Metabolism & metabolic disorders I

(Medical Biochemistry I)
Lipid metabolism I
BCH 1212
Fatty Acid Metabolism
Lecturer:Milton A Wesuta
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 Introduction

SCOPE
• Synthesis of fatty acids
• Synthesis of TAGs
• Differences between Brown and White fat
• Fatty acid catabolism (β – Oxidation)
• Ketone body metabolism and related disorders

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 Introduction

• The major form of energy in our bodies is from Fatty acids

stored in TAGs
• Metabolic pathways interact in a complex way in order to
allow an adequate regulation.
• The switch from storage to mobilization of fatty acids is
therefore dependent on the energy requirements
• This interaction includes the enzymatic control of each
pathway, each organ's metabolic profile and hormonal
control.
• Fatty acids are obtained majorly in diet and from excess
carbohydrates and proteins.
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 Fatty Acid Synthesis

• Fatty acid synthesis in eukaryotic cells (e.g. mammals) takes

place:
a. predominantly in the cytoplasm of the liver
b. in the adipose tissue
c. in mammary glands during lactation
• It is energetically unfavourable
• Needs activation with ATP carboxylation acetyl CoA
followed by decarboxylation which releases energy to drive
the process

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 LIPID BIOSYNTHESIS

• Fatty acid biosynthesis-basic fundamentals

• Fatty acid biosynthesis-elongation and desaturation
• Triacylglycerols
• Cholesterol & Cholesterol metabolism
• Phospholipids

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 Fatty Acid Biosynthesis Vs Oxidation
The two are not the exact reversals of each other.
Synthesis Beta Oxidation
• Cytosol • Mitochondria
• Requires NADPH • NADH, FADH2
• Intermediates linked to –SH • Intermediates linked to –SH
of Acyl carrier protein of CoA
• D-isomer • L-isomer
• Major enzyme is a single • Many different enzymes
polypeptide with multiple involved
functions
• CO2 activation
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• No CO2
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 Rule:
Fatty acid biosynthesis is a stepwise assembly
of acetyl-CoA units (mostly as malonyl-CoA)
ending with palmitate (C16 saturated)

3 Phases
Activation

Elongation

Termination
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• Acetyl-CoA is generated in the mitochondria primarily from 2
sources, the pyruvate dehydrogenase (PDH) reaction & fatty acid
oxidation.
• The shift from fatty acid oxidation and glycolytic oxidation
occurs when the need for energy diminishes.
• This results in reduced oxidation of acetyl-CoA in the TCA cycle
and the oxidative phosphorylation pathway. Under these
conditions the mitochondrial acetyl units can be stored as fat for
future energy demands.
• In order for these acetyl units to be utilized for fatty acid
synthesis they must be present in the cytoplasm.

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 PROBLEM:
Fatty acid biosynthesis takes place in the
cytosol. Acetyl-CoA is mainly in the Mitochondria

acetyl-CoA

How is acetyl-CoA made available to the

cytosolic fatty acyl synthase?
SOLUTION
Acetyl-CoA is delivered to cytosol from the
mitochondria via the tricarboxylate transport system as
CITRATE
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 CH2COO HS-CoA
Acetyl-CoA
HO-C-COO
mitochondria Citrate lyase
CH2COO
COO
C=O
CH2COO OAA
CH2 Malate
HO-C-COO COO dehydrogenase
NADH
CH2COO
COO
L-malate HO-C-H L-malate
Acetyl-CoA
CH2
CO2
OAA COO Malic enzyme
NADP+
CO2
COO NADPH + H+
Pyr Cytosol
C=O Pyruvate
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CH3 Milton A Wesuta 11
 • In the cytoplasm, citrate is converted to OAA and acetyl-
CoA by the ATP driven ATP- citrate lyase reaction.
• The reaction is the reverse of that catalyzed by the TCA
enzyme citrate synthase except it requires the energy of
ATP hydrolysis to drive it forward. The resultant OAA is
converted to malate by malate dehydrogenase (MDH)
• Two potential fates for malate:
1. The malate undergoes oxidative decarboxylation by malic
enzyme. The co-enzyme for this reaction is NADP+
generating NADPH used for lipid synthesis. Pyruvate
formed is sent back to mitochondria via pyruvate
transporter & Converted back to oxaloacetate by
pyruvate carboxylase, requires ATP

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 2. Can be transported back to mitochondria via malate -
α-ketoglutarate transporter . Malate is reoxidized to
oxaloacetate

In this series of reactions for converting mitochondrial

acetyl-CoA into cytoplasmic acetyl-CoA, the NADPH
produced is an additional source of reducing co-factor
required for the Fatty Acid Synthase activities on top
of that produced in the PPP (50-60%).

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 ACTIVATION The synthesis of malonyl-
CoA is the first
CH3C~SCoA committed step of fatty
O acid synthesis and the
ATP HCO3- enzyme that catalyzes
ADP + Pi this reaction, acetyl-CoA
-OOC-CH C~SCoA
carboxylase (ACC), is the
major site of regulation
2

O
of fatty acid synthesis.
Like other enzymes that
active carbon transfer CO2 to
substrates, ACC requires
Acetyl-CoA carboxylase
a biotin co-factor (Vit B7).
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 Fatty Acid Synthesis
• Overall goal: attach acetyl unit from malonyl-CoA to a growing
chain & then reduce it
• Reaction involves cycles of four main enzyme-catalyzed steps
• Condensation of the growing chain with activated acetate
• Reduction of carbonyl to hydroxyl (alcohol)
• Dehydration of alcohol to trans-alkene
• Reduction of alkene to alkane
• The growing chain is initially attached to the enzyme via a
thioester linkage
• During condensation, the growing chain is transferred to the
acyl carrier protein (ACP)
• After the second reduction step, the elongated chain is
transferred back to fatty acid
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synthase
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 The mammalian Acetyl-CoA Carboxylase,
enzyme is regulated, by:
Phosphorylation/dephosphorylation
allosteric control by local metabolites.

o Acetyl-CoA is made in the mitochondria

o But fatty acids are made in the cytosol
o So Acetyl-CoA is transported into the cytosol
with a cost of 2 ATPs
o Therefore, cost of FA synthesis is 3 ATPs
per 2-C unit

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 Acyl Carrier Protein

Phosphopantetheine
H H HO CH3 O
HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-CH2-Ser- ACP
O OH H O
Cysteamine

Acyl carrier protein

10 kDa
H H HO CH3 O O
HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-P-O-CH2 Adenine
O
O OHH O O
O H
O-P-O OH
Coenzyme A
OH

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 Initiation Overall Reaction
Malonyl-CoA + ACP
Malonyl transacylase
CH3C~S E -OOC-CH C~S-
2 ACP + HS-CoA

O Malonyl ACP O
Acyl Carrier
Acetylated
synthase
CO2 HS-CoA Protein

CH3C-CH2C~S- ACP β – keto acyl synthase

O O

NOTE: The enzymes Acetyl CoA – ACP transacylase

Transfers an Acetyl group from Acetyl CoA to the SH group of the
β – keto acyl synthase to form the acylated enzyme
Similarly Malonyl transacylase transfers a Malonyl group from Malonyl
CoA to the SH group of the Phosphopantetheine prosthetic group of
ACP
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 Malonyl-CoA carbons become new COOH end

Nascent chain remains tethered to ACP

CO2, HS-CoA are released at each condensation

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 -Carbon Elongation
CH3C-CH2C~S- ACP
O O Reduction
NADPH
-Ketoacyl-ACP reductase
D isomer H
CH3C-CH2C~S- ACP
HO O Dehydration
-H2O  -Hydroxyacyl-ACP dehydratase
H
CH3C-= C- C~S- ACP
NADPH H Reduction
O
Enoyl-ACP reductase

CH3CH2CH2C~S- ACP
O
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 Fatty Acid Synthase O
S-C-CH2-CH2-CH3
-Ketoacyl
KS Acetyl-CoA-
-ACP synthase Acetyl-CoA ACP transacylase
KS
O HS
CH3-CH2-CH2-C-S CoA-SH O Initiation or
S -C-CH3 priming
NADP+
Enoyl-ACP
reductase NADPH KS -SH
H+ O
O
ACP
KS S-C-CH3
CH3-CH=CH-C-S
-Hydroxyacyl-ACP H2O SH
dehydrase Malonyl-CoA
Malonyl-CoA-
OH O CoA-SH ACP transacylase
CH3-CH -CH2-C-S O
S -C-CH2-COO-
NADP+ S
CO2 -Keto-ACP
NADPH C=O synthase (condensing enzyme)
H+ KS -SH
-Ketoacyl CH2
-ACP reductase Elongation
C=O
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CH3
 TERMINATION
Cys -SH O

ACP -S – C – (CH2)13 – CH2 – CH3

H2O
Palmitoyl thioesterase

Cys -SH

ACP -SH + CH3 – CH2 - (CH2)13 – COO-

Palmitate (16c)

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 Overall Reactions

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

7 Acetyl-CoA + 7CO2 + 7ATP

7 malonyl-CoA +7ADP + 7Pi + 7H+

8 Acetyl-CoA + 14NADPH + 7H+ + 7ATP

Palmitate + 14NADP+ + 8 HSCoA + 6H2O + 7ADP + 7Pi

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 Post-Synthesis Modifications
• C16 saturated fatty acid (Palmitate) is the product
• Elongation
• Unsaturation
• Incorporation into triacylglycerols
• Incorporation into acylglycerol phosphates

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 Elongation of Chain (two systems)
R-CH2CH2CH2C~SCoA Malonyl-CoA*
O (cytosol)
HS-CoA
OOC-CH2C~SCoA CH3C~SCoA
O O
CO2
Acetyl-CoA
R-CH2CH2CH2CCH2C~SCoA (mitochondria)
O O
1 NADPH
NADH
Elongation systems are
2 - H2O found in smooth ER and
mitochondria
3 NADPH
R-CH2CH2CH2CH2CH2C~SCoA
O
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 Desaturation
Rules:
The fatty acid desaturation system is
in the smooth membranes of the endoplasmic
reticulum
There are 4 fatty acyl desaturase enzymes in
mammals designated 9 , 6, 5, and 4 fatty
acyl-CoA desaturase
Mammals cannot incorporate a double bond
beyond 9; plants can.
Mammals can synthesize long chain unsaturated
fatty acids using desaturation and elongation
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 Palmitate
Desaturase 16:0
Palmitoleate Elongase
Stearate
16:1(9)
18:0 Permitted
Desaturase
Oleate transitions
Essential 18:1(9) in mammals
fatty acid Desaturase
Linoleate
Desaturase 18:2(9,12)
-Linolenate
-Linolenate Desaturase 18:3(6,9,12)
18:3(9,12,15) Elongase
Eicosatrienoate
20:3(8,11,14)
Other lipids Desaturase
Arachidonate
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20:4(5,8,11,14) 27
 Regulation of Acetyl-CoA Carboxylase (ACCase)

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 Regulation of FA synthesis:
Acetyl CoA Carboxylase
• Allosteric regulation
• stimulated by citrate
– feed forward activation
• inhibited by palmitoyl CoA
– high β-oxidation (fasted state)
– or esterification to TAG (limiting)
• Inducible enzyme
– Induced by insulin
– Repressed by glucagon

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 Regulation of FA synthesis:
Acetyl CoA Carboxylase
• Covalent Regulation
• Activation (fed state)
– insulin induces protein
phosphatase
– activates ACC
• Inactivation (starved
state)
– glucagon increases cAMP
– activates protein kinase
A
– inactivates ACC
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 Lipid Metabolism in Fat Cells:
Fed State Starved or Exercising State
• Insulin • Glucagon (“GLUCOSE is
• stimulates LipoProtein GONE”)
Lipase(LPL) • Epinephrine (“We need
–increased uptake of FA energy now”)
from chylomicrons and VLDL activates adenylate
• stimulates glycolysis cyclase
– increased glycerol –increases cAMP
phosphate synthesis
– activates protein kinase A
– increases esterification
– activates HSL
• induces HSL-phosphatase
• net effect: TG
– inactivates HSL
mobilization and
• net effect: TG storage increased FFA
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 TAG BIOSYNTHESIS & β – OXIDATION OF
FATTY ACIDS
• Triacylglycerol (TAG) biosynthesis
• β – Oxidation of Fatty acids

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 Brown Fat Vs White fat
• Two types of fat found in mammals.
i. Brown adipose tissue (the other being
ii. White adipose tissue)
Brown Fat is especially abundant in newborns and in hibernating
mammals.

• fuel oxidation does not produce ATP but generate heats to keep
the newborn and hibernating mammals warm
• White fat serves many functions including insulating the viscera,
storing excess carbon energy in the form of triglycerides and
mediating glucose homeostasis
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 Brown Fat Vs White fat
• Brown fat also contains more capillaries than white fat,
since it has a greater need for oxygen than most tissues
• In contrast to white adipocytes (fat cells), which
contain a single lipid droplet, brown adipocytes contain
many smaller droplets and a much higher number of
(Fe3+ containing) mitochondria, thus large amounts of
cytochromes, with heme groups which make it brown.
• Brown fat cells have a unique protein Thermogenin, also
called the uncoupling protein in their inner membrane
which promotes thermogenesis

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 Comparison of Energy Sources
Glycogen Fat

• Typically store a one • Usually store a one

day energy supply month energy supply
• Less energy per gram • May store much more
produced than for fat energy
• About 70% by weight • More energy produced
is water per gram
• No water
• Released after glycogen
is gone
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 Storage and Mobilization of Triacylglycerols

• Fatty acids are stored in adipocytes as components of TAGs

• Triacylglycerol Synthesis in vivo
• The reactants in triacylglycerol synthesis are glycerol 3-
phosphate and fatty acid acyl groups carried by coenzyme A.
• TAG synthesis starts with synthesis of the precursor
molecules Glycerol – 3 - phosphate and Fatty acyl CoA.
• After a meal, blood glucose levels increase, insulin levels rise,
and glucagon levels drop.

Milton A Wesuta
 • Glucose enters cells, the rate of glycolysis increases,
and insulin activates the synthesis of triacylglycerols
for storage.
• Triacylglycerol synthesis proceeds by transfer of first
one and then another fatty acid acyl group from
coenzyme A to glycerol 3-phosphate.
• The reaction is catalyzed by acyl transferase, and the
product is phosphatidic acid.

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 Sources of Glycerol – 3 - Phosphate
1. Glycolysis to DHAP
in the liver &
adipose tissue

2. From free glycerol

in the liver

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 Source of Fatty acyl CoA

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 • Pathway has 4 reactions:
(i) Sequential addition of two fatty acyl CoA
units
(ii) the two free hydroxyl groups of L-glycerol 3-
phosphate are acylated to yield diacylglycerol
3-phosphate, more commonly called
phosphatidic acid or phosphatidate
(iii)Removal of phosphate from the phosphatidate

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 (iv) Addition of another of the third fatty acyl CoA
Phosphatidic acid, present in only trace amounts in
cells, is a central intermediate in lipid biosynthesis;
(a)it can be converted either to a triacylglycerol or
(b)to a Phosphoglyceride.
(c)In the pathway to triacylglycerols, phosphatidic
acid is hydrolyzed by phosphatidic acid phosphatase
to form a 1,2-diacylglycerol. Diacylglycerols are
then converted to triacylglycerols by
transesterification with a third fatty acyl–CoA.

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 Triacylglycerol Biosynthesis in Animals Is
Regulated by Hormones
• Carbohydrate, fat, or protein consumed in excess of energy
needs is stored as TAGs from which energy can be drawn to
withstand periods of fasting.
• Biosynthesis and degradation are regulated depending on the
metabolic resources and requirements of the moment.
• Insulin, promotes the conversion of carbohydrate to TAGs
• In severe diabetes mellitus, due to failure of insulin secretion
or action, glucose utilisation is hampered as well as synthesis
of fatty acids from carbohydrates or amino acids. If the
diabetes is un-treated, these individuals have increased rates
of fatty oxidation and ketone body formation and therefore
lose weight.
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