1

IV: Mitochondrial function (e.g. hepatocytes)

1) citric acid cycle as an energy source a) pyruvate or -ketoglutarate dehydrogenase b) lipoic acid therapy2) the respiratory chain as an energy source3) oxidative phosphorylation and uncouplers4) membrane transporters and shuttles a) cytosolic NADH oxidation b) acetyl CoA (NADPH export) c) transport systems in the mitochondria d) gluconeogenesis and glucose transport5) mitochondrial diseases and treatment a) creatine therapy b) coenzyme Q10 therapy

6) -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) toluene8) hemoprotein mediated diseases a) rhabdomyolysis b) kernicterus9) Heme biosynthesis & porphyria a) Heme biosynthesis b) Porphyria c) Oxidative degradation of heme to bilirubin

2

Stryer

An overview of the citric acid cycle

CITRIC ACID CYCLE AS AN ENERGY SOURCECITRIC ACID CYCLE AS AN ENERGY SOURCE

3

Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA

120uM plasma citrate120uM plasma citratecomplexes Fecomplexes Fe

toxic!toxic!

4

The citric acid cycle is a source of biosynthetic precursors

Stryer Fig. 20-17.Biosynthetic roles of thecitric acid cycle. Intermediates drawn offfor biosyntheses are replenished by the formation of oxaloacetatefrom pyruvate.(Anaplerotic)

Pyruvate

Acetyl CoA

Citrate

keto-glutarate

Amino acids

SuccinylCoA

Porphyrins

Oxaloacetate

Aminoacids

ADP, Pi

ATP, CO2

Phosphoenolpyruvate

Glucose

5

Control of the citric acid cycle

Stryer Fig. 20-22.Control of the citric acid cycle andthe oxidative decarboxylation ofpyruvate: * indicatessteps that require anelectron acceptor (NAD+ or FAD) that is regenerated by therespiratory chain.

6

2. THE RESPIRATORY CHAIN AS AN ENERGY SOURCE

7

The mitochondrial respiratory chain

Sequence of electroncarriers in therespiratory chain

Chemiosmotic theory of oxidative phosphorylation

Diagram of a mitochondrionNADH

NADH-Q reductase

Q

Cytochrome reductase

cyt c

Cytochrome oxidase

O2

complex I

complex III

complex IV

FADH2in flavoproteinssuccinate:Q reductase (complex II)

FMNH2

2Fe-2S

4Fe-4S

8

NADH coenzyme Q reductase: complex I

The reduction of ubiquinone to ubiquinol proceeds through a semiquinoneanion intermediate.

NADH

NAD+

FMN

FMNH2

Reduced Fe-S

Oxidised Fe-S

Q

QH2

NADH-Q reductase

C

CC

C

CC

O

O

H3CO

H3CO

CH3

(CH2CH

C

CH3

CH2)10 HC

CC

C

CC

O

OH

H3CO

H3CO

CH3

R

C

CC

C

CC

OH

OH

H3CO

H3CO

CH3

R

e- + H+ e- + H+

Coenzyme Q10(UBIQUINONE

Semiquinone Intermediate (Q )

Reduced Coenzyme Q10 ( UBIQUINOL)

9

Model of NADH-Q reductase

Stryer Fig 21-9

10

Q:Cytochrome c reductase (Complex III)

Q

QH

cyt b (+2)

cyt b (+3)

QH

QH2

Fe-S(+2)

Fe-S(+3)

cyt c1(+3)

cyt c1(+2)

cyt c(Fe+2)

cyt c(Fe+3)

cytochrome c reductase

Stryer Fig. 21-11Model of a portion ofQ: cytochrome c reductase

Stryer p. 537

11

Cytochrome oxidase (Complex IV)

Lodish Fig. 17-30

12

Electron transport can be blocked by specific inhibitorpoisons

NADH

NADH-QReductase

QH2

Cytochrome b

Cytochrome c1

Cytochrome c

Cytochrome Oxidase

O2

Blocked by rotenone and amytal

Blocked by antimycin

Blocked by

CN-, N3-, and CO

Sites of action of someinhibitors of electron transport

13

Cytochrome C - catalytic site

The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chainsby thioether linkages

The iron atom of the heme group incytochrome c is bonded to a methioninesulfur atom and a histidine nitrogen atom

R CH

CH2

HSCH2 R'R C

H

CH3

CH2

R'S+

Vinyl groupof the heme

Cysteine residueof the protein Thioether linkage

14

Cytochrome C - soluble NOT membrane bound

1. 26/104 amino acids residues have been invariant for > 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!

15

Origin of mitochondria: the endosymbiont hypothesis

The endosymbiont hypothesis suggests that mitochondria have evolvedfrom 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.

16

3. OXIDATIVE PHOSPHORYLATION AND

UNCOUPLERS

17

Oxidative phosphorylation

18

4.Mitochondrial MEMBRANE TRANSPORTERS

A) Cytosolic NADH oxidation

B) Acetyl CoA (NADPH export)

C) Transport systems in the mitochondria

D) Gluconeogenesis and glucose transport

19

Compartmentalization of the major pathways of metabolism

20

a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle)

Glucose

Glyceraldehyde - 3 - phosphate

1,3 - Bisphosphoglycerate

NAD+

NADH, H+Dihydroxyacetone phosphate

Glycerol - 3 - phosphate

Glycerol - 3 - phosphate

Dihydroxyacetone phosphate

Cytosol Outer membrane Inner Membrane

FAD

FADH2

RE

SP

IRA

TO

RY

C

HA

IN

Q

QH2

b

c1

c

a, a3

Glycolysis

1

2

1. Glycerol phosphate dehydrogenase2. Glycerophosphate oxidase

H2C

CHH2C

HO

OH

OP

H2C

CH2CO

OH

OP

See figure 21-30 Stryer 4th Ed.

21

b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/drug metabolismGlucose

Pyruvate

Acetyl CoA

Oxaloacetate

Pyruvate

Citrate

CitrateSynthase

Citrate

Oxaloacetate

Acetyl CoA fatty acid synthesisor drug metabolism(N-acetylation)

+ATP+CoA

ATP citrate lyaseat high concentration

Malate

Pyruvate

NADH

NAD+

NADP+

NADPH

CO2

malate dehydrogenase

malic enzyme

fatty acid synthesisor P450 catalyzed drug metabolism

Pentose Phosphate Pathway NADPH

Therefore malic enzyme supplies NADPHCitrate Lyase supplies acetyl CoA.

ATP

ADP CO2

Mitochondrial Matrix

Cytosol

22

Isocitrate as an NADPH shuttle for drug metabolismGlucose

Pyruvate Acetyl CoA

Citrate

Isocitrate

-ketoglutarate

NAD+

NADH

isocitratedehydrogenase

CO2

Succinyl CoA

NAD+NADH

CO2

Succinate

Fumarate

Malate

Oxaloacetate

NAD+

NADH

Isocitrate

-ketoglutarate

NADP+

NADPH

P450 catalyzedDRUG METABOLISM

isocitratedehydrogenase

CITRICACIDCYCLE

MITOCHONDRIAL MATRIXCYTOSOL

23

d) Gluconeogenesis and glucose export by the liver ! 3 irreversible steps

Major antidiabetic drug METFORMINInhibits gluconeogenesisDecr Hepatic Glucose Synth.

24

Glucagon 51aa & Insulin 29aa

• 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, antidiabetic drug inhibits gluconeogenesis but also can inhibit mitoch.complex I causing lactic acidosis.

• Insulin required for cells (e.g.liver,muscle,fat) to take up glucose and synthesise glycogen.

25

5. MITOCHONDRIAL DISEASES(e.g. DEFECTIVE ELECTRON

TRANSPORT) AND TREATMENT

A) Creatine therapyB) Coenzyme Q10 therapy

26

Mitochondrial Myopathies

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

27

CREATINE THERAPY (an ergogenic aid effective againstmitochondrial myopathies?) stored in muscles (makes ATP)

• daily intake is 2g including1g (meat, fish, animal products)• 1g formed in liver, kidneys, pancreas from glycine,arginine,methionine• plasma levels incr. in kidney,heart,liver damage or rhabdomyolysis • 5-7g x 4 per day for 5-7 days increases muscle creatine stores by 18%

(bigger increase 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).

28

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)

29

6. -OXIDATION OF FATTY ACIDS AS THE BEST ENERGY SOURCE

a) Starvation/diabetes/endstage renal diseaseb) Carnitine therapyc) Ketogenic diet therapyd) Drug induced non alcoholic steatohepatitis , NASH e) Alcohol induced steatohepatitis , ASH

30

Stages in theextraction ofenergy fromfood stuffs.

FATS POLYSACCHARIDES PROTEINS

Fatty acids and glycerol

Glucose andother sugars

Amino Acids

Acetyl CoA

CoA

ATP ADP

O2

CitricAcidCycle

2 CO2

e

STAGE I

STAGE II

STAGE III Oxidative

Phosphorylation

31

-Oxidation of fatty acids - transport of acyl carnitine into themitochondrial matrix

Stryer Fig 24-4

32

The -oxidation pathway as an energy source

R CH2

CH2

CH2

C S CoA

O

Acyl CoA

R CH2

CH

CH

C S CoA

O

R CH2

C C C S CoA

OOHH

HHR C

H2C C C S CoA

OO H

H

R CH2

C S CoA

O+

H3C C S CoA

O

trans- -Enoyl CoA

3-L-hydroxyacyl CoA-Ketoacyl CoA

Thiolysis

CoA-SH

H2OFAD FADH2

oxidation

Hydration

NAD+H+ + NADH

oxidation

Acyl CoA shortenedby 2 carbon atoms

Acetyl CoA

Citric acid cycle

33

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

R C

O

O

ATP HS-CoA R C

O

S CoA AMP PPi+ + + +acyl CoA synthetase(aka fattyacid thiokinase)

Outer Mitochondrial Membrane

R C

O

S CoA + H3C N

CH3

CH3

CH2

C

OH

H

CH2

C

O

OHS-CoA + H3C N

CH3

CH3

CH2

C

O

H

CH2

C

O

O

C O

R

Acyl CoA Carnitine acyl carnitine

carnitine acyl transferase I

Carnitine therapy for mitochondrial diseasesCarnitine therapy for mitochondrial diseases

34

The -oxidation pathway as an energy source

R CH2

CH2

CH2

C S CoA

O

Acyl CoA

R CH2

CH

CH

C S CoA

O

R CH2

C C C S CoA

OOHH

HHR C

H2C C C S CoA

OO H

H

R CH2

C S CoA

O+

H3C C S CoA

O

trans- -Enoyl CoA

3-L-hydroxyacyl CoA-Ketoacyl CoA

Thiolysis

CoA-SH

H2OFAD FADH2

oxidation

Hydration

NAD+H+ + NADH

oxidation

Acyl CoA shortenedby 2 carbon atoms

Acetyl CoA

Citric acid cycle

35

a) Starvation/Diabetes/Endstage renal diseaseFat breaks down to acetyl CoA which formketone bodies• Under low carbohydrate condition, oxaloacetate is converted to glucose (gluconeogenesis).

KETOGENESIS

2 Acetyl CoA

CoA

C

CH2

C

CH3

O

S CoA

Acetyl CoA

+H2O CoA

CH3

C

CH2

COO

O

C

CH2

CH

CH2

COO

O

S CoA

O HO

AcetylCoA

CH3

HC

CH2

COO

OH

CH3

C

CH3

O

H+ + NADH

NAD+

H+

CO2

D--Hydroxybutyrate

Acetone

Acetoacetate

-Hydroxy--methylglutaryl CoA

Acetoacetyl CoA

thiolase HMG-CoAsynthase

HMG CoAlyase

citric acidcycle

succinyl CoA

succinate

CoA transferase

Acetoacetate -hydroxybutyrate

(METABOLISM of ketone bodies)i.e., act as fuel and spares glucose

36

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 <0.5mM) and acetone breath smell( rotten apples or pear-drop smell)

• LIFE THREATENING: ketogenesis faster than ketone body metabolism-hydroxybutyric acid ↑↑> acetoacetic acid ↑& causes severe ACIDOSIS.

Antidote – insulin , water, base therapy (bicarbonate), carnitine• urinary excretion of Na+, K+, Pi, H2O, H+ dehydration, blood volume

37

b) Carnitine TherapyCarnitine alleviates acetyl-CoA mediated inhibition of pyruvatedehydrogenase.• 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.

FATTY ACIDOXIDATION

GLYCOLYSIS

ACETYL CoAPYRUVATE

pyruvatedehydrogenase

Carnitine

Acetylcarnitine + Coenzyme A

LACTATE

H3C N+

CH3

CH3

CH2

C

H

OH

CH2

C

O-

O

TCA (Krebs)

38

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)

Carnitine supplement

39

Sources

Meat and dairy products exported and synthesized byliver > kidney from lysine + methionine. Highest levels inskeletal muscle, heart, adrenal gland but can’t synthesise itso take it up from the plasma.

- total body store = 20-25gms.

Oral Bioavailability 5-15%

But over-the-counter formulations have low carnitine content andpoor dissolution.- plasma acylcarnitines accumulate

Journal of the American College of Nutrition, 17, 207-215 (1998)Progress in Cardiovascular Diseases, 40, 265-286 (1997)

Carnitine supplement (cont)

40

c) Ketogenic diet therapy (results 10-25% seizure free & 60% better) for epileptic children resistant to phenytoin or valproate

Energy Source Normal Diet Ketogenic DietProtein 27% 10.4% adequateCarbohydrate 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 notcarbohydrates.

Ketone Bodies

fasting, diabetiesKetogenic diet

Brain uses eitherglucose or ketone bodies as fuel

Liver produces ketonebodies

d) Drug induced Fatty liver by inhibiting fatty acid oxidation.

Liver (steatosis) and NASH (nonalcoholic

steatohepatitis & whilst 5% of these get 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 rarely.

Drugs that inhibit mitochondrial β-fatty acid oxidation

1)Tetracycline, valproic acid,oestrogens,glucocorticoids

2) Amiodarone,perhexiline are charged lipophilic drugs concentrate in liver mitochondria & inhib. β-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 41

e) Ethanol induced steatohepatitis (ASH) proposed endotoxin fatty liver mechanism

1) Ethanol causes lipogenesis and

fatty liver (caused by inhibition of LDL synth. & export).

2) Ethanol oxidised by CYP2E1 to form hydroxyethyl radicals

AND ethanol oxidised by ADH to form acetaldehyde which cause oxidative stress and hepatocyte/gut cytotoxicity.

3) 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) 42