) Ca2+ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca2+ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca2+ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca2+ leak or preventing mitochondrial Ca2+ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that trigg...
The large number of homoplasmic variants identified in the NMRI strain does not have a major impa... more The large number of homoplasmic variants identified in the NMRI strain does not have a major impact on the mpileup reported coverage. The relative coverage reported by mpileup was plotted against each single position of the mouse reference genome for both the B6D2F1 and the NMRI datasets. The only noticeable differences are two NMRI specific "extreme" drops of coverage (positions 5'205 and 9'821) resulting from near homoplasmic indels (see the Additional file 2 for details on "extreme" coverage drops). (PPTX 264 kb)
MitoRS output file for the two plasmids sequenced for the benchmark of RCA. Tab1: Raw MitoRS outp... more MitoRS output file for the two plasmids sequenced for the benchmark of RCA. Tab1: Raw MitoRS output for plasmid1 (P1). Sample names are prefixed "Crude" when no amplification was done and "RCA" when rolling circle amplification was performed. The four replicates are named A, B, C and D. The data for the eight sequencing experiments are populated in consecutive columns. Column headers are detailed in the Additional files 14: Table S1 legend. Tab2: Raw MitoRS output for plasmid2 (P2). Same as for Plasmid1. (XLSX 6141 kb)
Mouse SNV frequencies are homogenous within the 88 position analyzed. A. Individual SNV frequency... more Mouse SNV frequencies are homogenous within the 88 position analyzed. A. Individual SNV frequency. For the 12 mouse mtDNA mixture ratios tested, the frequencies measured for each SNV (88 in total) were plotted and summarized as a boxplot. The boxplot whiskers highlight the extreme values (min and max). Note that the scale is different for each plot. The eight positions showing the highest frequency underestimation are highlighted in blue. These data were used to build the Fig. 4. The raw data are available from the Additional file 7. B. The eight SNV with underestimated frequency are located into two dense clusters. For each 88 SNV, the deviation from the theoretical frequency was calculated and plotted versus their position in the mitochondrial genome. The eight positions showing a systematic frequency underestimation highlighted in A. are also shown in blue. They cluster into two very short genomic regions. (PPTX 484 kb)
Supporting Information. (DOCX 56Â kb)
Figure S3. Glucose-dependent regulated proteins. A) Volcano plots displaying the distribution of ... more Figure S3. Glucose-dependent regulated proteins. A) Volcano plots displaying the distribution of significant regulated proteins overtime. Proteins significantly changed (p-value 0.3 or
Figure S1. Randomization of the samples and conditions for the proteomic analysis. TMT labelling ... more Figure S1. Randomization of the samples and conditions for the proteomic analysis. TMT labelling was performed as indicated with the code of colors. Stimulation with PMA was also included in the experiments but the results were previously reported [28]. We present here the results of the glucose stimulation for the time series. (PDF 40 kb)
Figure S5. Kinase-substrate enrichment analysis upon 5, 30 and 60 min of continuous glucose stimu... more Figure S5. Kinase-substrate enrichment analysis upon 5, 30 and 60 min of continuous glucose stimulation. A) Heatmaps containing KSEA scores for every experimental replicate at 5, 30 and 60 min (from left to right). For kinases higher KSEA positive scores (in red) indicates higher activity whereas negative scores (in blue) indicates lower activity. Conversely, for phosphatases higher KSEA positive scores (in red) indicates lower activity whereas negative scores (in blue) indicates higher activity. The statistical significance of the KSEA score was evaluated, p-value (*** p
Figure S4. Gene Ontology enrichment analysis in phosphoproteins exclusively regulated either at 5... more Figure S4. Gene Ontology enrichment analysis in phosphoproteins exclusively regulated either at 5, 30 or 60 min. Heatmap displaying the top 30 differentially enriched ontology terms overtime considering proteins containing p-sites exclusively regulated at specific time points. (PDF 1764 kb)
The FASEB Journal, 2018
In pancreatic b-cells, mitochondria generate signals that promote insulin granule exocytosis. Her... more In pancreatic b-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca 2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in b-cell energy metabolism and Ca 2+ signaling.
Table S9. List of drugs tested for their ability to regulate mitochondrial respiration induced by... more Table S9. List of drugs tested for their ability to regulate mitochondrial respiration induced by glucose. The table displays information on the drug tested, targeted mechanism, effect of the interaction and the range of concentrations tested. (XLSX 9 kb)
Journal of the International Society of Antioxidants in Nutrition & Health, 2015
The pancreatic beta-cells possess a unique signal transduction system linking metabolism of nutri... more The pancreatic beta-cells possess a unique signal transduction system linking metabolism of nutrients to the secretion of insulin. Mitochondria play a crucial role in this process through the generation of ATP and other factors, raising intracellular Ca2+ for the stimulation of insulin secretion. Here we have compared the signals generated by glucose and the mitochondrial complex-II substrate methylsuccinate, in pancreatic INS-1E beta-cells. Glucose and methylsuccinate promoted distinct patterns of oxygen consumption and induced nutrient-stimulated insulin secretion. Surprisingly, only glucose was able to initiate intracellular [Ca2+] rises. In contrast, methylsuccinate-stimulated INS-1E cells showed little Ca2+ rises and even depression of the signals. Glucose was further associated with an increase in the autofluorescence of NAD(P)H. Consistently, the redox-sensitive roGFP1 signal revealed a net reduction of the mitochondrial matrix in response to glucose. This redox response was ...
International Journal of Molecular Sciences
Accumulation of calcium in energized mitochondria of pancreatic β-cells is emerging as a crucial ... more Accumulation of calcium in energized mitochondria of pancreatic β-cells is emerging as a crucial process for pancreatic β-cell function. β-cell mitochondria sense and shape calcium signals, linking the metabolism of glucose and other secretagogues to the generation of signals that promote insulin secretion during nutrient stimulation. Here, we describe the role of mitochondrial calcium signaling in pancreatic β-cell function. We report the latest pharmacological and genetic findings, including the first mitochondrial calcium-targeted intervention strategies developed to modulate pancreatic β-cell function and their potential relevance in the context of diabetes.
Structure of the output csv table. Example of an output file generate by the analysis pipeline. T... more Structure of the output csv table. Example of an output file generate by the analysis pipeline. The table is populated for all positions of the reference genome. Chrom: Reference genome used, Position: Position in the reference genome, Covmp: Absolute depth of coverage, PercentCov: relative depth of coverage expressed as a percentage of the average coverage obtained for the sample, FilterCov: Flagging for insufficient relative coverage, Ref: Nucleotide from the reference genome, Var: Alternative nucleotide identified by VarScan, Cons: Consensus nucleotide kept by VarScan, FastA: Nucleotide kept in the exported fastA file, QDepth: Absolute depth of coverage, Reads1: Reference nucleotide coverage by mpileup, Reads2: Alternative nucleotide coverage by mpileup, Freq: Variant frequency, P-value: VarScan p-value, StrandFilter: VarScan strand filter, R1+: Reference nucleotide coverage from the positive strand, R1-: Reference nucleotide coverage from the negative strand, R2+: Alternative nu...
Sanger sequencing validation for the inheritance of high level heteroplasmy SNV. Slide 1. CEPH fa... more Sanger sequencing validation for the inheritance of high level heteroplasmy SNV. Slide 1. CEPH family 1463 pedigree chart. Slide 2. Level of heteroplasmy calculated from the MitoRS data, and the Sanger sequencing data in both forward and reverse orientation. MitoRS calculated frequencies are highlighted in red (homoplasmy, > 98%) or orange (high frequency heteroplasmy, between 10% and 98%). The corresponding Sanger frequencies are highlighted in green for easier visualization. Slides 3 to 10. Chromatograms highlighting a difference between the two family members. The positions considered are shown with a black arrowhead. Slide 10. Virtual gel visualization (on a LabChip GX - Perkin Elmer) of the PCR amplification products analyzed by Sanger sequencing. (PPTX 513Â kb)
MitoRS output file for the analysis of the CEPH family 884. Legend similar to the Additional file... more MitoRS output file for the analysis of the CEPH family 884. Legend similar to the Additional file 8 but for the CEPH family 884. (XLSX 28519Â kb)
Overview of the MitoRS analysis methods. FastQ files are aligned with BWA to the original, and an... more Overview of the MitoRS analysis methods. FastQ files are aligned with BWA to the original, and an origin-shifted version, of the DNA reference sequence (refer to Additional file 2 for details). Variant frequency is evaluated by samtools mpileup. Low frequency variants are finally identified using VarScan 2. Datasets generated from both the original reference and the shifted reference are merged, keeping only the per position values from the dataset with the highest coverage. Data are further processed to generate 1) a csv file summarizing the sequencing results observed at each individual mitochondrial DNA position and 2) a fastA file representing the corresponding consensus sequence. More details can be found in the Methods section. (PPTX 264Â kb)
Haplogroup determination for the CEPH family 1463. The fastA file generated for each individual o... more Haplogroup determination for the CEPH family 1463. The fastA file generated for each individual of the CEPH family 1463 was submitted to the Haplofind tool. Note that N are considered as deletion. When the completion status is "No", Haplofind was not able to determine the exact subhaplogroup. (DOCX 39 kb)
RCA enriches circular versus linear templates. A. Principle of circular DNA enrichment. A single ... more RCA enriches circular versus linear templates. A. Principle of circular DNA enrichment. A single priming event will generate several concatenated copies of a circular template. At the opposite, a single copy will be amplified if the template DNA is linear. B. The RCA amplified material is mostly mtDNA. Digestion of the mouse DNA RCA product with the SpeI restriction endonuclease results in the expected mtDNA digestion product with only low amount of undigested DNA left. SpeI restriction digest is expected to result in four fragments for the B6D2F1 strain (7'398, 3'759, 3'105, and 2'035 bp) and five fragments for the NMRI strain (7'398, 3'105, 2'150, 2'037, and 1609 bp). Ladder sizes imprecision is in accordance with the Agilent Genomic DNA ScreenTape specifications. (PPTX 133 kb)
Coverage drop at the position 310 human C-stretch. The human sample #12878 from the CEPH family 1... more Coverage drop at the position 310 human C-stretch. The human sample #12878 from the CEPH family 1463 was sequenced either following the pipeline described in this paper or from a whole genome PCR free library (generated in our lab). The relative coverage reported by mpileup was plotted against each single position of the reference genome with a zoom in the C-stretch at position 310. (PPTX 233Â kb)
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Papers by Umberto De Marchi