Differential requirements for mitochondrial electron transport chain components in the adult murine liver
- Children's Medical Center Research Institute, University of Texas Southwestern Medical Center, United States
- Department of Biochemistry, University of Texas Southwestern Medical Center, United States
- Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, United States
- Advanced Imaging Research Center, University of Texas Southwestern Medical Center, United States
- Department of Pediatrics, University of Texas Southwestern Medical Center, United States
- Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 3 supplements
Mitochondrial complex I is dispensable in adult murine hepatocytes.
(A) Ndufa9 mRNA levels (relative to β2-microglobulin; normalized) in isolated hepatocytes of the indicated genotype, assessed by qRT-PCR. n=3 animals per group. The same color scheme is used …
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Figure 1—source data 1
Full gel images for Figure 1.
Full gel images and original image files for western blots in Figure 1.
- https://cdn.elifesciences.org/articles/80919/elife-80919-fig1-data1-v2.zip
Figure 1—figure supplement 1
Metabolic flux analysis of Ndufa9f/f and Ndufa9-/- MEFs.
(A) Oxygen consumption rates in Ndufa9f/f and Ndufa9-/- mouse embryonic fibroblasts (MEFs). n=22–24 wells per group. The same color scheme is used throughout this figure. (B) Normalized NAD+ / NADH …
Figure 1—figure supplement 2
Analysis of Ndufa9-/- livers at 2 weeks post-AAV administration.
(A) Representative histology (H&E, Oil Red O, PAS staining) images of liver cross sections from animals of the indicated genotype at 2 weeks post-AAV administration. Scale bar: 50 μm. (B) Liver …
Figure 1—figure supplement 3
Analysis of Ndufa9-/- livers at 8 weeks post-AAV administration.
(A) Representative histology (H&E, Oil Red O, PAS staining) images of liver cross-sections from animals of the indicated genotype at eight weeks post-AAV administration. Scale bar: 50 μm. (B) …
Figure 2 with 2 supplements
Complex I deficiency in adult liver does not impact redox status.
(A) ATP levels from livers of the indicated genotype. n=3 mice per group. The same color scheme is used throughout this figure. (B) Reduced glutathione to oxidized glutathione ratio (GSH/GSSG) in …
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Figure 2—source data 1
Full gel images for Figure 2.
Full gel images and original image files for western blots in Figure 2.
- https://cdn.elifesciences.org/articles/80919/elife-80919-fig2-data1-v2.zip
Figure 2—figure supplement 1
Analysis of mitochondrial proteomes in complex I deficient livers.
(A) Volcano plot of protein abundances changes in Ndufa9-/- vs. Ndufa9f/f livers, based on proteomics analysis of enriched mitochondria. Mitochondrial proteins (based on MitoCarta 3.0) are shown. …
Figure 2—figure supplement 2
Immunofluorescence of hepatic sections.
Representative immunofluorescent images of liver sections at 20 x (left; scale bar 50 μm) and 63 x (right; scale bar 5 μm) of the indicated genotype. TOMM20 (mitochondrial) staining is indicated in …
Figure 3 with 1 supplement
Complex I deficiency in adult liver does not impact glucose utilization or oxygen status.
(A) Schematic of potential fates of glucose and lactate within central carbon metabolism. Carbon atoms are indicated with circles for each compound. G1P, glucose-1-phosphate; 3 PG, …
Figure 3—figure supplement 1
Metabolic analysis of complex I deficient livers.
(A) Mass isotopomer distributions in liver tissue from [U-13C]glucose infusions in animals of the indicated genotype. n=3 animals per group; 4 weeks post-AAV administration. Statistical significance …
Figure 4 with 1 supplement
Complex IV deficiency in adult hepatocytes induces liver dysfunction.
(A) Cox10 mRNA levels (relative to β2-microglobulin; normalized) in livers of the indicated genotype, assessed by qRT-PCR at 4 weeks post-AAV administration. n=4–5 animals per group. The same color …
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Figure 4—source data 1
Full gel images for Figure 4.
Full gel images and original image files for western blots in Figure 4.
- https://cdn.elifesciences.org/articles/80919/elife-80919-fig4-data1-v2.zip
Figure 4—figure supplement 1
Lactate/pyruvate tolerance test in complex IV deficient livers.
(A) Mass isotopomer distributions related to lactate/pyruvate tolerance test (Figure 4H, I), for plasma lactate and glucose. n=4–5 mice per group; 8 weeks post-AAV administration. Numerical data for …
Figure 5 with 8 supplements
Complex IV deficiency alters metabolism and cellular redox status in adult liver.
(A) ATP levels in livers of the indicated genotype. n=3 animals per group. The same color scheme is used throughout this figure. (B) Reduced glutathione to oxidized glutathione ratio (GSH:GSSG) in …
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Figure 5—source data 1
Full gel images for Figure 5.
Full gel images and original image files for western blots in Figure 5.
- https://cdn.elifesciences.org/articles/80919/elife-80919-fig5-data1-v2.zip
Figure 5—figure supplement 1
Functional annotation (DAVID) analysis of upregulated proteins in complex IV deficient livers.
(A) Significantly enriched biological pathways (FDR <0.05) from gene ontology analysis of all upregulated proteins (log2(fold change)>1; p-value <0.05) in Cox10-/- livers, based on proteomic …
Figure 5—figure supplement 2
Functional annotation (DAVID) analysis of downregulated proteins in complex IV deficient livers.
(A) Significantly enriched biological pathways (FDR <0.05) from gene ontology analysis of all downregulated proteins (log2(fold change) < –1; p-value <0.05) in Cox10-/- livers, based on proteomic …
Figure 5—figure supplement 3
Analysis of mitochondrial proteomes in complex-IV-deficient livers.
(A) Volcano plot of protein abundances changes in Cox10-/- vs. Cox10f/f livers, based on proteomics analysis of enriched mitochondria. Mitochondrial proteins (based on MitoCarta 3.0) are shown. Log2(…
Figure 5—figure supplement 4
Metabolic analysis of complex-IV-deficient livers.
(A) Relative abundance of cholate species in livers of the indicated genotype. n=4–6 mice per group; 8 weeks post-AAV administration. (B) Relative abundance of electron acceptors/donors from livers …
Figure 5—figure supplement 5
Metabolic / proteomic analysis of fatty-acid-related pathways in complex-IV-deficient livers.
(A) Left, volcano plot of identified proteins from the Fatty Acid Synthesis (GO:0006633) pathway, based on proteomics performed on Cox10-/- vs. Cox10f/f livers at 8 weeks post-AAV administration. …
Figure 5—figure supplement 6
Metabolic / proteomic analysis of bile-acid-related pathways in complex-IV-deficient livers.
(A) Left, volcano plot of identified proteins from the Bile Acid Metabolic Process (GO:0008206) pathway, based on proteomics performed on Cox10-/- vs. Cox10f/f livers at 8 weeks post-AAV …
Figure 5—figure supplement 7
Metabolic / proteomic analysis of oxidoreductase-related pathways in complex-IV-deficient livers.
(A) Left, volcano plot of identified proteins from the Oxidoreductase (GO:0016491) pathway, based on proteomics performed on Cox10-/- vs. Cox10f/f livers at 8 weeks post-AAV administration. …
Figure 5—figure supplement 8
Immunofluorescence of hepatic sections.
Representative immunofluorescent images of liver sections at 20 x (left; scale bar 50 μm) and 63 x (right; scale bar 5 μm) of the indicated genotype. TOMM20 (mitochondrial) staining is indicated in …
Figure 6 with 1 supplement
Complex IV deficiency impairs electron transport chain function and oxygen status in the adult liver.
(A) Perfusion values (reported as institutional units (I.U.)) based on arterial spin labeling in animals of the indicated genotype at 4 weeks post-AAV administration. n=3 animals per group. The same …
Figure 6—figure supplement 1
Isotope tracing in complex-IV-deficient livers.
(A) Mass isotopomer distributions in liver tissue from [U-13C]glucose infusions of the indicated genotype. n=3 animals per group; 4 weeks post-AAV administration. Statistical significance was …
Author response image 1
BN-PAGE analysis to analyze ETC complexes and supercomplexes from murine liver lysate.
(A) BN-PAGE, followed by Coomassie staining, of isolated mitochondria extracted with 5g/g digitonin/protein ratio. 50mg of protein per lane were assessed by BN-PAGE, and bands are annotated based on …
Author response image 2
Quantitation of DHODH abundance in ETC-deficient livers.
(A) DHODH abundance was assessed by western blot in ndufa9f/f and ndufa9-/- livers, and was quantitated relative to b2microglobulin (as a loading control). (B) Relative DHODH abundance based on …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, male and female) | Cox10f/f | The Jackson Laboratory | 024697 | |
| Strain, strain background (Mus musculus, male and female) | Ndufa9f/f | |||
| Other | pAAV.TBG.PI.eGFP. WPRE.bGH (AAV-GFP) | Addgene | 105535 | Adeno-associated virus |
| Other | pAAV.TBG.PI.Cre. rBG (AAV-Cre) | Addgene | 107787 | Adeno-associated virus |
| Antibody | Anti-Tom20 (rabbit polyclonal) | Proteintech | 11802 | WB, 1:2000 |
| Antibody | Anti-PC (rabbit polyclonal) | Proteintech | 16588 | WB, 1:1000 |
| Antibody | Anti-βActin (mouse monoclonal) | Proteintech | 66009 | WB, 1:5000 |
| Antibody | Anti-Ndufa9 (mouse monoclonal) | ThermoScientific | 459100 | WB, 1:2000 |
| Antibody | Anti-β2microglobulin (rabbit monoclonal) | ThermoScientific | 701250 | WB,:5000 |
| Antibody | Anti-Cox10 (rabbit polyclonal) | Abcam | ab84053 | WB,:1000 |
| Commercial assay or kit | Complex I Enzyme Activity Microplate Assay Kit (Colorimetric) | Abcam | ab109721 | |
| Commercial assay or kit | ATP Colorimetric/ Fluorometric Assay Kit | Biovision | K354 | |
| Commercial assay or kit | Deproteinizing Sample Preparation Kit | Biovision | K808 | |
| Commercial assay or kit | Luna Universal One- Step RT-qPCR kit | New England Biolabs | E3005S | |
| Commercial assay or kit | DC Protein Assay | Biorad | 5000112 | |
| Chemical compound, drug | [U-13C]glucose | Cambridge Isotopes Laboratories | CLM-1396 | |
| Chemical compound, drug | [U-13C]lactate | Cambridge Isotopes Laboratories | CLM-1579 | |
| Chemical compound, drug | [U-13C]pyruvate | Cambridge Isotopes Laboratories | CLM-2440 |
Additional files
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Supplementary file 1
Excel spreadsheet containing raw data and statistical analysis of proteomics and metabolomics data for Ndufa9-/- vs. Ndufa9f/f livers at 4 weeks post-AAV administration.
- https://cdn.elifesciences.org/articles/80919/elife-80919-supp1-v2.xlsx
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Supplementary file 2
Excel spreadsheet containing raw data and statistical analysis of proteomics and metabolomics data for Cox10-/- vs. Cox10f/f livers at 8 weeks post-AAV administration.
- https://cdn.elifesciences.org/articles/80919/elife-80919-supp2-v2.xlsx
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Supplementary file 3
Excel spreadsheet containing raw data for panels presented in the figures and figure supplements.
- https://cdn.elifesciences.org/articles/80919/elife-80919-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/80919/elife-80919-transrepform1-v2.docx
