There are many causes of liver disease however the aetiology is often undetermined. It is speculated that mitochondrial dysfunction has a role in liver disease and this presentation explores possible causes and techniques for investigating mitochondrial dysfunction in liver.
2. ◦ Mitochondria are tiny cellular organelles responsible
for energy production via oxidative phosphorylation
◦ Complexes I-IV form the electron transport chain
and create a proton gradient
◦ Complex V uses this gradient to make ATP from ADP
3. The liver is a vital organ
◦ Has a high energy requirement
◦ Has a role in metabolism of macromolecules,
protein biosynthesis and clearing of toxic
compounds
◦ Highly dependent on ATP and functioning
mitochondria
◦ Mitochondrial disease affects about 1 in 5,000 to
1 in 10,000 of the population and the liver can be
affected in as many as 20% of these cases
4. Many types of liver disease including alcoholic
liver disease, hepatitis, acute liver failure
Symptoms are varied – lactic acidosis,
hypoglycaemia, jaundice, anaemia,
coagulopathy
Treatment is symptomatic
Transplantation is life saving in severe cases
5. Often unknown
Drugs such as valproate
Viruses such as hepatitis B and C
However it is speculated that mitochondrial
dysfunction has a role in liver disease
Several studies have associated liver disease with
mitochondrial defects
6. Defects in assembly of respiratory chain
complexes
◦ BCS1L
Missense mutations
Chaperone for Rieske Fe-S subunit of Complex III
Hepatopathy, tubulopathy and encephalopathy
◦ SCO1
Copper insertion into Complex IV
Hepatopathy and ketoacidic coma
7. Alpers’ Syndrome
◦ Polymerase gamma mutations
◦ Affects the liver and nervous system
DGUOK deficiency
◦ Deoxyguanosine kinase maintains a stable
ribonucleotide pool for mtDNA synthesis
◦ Affects the liver and nervous system
8. TSFM
◦ Encodes elongation factor EFTs
◦ A mutation causes a severe hepatopathy with a
respiratory chain deficiency
TRMU
◦ Encodes a tRNA modifying enzyme
◦ Required for thiol addition to tRNAs for lysine,
glutamate and glutamine
◦ A mutation causes acute liver failure with reduced
activity of complexes I, III and IV
9. Isolation of mitochondria from frozen liver
tissue
◦ Homogenisation of tissue
Cut and washed
Homogenised in a glass Elvehjem potter
◦ Differential centrifugation
Sucrose gradient
◦ Addition of detergents and sample buffer
containing aminocaproic acid for BN PAGE only
10. Blue native polyacrylamide gel electrophoresis
◦ Native separation of protein complexes on a gradient gel
◦ Uses Coomassie G250
Attaches a negative charge to protein complexes
Doesn’t dissociate complexes into subunits
◦ Digitonin and n-dodecyl B-D-maltoside
Detergents
Break down mitochondrial membrane
Dissociate some supercomplexes
◦ Immunodetection of proteins with antibodies to complexes
I-V
11. ◦ Biochemical assays of the catalytic activity of
enzymes
◦ Complex II
reduction of ubiquinone at 595 nm
◦ Complex IV
oxidation of cytochrome c at 550 nm
◦ Citrate Synthase
formation of citrate from oxaloacetate at 405 nm
Indicator of number of functional mitochondria
12. Confocal microscopy
Immunocytochemistry
Study mtDNA transcription or translation
Sequencing of genes commonly affected in
mitochondrial disease e.g. TRMU, DGUOK, GFM1
Whole exome sequencing
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hepatology. 2005 Aug; 43(2):207-9
Fernandez-Vizarra E, Ferrin G, Perez-Martos A, Fernandez-Silva P,
Zeviani M, Enriquez JA. Isolation of mitochondria for biogenetical
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Lee WS, Sokol RJ. Liver disease in mitochondrial disorders. Seminars
in liver disease. 2007 Aug; 27(3):259-73
Rahman S. Gastrointestinal and hepatic manifestations of
mitochondrial disorders. Journal of inherited metabolic disease.
Epub 2013/05/16
Schagger H, von Jagow G. Blue native electrophoresis for isolation of
membrane protein complexes in enzymatically active form.
Analytical biochemistry. 1991 Dec; 199(2):223-31
Notas del editor
Translation – for example by in vivo metabolic 35S-methionine labelling of mitochondrial protein synthesisTranscription – for example by Northern blotting or PCR for mRNA
