Fatty acid oxidationKetone bodies

Fatty acid synthesis

AV. Lipid metabolism 1-2. 2011

Triacylglycerols

Major energy reserveOxidation: 9 kcal/g

(for carbohydrates: 4 kcal/g)

11 kg of 70 kg total body weight

Site of accumulation: cytoplasm of ADIPOSE CELLS

Adipose tissue

-specialized for synthesis, storage, mobilization of lipids

Lipases

Number of carbons

Number of double bonds

Name

16 0 Palmitate

18 0 Stearate

20 0 Arachidate

16 1 Palmitoleate Cis 9

18 1 Oleate Cis 9

18 2 Linoleate Cis 9, 12

18 3 Linolenate Cis 9, 12, 15

20 4 Arachidonate Cis 5, 8, 11, 14

The most important fatty acids

Mobilization of triacylglycerols stored in adipose tissueGlukagon, adrenalin, ACTH

↓ receptor activation

↓protein kinase A

↓phosphorylation of Perilipin

(when dephosphorylated it inhibits the access of lipases to TG and DG

+phosphorylation of HSL

(hormon-sensitive lipase)activation

↓CGI dissociates from Perilipin

then associates with ATGL (adipocyte trigliceride lipase)

↓TG→DG + fatty acid

↓HSL: DG→MG + fatty acid

↓MGL (monoacylglycerol lipase):

MG→G + fatty acid Lehninger, Principals of Biochemistry, 2013

ADIPOSE TISSUE

TG Fatty acids

Fatty acids - bound to albumin

(10 fatty acids/albumin monomer)

(free fatty acids, FFA)

MUSCLE, HEART MUSCLE, RENAL CORTEX

Fatty acid activation, transport into the mitochondria, β-oxidation

CIRCULATION

Site of β-oxidation: mitochondria

Activation of fatty acids:

Fatty acid + ATP + CoA <------> Acyl-CoA + PPi + AMP Acyl-CoA synthetase

ATP

R C

O

S CoA

R C

O

AMP PPi

R C

O

P O

O

O

Ribose Adenin

R C

O

AMP HS-CoA

R-COOH + +

acyl-adenylate

+ AMP+

1.

2.

acyl-CoA

Fast PPi hydrolysis reaction is irreversible in vivo

Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane

Triacylglycerols

Major energy reserveOxidation: 9 kcal/g

(for carbohydrates: 4 kcal/g)

11 kg of 70 kg total body weight

Site of accumulation: cytoplasm of ADIPOSE CELLS

Adipose tissue

-specialized for synthesis, storage, mobilization of lipids

Lipases

Number of carbons

Number of double bonds

Name

16 0 Palmitate

18 0 Stearate

20 0 Arachidate

16 1 Palmitoleate Cis 9

18 1 Oleate Cis 9

18 2 Linoleate Cis 9, 12

18 3 Linolenate Cis 9, 12, 15

20 4 Arachidonate Cis 5, 8, 11, 14

The most important fatty acids

Lipid droplet

Mobilization of fatty acids from the adipose tissue

Glukagon, adrenalin, ACTH

Receptor activation

Protein kináz A

Phosphorylation of Perilipin (when dephosphorylated it inhibits the

access of lipase to TG,phosphorylated perilipin has no such

effect)+

Phosphorylation of Hormon-sensitive lipase

(activation)

Mobilization of fatty acids from TG

ADIPOSE TISSUE

TG Fatty acids

Fatty acids - bound to albumin

(10 fatty acids/albumin monomer)

(free fatty acids, FFA)

MUSCLE, HEART MUSCLE, RENAL CORTEX

Fatty acid activation, transport into the mitochondria, β-oxidation

CIRCULATION

Site of β-oxidation: mitochondria

Activation of fatty acids:

Fatty acid + ATP + CoA <------> Acyl-CoA + PPi + AMP Acyl-CoA synthetase

ATP

R C

O

S CoA

R C

O

AMP PPi

R C

O

P O

O

O

Ribose Adenin

R C

O

AMP HS-CoA

R-COOH + +

acyl-adenylate

+ AMP+

1.

2.

acyl-CoA

Fast PPi hydrolysis reaction is irreversible in vivo

Site of fatty acid activation: cytosolic side of the mitochondrial outer membrane

Transport of fatty acids into the mitochondria

Transport of Long-chain (12-18 C atom) fatty acids with carnitine

Transport of carnitine-acylcarnitine is the rate-limiting step and most important control point in fatty acid oxidation

carnitine-acyltransferase I (CPT I)

carnitine-acyltransferase II (CPT II)

Fatty acids with <12 C enter mitochondria without carnitine and are activated in the mitochondria

Transport of fatty acids into the mitochondria

Three isoenzymes: -long-chain fatty acids (C 12-18) -medium chain fatty acids (MCAD, 4-14) -short-chain fatty acids (4-8)

MCAD deficiency is relatively frequent

Specific for L-stereoisomere

Oxidation of fatty acids with >12 C is carried out by a multienzyme bound to the mitochondrial inner membrane, in which the last three enzymes are tightly associated (trifunctional protein),when the chain is < 12 C soluble enzymes in the matrix continue the oxidation

β-oxidation of fatty acids

Conversion of glycerol to glycolysis intermediate – in the liver

glycerol kinase Glycerol-P dehydrogenase

hormones(adrenaline, glucagon)

Malonyl-CoA

Increased level of free fatty acids

Oxidation

Inhibiton of PerilipinActivation of hormone-sensitive lipase

NADH

Inhibition of carnitineacyltransferase I

Entry of fatty acids into mitochondria is

inhibited

Oxidation

Inhibition of 3-hydroxyacyl CoA

dehydrogenase

thiolase

Inhibition of ß-oxidation

High energy state

Regulation of fatty acid oxidation

Acetyl-CoA

Long-term regulation:

PPAR (peroxisome proliferator/activated receptors)nuclear receptor – transcription factors

PPARα – muscle, adipose tissue, liver regulate the transcription of fatty acid transporters, CPT I és CPT II,

and acyl-CoA dehydrogenase

- energy need (fasting, between-meal periods)

PPARα activation

transcription of enzymes of fatty acid oxidation

- fetus - principal fuels for heart: glucose and lactate -neonatal – fatty acid

Regulation of metabolic transition by PPARα

- sustained exercise – PPARα expression in muscles

The most common genetic defect in fatty acid oxidation Acyl-CoA dehydrogenase deficiency

For the medium length acyl-CoA dehydrogenase

Prevalence: 1:40 – mutation in one of the chromosomes 1:10000 – two mutant copies – disease manifestation

symptoms in the first years

-hypoglycemia – with decreased ketone body formation (decreased fatty acid oxidation and gluconeogenesis in the liver)

-accumulation of lipids in the liver

-vomiting, drowsiness

Therapy: frequent carbohydrate-rich meals + carnitine supply

Deficiency of carnitine transport into the mitochondria

Long-chain fatty acid transport

Carnitine deficiency

-high-affinity plasma membrane transporter (heart, kidney, muscle – but not liver) muscle cramps – weakness – death

addition of carnitine

-secondary carnitine deficiency due to deficiency on β-oxidation acyl-carnitine in the urine

Carnititne acyltransferase deficiency

most common - CPT II gene mutation – partial loss of enzyme activity muscle weakness when more serious – hypoglycemia with decreased ketone body formation

GLUCOSE

PYRUVATE

OXALOACETATE

CITRATE

GL

UC

ON

EO

GE

NE

SIS

ACETYL-CoA

FATTY ACID

ß-oxidation

Ketone bodies

FORMATION OF KETONE BODIES

ANAPLEROTIKUS REAKCIÓ

Fatty acid oxidation + lack of oxaloacetate

fastinguntreated diabetes

Synthesis of ketone bodies in the liver

Ketone bodies as fuels

Oxidation of ketone bodies in the extrahepatic tissues

heart musclestriatal musclekidneybrain

Ketone bodies can be regarded as a transport form of acetyl groups

Important sources of energy: heart muscle, renal cortex (preference to glucose, 1/3 of the energy)

brain - glucose is the major fuel but in starvation and diabetes brain uses acetoacetate

Ketone bodies

Fasting

Diabetes

high level of ketone bodies in the blood

KETOSIS

Formation in the liver exceeds the use in the periphery.

Level of ketone bodies after an overnight fast: ~0.05 mM

2 days starvation: 2 mM (40-fold increase!)

40 days: 7 mM

Fatty acid synthesis - repeated cycles – in each cycle the chain is extended by two carbons – four steps in each cycle Enzyme:

fatty acid synthase

Seven active site for different reactions in separate domains of

a single large polypeptide

Fatty acid synthesis –Lipogenesis-not a reversal of degradation

SYNTHESIS BREAKDOWN

Site Cytosol Mitochondrial matrix

Intermediates bound to Acyl-carrier protein CoA

Enzymes Joined in a single polypeptide chain (fatty acid synthase)

Not associated

Reducing equivalents NADPH NAD, FAD

Units Malonyl-CoA Acetyl-CoA

3-hydroxyacyl-derivative D-enantiomer L-enantiomer

Fatty acid synthesis:

*Liver*Adipose tissue*Lactating mammary gland

Committed step in fatty acid synthesis: formation of malonyl-CoA from acetyl-

CoA

acetyl-CoA carboxylase - prosthetic group: biotin

Acetyl-CoA carboxylase has three activities in a single polypeptide

Biotin – covalently bound to Lys έ-amino group

1. transfer of carboxyl group to biotin

ATP-dependent

move of activated CO2 from the biotin carboxylase region to the

transcarboxylase active site

2. transfer of the activated carboxil group from biotin to acetyl-CoA

Acyl carrier protein

SH group is the site of ently of malonyl group during fatty acid synthesis

CH2 SH

SHCondenzing enzyme - cys

Critical SH-groups carry the intermediates during the synthesis of fatty acids

β-ketoacyl-ACP synthase

Fatty acid synthase

Acetyl group from Acetyl-CoA is transferred to

Cys-SH of β-ketoacyl-ACP synthase (KS) by MAT

Malonyl group is transferred to ACP-SH by MAT

Acetyl group (from Acetyl-CoA) is transferred to the

malonyl group on ACP (methyl terminal)

Acetoacetyl-ACP

by β-ketoacyl-ACP reductase

Reduction

Dehydration

by β-hydroxyacyl-ACP dehydratase

Reduction

by enoyl-ACP reductase

ACP is recharged with another malonyl group by MAT

Second round of fatty acid synthesis cycle

The overall process of palmitate synthesis

Seven cycles for the synthesis of palmitate

Palmitate is released from ACP by thioesterase (TE)

STOICHIOMETRYSeven cycles for the synthesis of palmitate

Ac-CoA + 7 malonyl-CoA + 14 NADPH + H+ Palmitate + 7 CO2 + 14 NADP+ + 8 CoA + 6 H2O

7 Ac-CoA + 7 CO2 + 7 ATP 7 malonyl-CoA + 7 ADP + 7 Pi

Overall: 8 Ac-CoA + 7 ATP + 14 NADPH + H+ palmitate + 14 NADP+ + 8 CoA + 6 H2O + 7 ADP + 7 Pi

Fatty acid synthase is present exclusively in the cytosol

In adipocytes and hepatocytes cytosolic [NADPH]/[NADP] ratio is high (~75) – strongly reducing environment

In hepatocytes and lactating mammary gland cytosolic NADPH is generated largely by pentose phosphate pathway but malic enzyme is also significant

MITOCHONDRION CYTOSOL

CITRATE CITRATE

OXALOACETATEOXALOACETATE

PYRUVATEPYRUVATE

MALATE

AcCoA

AcCoA

NADH

NADPH

Citrate carries Acetyl-CoA from mitochondria to the cytosol for

fatty acid synthesisATP:citrate lyase

malic enzyme

Source of NADPH for fatty acid synthesis

Regulation of fatty acid synthesis

-regulation of Acetyl-CoA carboxylase-

- allosteric stimulation by citrate

-regulation by covalent modification

Dephosphorylated form – active-Polymerizes into long filaments insulin

Active dephosphorylated ACC

Phosphorylation inactivates the enzyme

-negative feed-back inhibition by

palmitate

Control of fatty acid synthesis

Short term regulation

• Ac-CoA • ATP isocitrate dehydrogenase inhibited

citrate – stimulates Ac-CoA carboxylase +carries the substrate (Ac-CoA)(indicates that two-carbon units & ATP are available

for synthesis)

Glucagon cAMPactivation of protein kinases

phosphorylation of Ac-CoA carboxylase - switch off

Palmitoyl-CoA*Inhibits Ac-CoA carboxylase

*Inhibits translocation of citrate from mitochondria to cytosol

*Inhibits glucose 6-P dehydrogenase NADPH

Control of fatty acid synthesis

Long term regulation

– low fat, high carbohydrate diet the amount of Acetyl-CoA carboxylase & fatty acid synthase is

increased

- fasting and high fat diet the amount of Acetyl-CoA carboxylase is decreased

Control of fatty acid synthesis

High-fat low carbohydrate diet – Atkins dietfatty acid mobilization - ketone body formation (loss in the urine)