IV: Mitochondrial function (e.g. hepatocytes)

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IV Mitochondrial function (e.g. hepatocytes)
1) citric acid cycle as an energy source a)
pyruvate or a-ketoglutarate dehydrogenase b)
lipoic acid therapy 2) the respiratory chain as
an energy source 3) oxidative phosphorylation and
uncouplers 4) membrane transporters and shuttles
a) cytosolic NADH oxidation b) acetyl CoA
(NADPH export) c) transport systems in the
mitochondria d) gluconeogenesis and glucose
transport 5) mitochondrial diseases and
treatment a) creatine therapy b) coenzyme
Q10 therapy
6) b-oxidation of fatty acids as an energy
source a) starvation/diabetes/endstage renal
disease b) carnitine therapy c) ketogenic
diet therapy d) drug induced fatty liver and
NASH e) alcohol induced fatty liver and ASH
7) hepatic detoxification of a) monoamines
b) alcohols c) toluene 8) hemoprotein
mediated diseases a) rhabdomyolysis b)
kernicterus 9) Heme biosynthesis porphyria
a) Heme biosynthesis b) Porphyria c)
Oxidative degradation of heme to bilirubin 10)
Nitrogen metabolism a) N-catabolism of
amino acids b) N-catabolism of purines
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1. CITRIC ACID CYCLE AS AN ENERGY SOURCE

• Pyruvate or a-ketoglutarate dehydrogenase
• Requires Thiamine (Vit B1) and lipoic acid
• B) Lipoic acid therapy

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An overview of the citric acid cycle
Stryer
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ROS
toxic!
120uM plasma citrate complexes Fe
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Pyruvate dehydrogenase complex
Pyruvate decarboxylase inhibited by branched
chain aminoacids that accumulate in maple syrup
disease. Babies born with mitochondrial diseases
usually have cytochrome deficiencies but
some have low E1 that causes lactic acidosis .
Therapy includes thiamine or CoQ supplements,
dichloroacetate or a ketogenic diet.
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Pyruvate dehydrogenase complex
Stryer Fig. 20-12. Summary of the
reactions catalyzed by the pyruvate dehydrogenase
complex. L. refers to the lipoyl group.
Pyruvate CoA NAD ? acetyl CoA CO2 NADH
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Lipoic acid (thioctic acid) therapy

• Scavenges reactive oxygen and nitrogen oxide
  species
• restores other cellular antioxidants
• decreases oxidative stress toxicity
• prevents diabetes complications, cataracts and
  alcoholic liver
• damage
• prevents Cis Pt chemotherapy induced
  nephrotoxicity,
• hypertension and aging in rats
• Free Rad. Biol. Med. 24, 1023-39 (1998).

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The citric acid cycle is a source of biosynthetic
precursors
Stryer Fig. 20-17. Biosynthetic roles of
the citric acid cycle. Intermediates drawn
off for biosyntheses are replenished by the
formation of oxaloacetate from
pyruvate. (Anaplerotic)
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Control of the citric acid cycle Stryer Fig.
20-22. Control of the citric acid cycle and the
oxidative decarboxylation of pyruvate
indicates steps that require an electron acceptor
(NAD or FAD) that is regenerated by
the respiratory chain.
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2. THE RESPIRATORY CHAIN AS AN ENERGY SOURCE
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The mitochondrial respiratory chain
Diagram of a mitochondrion
Chemiosmotic theory of oxidative phosphorylation
Sequence of electron carriers in the respiratory
chain
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The reduction of flavin mononucleotide (FMN) to
FMNH2 proceeds through a semiquinone intermediate
Molecular models of iron-sulfur complexes iron
red cysteine sulfur yellow inorganic sulfur
green
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NADH coenzyme Q reductase complex I
The reduction of ubiquinone to ubiquinol proceeds
through a semiquinone anion intermediate.
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Model of NADH-Q reductase
Stryer Fig 21-9
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QCytochrome c reductase (Complex III)
Stryer p. 537
Stryer Fig. 21-11 Model of a portion of Q
cytochrome c reductase
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Cytochrome oxidase (Complex IV)
Lodish Fig. 17-30
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Electron transport can be blocked by specific
inhibitor poisons
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Cytochrome C - catalytic site
The iron atom of the heme group in cytochrome c
is bonded to a methionine sulfur atom and a
histidine nitrogen atom
The heme in cytochromes c and c1 is covalently
attached to 2 cysteine side chains by thioether
linkages
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Spectroscopic characteristics of cytochrome c
Http//www.princeton.edu/macase/cytochrome_c.html
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Evolutionary Tree of Cytochrome C
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Cytochrome C - soluble NOT membrane bound
1. 26/104 amino acids residues have been
invariant for gt 1.5 x 109 years. 2. Met 80 and
His 18 - coordinate Fe. 3. 11 residues from
number 70 - 80 lining a hydrophobic crevice have
remained virtually unchanged throughout all
cytochrome c regardless of species or even
kingdom. 4. A number of invariant arginine and
lysine clusters can be found on the surface
of the molecule. Cytochrome c has a dual
function in the cell. Electron transport for ATP
production AND the major cause of most
programmed cell death (apoptosis) is initiated
by the release of cytochrome c into the cytosol!
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Origin of mitochondria the endosymbiont
hypothesis

• The endosymbiont hypothesis suggests that
  mitochondria have evolved
• from anaerobic bacteria which were phagocytosed
  by eukaryote cells
• at the time oxygen appeared on earth,
• Similarities between mitochondria and bacteria
  include the presence of
• cardiolipin
• transporters
• ribosomes
• circular RNA and DNA
• Therefore mitochondria protein synthesis should
  be inhibited by
• TETRACYCLINE
• CHLORAMPHENICOL.
• E.g. The extensive use of these drugs can inhibit
• 1. Bone marrow mitochondrial protein
  synthesis leading to a
• decline in the production of white or
  red cells.
• 2. Intestinal epithelial cells causing
  them to cease dividing.

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3. OXIDATIVE PHOSPHORYLATION ANDUNCOUPLERS
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Oxidative phosphorylation
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Uncouplers Physiological use of
uncoupling oxidative phosphorylation
Brown fat which is very rich in
mitochondria/chloroplast supply heat for
newborns, hibernating animals, flowering
plants. Thermogenin is a 33Kd protein which
uncouples mitochondria to generate heat.
thermogenin
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Uncouplers of oxidative phosphorylation
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Mitochondria and weight loss/poison uncouplers. Ox
ygen electrode shows DNP is a protonophore (short
circuits proton gradient) markedly increases
oxygen uptake causing heat without ATP
formation. Oligomycin is an ATPase inhibitor.
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4. MEMBRANE TRANSPORTERS

• A) Cytosolic NADH oxidation
• B) Acetyl CoA (NADPH export)
• C) Transport systems in the mitochondria
• D) Gluconeogenesis and glucose transport

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Compartmentalization of the major pathways of
metabolism
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a) Cytosolic NADH oxidation membrane
transporters glycerol phosphate shuttle
(Bucher shuttle)
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Malate-aspartate shuttle
NADH cant permeate mitochondrial membrane but
cytosol NADH must be oxidised by mitochondria to
sustain glycolysis
Vitamin B6
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b) Acetyl CoA/NADPH export to cytosol for fatty
acid synthesis/ drug metabolism
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Isocitrate as an NADPH shuttle for drug metabolism
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c) Transport systems in the mitochondria
Pyruvate transporter inhibited by hydroxycinnamate
Malate transporter inhibited by butylmalonate
Harpers Biochemistry.
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d) Gluconeogenesis and glucose export by the
liver !
Mitochondria/ cytosolic enz., 6 ATP,2NADH
required by pyruvate carboxylase, PEP
carboxykinase, Pglycerate kinase,GAPDH for
2pyruvate?glucose
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Glucagon Insulin

• Pancreas synthesises both peptide hormones
• Glucagon hepatocyte receptors signals
  glycogenolysis (glycogen breakdown to glucose
  then increases gluconeogenesis pyruvate --?
  glucose)
• Drugs. Dipeptidyl peptidase-4 inhibitor (Januvia,
  new anti type 2 diabetes) increases incretin , a
  GI hormonal peptide inhibitor of glucagon which
  lowers plasma glucose.
• Metformin,major antidiabetic but rarely inhibits
  mitoch.complex I causing lactic acidosis.
• Insulin hepatocyte/adipose receptor signals cell
  glucose uptake and glycogen synthesis.

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5. MITOCHONDRIAL DISEASES (e.g. DEFECTIVE
ELECTRON TRANSPORT) AND TREATMENT
A) Creatine therapy B) Coenzyme Q10 therapy
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Mitochondrial Myopathies

• Genetic defects in mitochondrial structure
  function leading to defective aerobic energy
  transduction and resulting in exercise
  intolerance, lactic acidosis, stroke/seizure,
  headaches.

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a) CREATINE THERAPY Creatine supplementation (an
ergogenic aid and effective against mitochondrial
myopathies?)

• daily requirement is 2g
• daily dietary intake is 1g (meat, fish, animal
  products)
• also formed in liver, kidneys, pancreas from the
  amino acids glycine,
• arginine and methionine
• 5-7g x 4 per day for 5-7 days increases muscle
  creatine stores by 18
• (particularly in vegetarians) enhances
  performance in certain
• repetitive, high intensity, short-term
  exercise tasks in healthy
• individuals, offsets fatigue in mitochondrial
  myopathy patients and
• improves the mobility of the elderly. J. Amer.
  Coll. Nutr. 17, 216-234 (1998).

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b) Ubiquinone (Coenzyme Q10) as a Food Supplement
or Therapy

• An essential electron and proton carrier in the
  mitochondrial respiratory chain.
• Found in all intracellular membranes (acts as a
  mobile lipid soluble antioxidant
• that prevents membrane lipid peroxidation)
• Better antioxidant if reduced to ubiquinol
  (UQH2) by NADH dehydrogenase
• of the respiratory chain.
• Synthesised in mitochondria
• Contributes to the fluidity of the phospholipid
  bilayer in membranes
• Prevents plasma lipoprotein oxidation
• Is a dietary supplement that protects liver from
  hepatotoxins (e.g. ethanol)
• and partly prevents mitochondrial myopathies (J.
  Neurol. Neurosurg. Psych. 50,
• 1475-81)
• Deficiency may occur in patients taking
  cholesterol lowering drugs (the
• statins) which act by inhibiting HMG-CoA
  reductase (e.g. lovastatin) Proc. Nat.
• Acad. Sci. 87, 8931 (1990)

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6. b-OXIDATION OF FATTY ACIDS AS AN ENERGY SOURCE

• Starvation/diabetes/endstage renal disease
• b) Carnitine therapy
• c) Ketogenic diet therapy
• d) Drug induced non alcoholic steatohepatitis ,
  NASH
• e) Alcohol induced steatohepatitis , ASH

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Stages in the extraction of energy from food
stuffs.
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b-Oxidation of fatty acids - transport of acyl
carnitine into the mitochondrial matrix
Stryer Fig 24-4
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Fatty acid Metabolism

• Fatty acids are linked to coenzyme A (CoA)
  before they are oxidised

• Carnitine carries long-chain activated fatty
  acids into the mitochondrial
• matrix

Carnitine therapy for mitochondrial diseases
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The b-oxidation pathway as an energy source
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• a) Starvation/Diabetes/Endstage renal disease
• Fat breaks down to acetyl CoA which form
• ketone bodies
• Under low carbohydrate condition,
• oxaloacetate is converted to
• glucose (gluconeogenesis).

KETOGENESIS
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• Diabetic ketoacidosis weakness, dehydration,
  thirst, drowsiness,coma
• Usually precipitated by infection
• lipolysis is the major energy source increases
  acetyl CoA levels which
• increases ketone body formation.Acetone
  excreted by the lungs/kidney.
• e.g. by starvation or diabetes mellitus
  (insulin-stimulated glucose entry
• into cells is impaired? fatty acids are
  oxidised to maintain ATP levels.
• if citric acid cycle is slowed by thiamine
  deficiency.
• disease state plasma ketone levels 10-25 mM
  (normal lt0.5mM) and
• acetone breath smell( rotten apples or
  pear-drop smell)
• LIFE THREATENING ketogenesis faster than
  ketone body metabolism
• ?-hydroxybutyric acid ??gt acetoacetic acid ?
  causes severe ACIDOSIS.
• Antidote insulin , water, base therapy
  (bicarbonate?), carnitine
• ? urinary excretion of Na, K, Pi, H2O, H
• ? dehydration, ? blood volume

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• b) Carnitine Therapy
• Carnitine alleviates acetyl-CoA mediated
  inhibition of pyruvate
• dehydrogenase.
• Both glycolysis and fatty acid metabolism
  produce acetyl CoA
• Accumulation of acetyl CoA can inhibit pyruvate
  dehydrogenase, the
• enzyme responsible for producing acetyl CoA
  from pyruvate.
• Pyruvate will then be converted to lactic acid
• Carnitine can temporarily scavenge acetyl CoA to
  form acetylcarnitine
• thus alleviating lactic acidosis in the muscle.

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Carnitine supplement
Uses 1. Improves quality of life and walking
performance in patients with limited
walking capacity e.g., from end-stage renal
disease and peripheral arterial disease. 2.
Neurodegenerative diseases and recovery from
cerebral ischemia. 3. Possible ergogenic aid but
can cause an unpleasant body odour
likened to rotting fish. 4. Improves memory of
old rats (PNAS 99, 1876-81 (2002)) Biochemistry
1. Increases carnitine content, carries activated
fatty acids across mitochondrial membrane
and required for mitochondrial fatty acid
oxidation. 2. Prevents acetyl CoA accumulation
which inhibits pyruvate dehydrogenase. 3.
Chelates iron and stabilizes membranes
(antioxidant properties)
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Carnitine supplement (cont)
Oral Bioavailability 5-15 But over-the-counter
formulations have low carnitine content and poor
dissolution. - plasma acylcarnitines accumulate
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c) Ketogenic diet therapy (results 10-25 seizure
free 60 better) for epileptic children
resistant to phenytoin or valproate
Energy Source Normal Diet Ketogenic
Diet Protein 27
10.4 adequate Carbohydrate 56
- Fat 17 89.6
Ketogenic diet consists of an egg nog that tastes
like a mild shake (or frozen like ice
cream) Supplying the body with fuel in the form
of fat and proteins but not carbohydrates.
Liver produces ketone bodies
Brain uses either glucose or ketone bodies as
fuel
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d) Drugs that inhibit fatty acid oxidation cause
fatty liver (steatosis) and 10 get NASH
(nonalcoholic steatohepatitis liver cancer)

• Steatosis (fatty liver) in 33 population 80
  of obese patients. Higher also in diabetes ,
  high plasma triglycerides. NASH in 2-9 patients
  undergoing routine liver biopsy. Hepatocellular
  carcinoma.
• Drugs that inhibit mitochondrial ß-fatty acid
  oxidation
• 1)Tetracycline, valproic acid,oestrogens,glucocort
  icoids
• 2) Amiodarone,perhexiline are charged lipophilic
  drugs concentrate in liver mitochondria inhib.
  ß-fatty acid oxidn respiration, cause lipid
  peroxidn. reactive oxygen species (ROS).
  Steatosis and steatohepatitis are independent.
  Fibrosis occurs.
• 3) Drugs induce sporadic events of both e.g.
  carbamazepine
• 4) Latent NASH e.g. tamoxifen

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e) Ethanol induced steatohepatitis (ASH) proposed
endotoxin mechanism

• Ethanol causes lipogenesis and
• fatty liver (caused by inhibition of LDL synth.
  export).
• 2) Ethanol oxidised by CYP2E1 to form
  hydroxyethyl radicals and oxidised by ADH to form
  acetaldehyde which cause oxidative stress and
  hepatocyte gut cytotoxicity.
• Oxidative stress disrupts intestinal mucosal cell
  actin cytoskeleton (prev. by oats supplement).
• 4) Intestine becomes leaky endotoxin enters
  blood liver which causes liver inflammation and
  ASH.
• JPET 329,952-8(2009)

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Drugs causing weight gain in some patients

• Tricyclic antidepressants imipramine,amitriptyline
  ,nortryptyline,
• Antipsychotics haloperidol,clozapine,olanzapine,ri
  speridone,chlorpromazine,paxil
• Corticosteroids prednisone. Also steroids
  estrogen,tamoxifen
• Antidiabetics rosiglitazone,pioglitazone,insulin
• Antirheumatism etanercept,enbrel
• Anti-seizure valproate,depakote,carbamazepine,carb
  amazepine
• Lithium
• MAO inhibitors phenelzine,tranylcypromine

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7. Hypoxia Ischemia Reperfusion
Injury Mitochondrial Toxins
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Mitochondrial Toxins

• 1. Cyanide, rotenone, amytal, CO, H2S, antimycin
  A
• 2. Drug reactive metabolites, e.g., acetaminophen
• 3. Toxin reactive metabolites, e.g., CCl4,
  cocaine,
• ethanol
• 
  
• 4. NSAID drugs

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Electron transport can be blocked by specific
inhibitors
Antimycin A Complex III inhibitor
CN-or CO Complex IV inhibitor
Rotenone Complex I inhibitor
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Ref Stryer or Voigt
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Stryer or Voigt
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Cytoprotective agents against ischemia
reperfusion injury or mitochondrial toxins

• Catalase, superoxide dismutase
• Superoxide scavenging antioxidants
• Nutrients that oxidize NADH and supply ATP
• Ferric chelators, e.g., desferoxamine
• Xanthine oxidase inhibitors, e.g., allopurinol
• Ca2 channel blockers
• Phospholipase or protease inhibitors

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Transplantation Surgery Cold storage/reperfusion
injury to liver
Ref Seminars in Liver Disease 13, 93 (1993)
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Cell death mechanismsAPOPTOSIS VS NECROSIS

• 2011

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8. Apoptosis vs Necrosis

• 2002 Nobel Prize in Physiology or Medicine
• Sydney Brenner (Salk Institute, La Jolla, Ca,
  USA), John
• Sulston (Wellcome Trust Sanger Institute,
  Hinxton, UK) and
• Robert Horvitz (Massachusetts Institute of
  Technology,
• Boston, MA, USA)
• Caspase mutations in the worm Caenorhabditis
  elegans
• prevented cell death (programmed for worm
  development
• from egg).

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Apoptosis targets cells that endanger organism

• Damaged DNA cells
• Cells growing too fast or too slow
• Senescent cells
• Obsolete cells as organisms grow and develop

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Part I Apoptosis versus Necrosis A comparison