Metabolism of Fatty Acids

-Oxidation
Florence Rajaretnam M.Sc., M.Phil.
 Learning Objectives
After reading this chapter you should be
able to:
Describe the mobilization of fatty acids from adiposites
Trace out the pathway followed by glycerol and Fatty acids
Outline the pathway for activation & transport of FAs to the
mitochondrion for catabolism.
Describe the sequence of rxns involved in oxidation of FAs in the
mitochondrion.
Explain the rationale for the pathway of ketogenesis & identify the
major intermediates & products of this pathway.

2
3
 When the energy supply from the diet is limited, the body responds to this
deficiency through the secretion of hormones like
glucagon, epinephrine or adrenocorticotropic hormones.
These Hormones trigger mobilization of stored TAGs:
When hormones signal the need for metabolic energy,
TAGs stored in adipose tissue are
mobilized (brought out of storage) &
transported to tissues (skeletal muscle, heart, & renal cortex)
in w/c FAs can be oxidized for energy production
The hormones epinephrine & glucagon; Fig-D9
secreted in response to low blood glucose levels,
activate the enzyme adenylyl cyclase
in the adipocyte plasma membrane
w/c produces the cAMP
4
5
6
 Cells with defective perilipin genes;
have almost no response to increases in cAMP conc.
their HSL does not associate with lipid droplets

HSL hydrolyzes the triacylglycerol at C#1 or C#3 to produce diacylglycerol and a

fatty acid.

Diacylglycerol lipases hydrolyze the diacylglycerol to monoacylglycerol and a fatty

acid.

Finally Monoacylglycerol lipases hydrolyze monoacylglycerol to fatty acid and

glycerol.

7
8
Fate of the glycerol and Fatty acids obtained during TAG
degradation
Glycerol:
Adipose tissue lacks glycerol kinase and therefore cannot use
this glycerol to synthesize fats.

This glycerol is transported through the blood to the liver

where it may undergo different fates:

1-By the reverse of the glycerol dehydrogenase reaction,

glycerol may be converted into dihydroxyacetone phosphate
which can be converted
to glucose by gluconeogenesis or
pyruvate by the glycolysis sequence of reactions. 9
2-By the action of glycerol kinase
glycerol may be phosphorylated to glycerol-3-phosphate,
which can be used to synthesize fats.

About 95% of the biologically available energy of TAGs

resides in their three long-chain FAs
only 5% is contributed by the glycerol moiety.

10
FATE OF FATTY ACIDS:
The FFA pass from the adipocyte into the blood,
where they bind to the blood protein serum albumin
Bound to this soluble protein, the otherwise insoluble FAs,
are carried to tissues such as skeletal muscle, heart, & renal
cortex
In these target tissues, FAs dissociate from albumin & are
moved;
by plasma membrane transporters into cells to serve as
fuel

11
 OXIDATION OF FATTY ACIDS
-oxidation of FA is:
the principal pathway for the catabolism of FA
two carbon fragments are successively removed from the
carboxyl end of the fatty acid, producing acetyl CoA.

Site: Mitochondrial matrix of tissues such as

Heart (80% of its fuel), Liver, lungs, muscles, kidney.

Human tissues in general Except for RBC & Brain

Brain cells have -oxidation ability but blood brain barrier:
prevent the entrance of FAs to these cells,
12
 Steps in -Oxidation

Step I-Activation of Fatty Acids in the Cytosol

As the priming step for their catabolism, the FAs are:
activated to their CoA derivative,
using adenosine triphosphate (ATP) as the energy
source.
The enzyme is Fatty acyl CoA synthetase
Fatty acid + ATP + CoA Acyl-CoA + PPi + AMP

the rest of the reactions of -Oxidation occur in the

mitochondria;
13
 Acyl CoA Transport & Role of Carnitine
The FAs with chain lengths of 12 carbons;
enter mitochondria without the help of membrane
transporters
Those with 14 carbons;
which constitute the majority of the FFA:
obtained in the diet or released from adipose tissue,
cannot pass directly through the mitochondrial
membranes

14
Therefore, a specialized carrier located in the inner
mitochondrial membrane transports the acyl group from the
cytoplasm into the mitochondrion.

This carrier is Carnitine, and the transport process is called the

Carnitine shuttle.

The Carnitine Shuttle

1. The CoA from fatty acyl CoA is exchanged for Carnitine by
Carnitine Palmitoyl transferase I (CPT I), (which resides in
the outer mitochondrial membrane) to form fatty acyl-
carnitine.

15
2. The fatty acyl-carnitine is transported into the mitochondrial
matrix by carnitine cayl carnitine translocase.

3. Carnitine palmitoyl transferase II (CPT II) catalyzes the

regeneration of the fatty acyl-CoA molecule (figure I.1).

16
Fig-1.1 Transport of Long-chain FAs into Mitochondria 17
Mitochondrial Oxidation of FAs
Takes place in three
stages Fig-1.2

18
In the 1st stage -oxidation Fig-1.3
2 carbons are cleaved at a time to form acyl-CoA
molecules,
starting from the carboxyl end
The chain is broken between the (2) & (3)
carbon atoms,
hence the name -oxidation
Formation of each acetyl-CoA requires
removal of 4 hydrogen atoms
from the fatty acyl moiety by dehydrogenases
19
In the 2nd stage of FA oxidation,
The acetyl groups of acetyl-CoA are
oxidized to CO2 in the TCA cycle mitochondrial
matrix
The first two stages of fatty acid oxidation produce
the reduced electron carriers NADH & FADH2,
w/c in the 3rd stage donate electrons to ETC;
through which the electrons pass to oxygen
with the concomitant phosphorylation of ADP to ATP
The energy released by fatty acid oxidation is
thus conserved as ATP

20
 Fig-1.3. Overview of mitochondrial
long-chain FA metabolism
1) FA binding proteins (FaBP)
transport FAs across
the plasma membrane & bind them
in the cytosol
2) Fatty acyl CoA synthetase
activates FAs to fatty acyl CoAs
3) Carnitine transports the activated
fatty acyl group
into mitochondria
4) -oxidation generates
NADH, FAD(2H), & acetyl CoA
5) In the liver, acetyl CoA is
converted to ketone bodies

21
 -Oxidation of Saturated FAs
Overview of - Oxidation Fig-2
Four Basic Steps:
4 enzyme-catalyzed rxns make up the first stage of FA
oxidation
Step I: Oxidation (unsaturation)- dehydrogenation:
Catalyzed by acyl-CoA dehydrogenase
Coenzyme for this rxn is
a flavoprotein FAD as a prosthetic group
FADH2 is oxidized;
in mitochondrial respiratory chain to give 2 ATP
22
Contd
Step II: Hydration:
Catalyzed by enoyl-CoA Hydratase (crotonase)
which helps the addition of H2O to saturate double
bond
Step III: Oxidation (-oxidation)- dehydrogenation
By -Hydroxyacyl CoA dehydrogenase & NAD+
Oxidation of NADH + H+ in respiratory chain gives 3
ATP

23
Step IV : Splitting of active acetate- (Chain breakage):
It is catalyzed by Thiolase (acyl-CoA acetyltransferase)
It splits acyl CoA into Acetyl CoA & Acyl CoA,
w/c is shorter than the first one by 2 carbon
atoms
The process is repeated Until the whole FA is
Broken into acetyl CoA;
w/c are then oxidized to CO2 & H2O in Krebs
cycle

24
25
How is the amount of ATP produced by the degradation of a fatty
acid calculated?
In order to obtain this information it would be very easy if we
follow thes steps:
1- The following formula may be used to calculate the number of
times the cycle of the reactions may occur till the total
degradation of a fatty acid to acetyl CoA

1. Number of cycles = n/2- 1, where n= number of carbon atoms

of the fatty acyl CoA.

2. For each cycle, 5 molecules of ATP are generated.

Multiply the results obtained in the above step by 5 to obtain the
number of ATP molecules generated during the oxidation
26
3.Subtract 2 ATP from the above to account for the two ATP that
was lost by the cell in the activation reaction of the fatty acid.

By these three steps we can calculate the amount of ATP obtained

by the degradation of a fatty acid till acetyl CoA.

27
If we want to know the total amount of ATP obtained by the total
degradation of the fatty acid to CO2 and H2O, another two steps
are necessary:
4-The total number of acetyl CoA molecules obtained from
oxidation of a fatty acid is obtained by the formula n/2
5-This last result is multiplied by 12
(every acetyl CoA molecule degraded by the Krebs cycle reactions
yields12 ATP).
Energy gain = [ (N/2 1) x 5 ATP] + [N/2 x 12 ATP] 2 ATP
where N = Number of carbons of a FA

For example:
The degradation of palmitoyl CoA (16 carbons atoms) till acetyl
CoA yields 33 ATP, but its total degradation till CO2 and H2O
produces 129 ATP.
28
Oxidation of FAs With Odd No of C-Atoms
 Formation of propionyl CoA from odd-chain FAs

Fig-9a: Oxidation of Propionyl-CoA produced by Oxidation of odd-No FAs

Fig-9b. Conversion of Propionyl CoA to Succinyl CoA
Importance of -Oxidation
Source of energy:
Oxidation of FA is a major source of energy
to the body during starvation
Production of acetyl CoA:
Acetyl CoA is converted to several useful compounds:
e.g. cholesterol, acetyl choline
Ketone bodies formation:
Formation of acetoacetyl CoA;
w/c is derived from oxidation of long chain acyl
CoA,
i.e. the last 4 carbon atoms, may be converted to;
32
Acetoacetic acid w/c is one of the ketone bodies
Regulation of -Oxidation
FAs are used as fuels principally when they are Fig-10a
Released from adipose tissue TAGs in response to
Hormones that signal Fasting or Increased demand
The process of - oxidation is regulated by:
the cells requirements for energy
i.e., by the levels of ATP & NADH
b/c FAs cannot be oxidized any faster than
NADH & FAD(2H) are reoxidized in the ETC
33
 Fig-10a.
Regulation of lipid
metabolism by
glucagon &
34 epinephrine .
 When energy increases (ATP), Fig-10b- step 3
-oxidation is inhibited & vice versa
Excess ATP moles in the cells Inhibit respiratory chain
Thus the reduced FADH2 & NADH + H+ can not undergo
Oxidation in respiratory chain, & remain as such.
This leads to Decreased energy & Inhibition of -oxidation
Fatty acid oxidation also may be restricted
by the mitochondrial CoASH pool size
Acetyl CoASH units must enter:
TCA cycle or another metabolic pathway
To regenerate CoASH required for formation of
Fatty acyl CoA derivative from fatty acyl carnitine

35
 Fig-10b. Regulation of -Oxidation
1. Hormones control the supply of fatty acids in the blood.
2.CPT- I is inhibited by malonyl CoA, w/c is synthesized by acetyl CoA
carboxylase
AMP-PK is the AMP-dependent protein kinase
3.The rate of ATP utilization controls the rate of the ETC,
w/c regulates the oxidative enzymes of -oxidation and the TCA cycle
36
An additional type of regulation occurs at CPTIFig-10b-Step2
CPT I is inhibited by malonyl CoA,
w/c is synthesized by Acetyl CoA carboxylase
Acetyl CoA carboxylase is regulated by a no of different
mechanisms some of w/c are tissue dependent
In skeletal muscles & liver,
it is inhibited when phosphorylated by protein kinase B an
AMP-dependent protein kinase
Thus, during exercise when AMP levels increase,
AMP-dependent protein kinase phosphorylates
Acetyl CoA carboxylase becomes inactive37
Consequently, malonyl CoA levels decrease:
CPTI is activated & -oxidation of FAs is able to;
Restore ATP homeostasis & decrease AMP levels
In liver, in addition to the regulation by
AMP-dependent protein kinase Fig-10b-Step 2
Acetyl CoA carboxylase is activated by
Insulin-dependent mechanisms
w/c promotes the conversion of malonyl CoA to
palmitate In the FA synthesis pathway
Thus, in the liver, malonyl CoA inhibition of CPTI;
Prevents newly synthesized FAs from being oxidized 38
-oxidation is strictly an aerobic pathway
Dependent on O2 & Mitochondria
Tissues that lack mitochondria, such as RBCs,
Cant oxidize FAs by -oxidation
FAs also do not serve as
a significant fuel for the brain
They are not used by adipocytes, whose function is;
to store TAGs to provide a fuel for other tissues
Those tissues that
Dont use FAs as a fuel, or use them only to a limited
extent, are able to use ketone bodies instead
39
 Metabolism of Ketone Bodies
In humans & most other mammals,
Acetyl-CoA formed in the liver during oxidation of FAs
can either enter the TCA cycle or
undergo conversion to the ketone bodies:
Acetoacetate, D--hydroxybutyrate, & acetone
for export to other tissues

Acetone, produced in smaller quantities than the others

which is excreted in urine or expired by the lungs

40
41
Synthesis of Ketone Bodies/Ketogenesis
In the liver, ketone bodies are synthesized
In the mitochondrial matrix
from acetyl CoA generated from FA oxidation
The thiolase rxn of FA oxidation, w/c converts;
acetoacetyl CoA to two molecules of acetyl CoA
is a reversible rxn,
although formation of acetoacetyl-CoA is not the favored
direction
It can, thus, when acetyl-CoA levels are high,
42
The acetoacetyl CoA will react with acetyl CoA Fig-15
to produce 3-hydroxy-3-methylglutaryl CoA (HMG-
CoA)
The enzyme that catalyzes this rxn is HMG-CoA
synthase
In the next rxn of the pathway,
HMG-CoA lyase catalyzes the cleavage of HMG-CoA to
form
acetyl CoA & acetoacetate
enters the blood

43
Acetoacetate can directly enter the blood or
it can be reduced by -hydroxybutyrate dehydrogenase
to
-hydroxybutyrate, which enters the blood

dehydrogenase rxn is readily reversible

interconverts these two ketone bodies

44
Under normal conditions,
the ratio of -hydroxybutyrate to acetoacetate in the
blood is 1:1
An alternate fate of acetoacetate is
spontaneous decarboxylation, a nonenzymatic rxn that
cleaves
acetoacetate into CO2 & acetone
Because acetone is volatile,
it is expired by the lungs or excreted in urine
45
Ketogenesis

46
Oxidation of Ketone Bodies as Fuels/Ketolysis
Acetoacetate and -hydroxybutyrate
can be oxidized as fuels in most tissues, including
skeletal muscle & brain,
certain cells of the kidney,
cells of the intestinal mucosa
Cells transport both acetoacetate & -hydroxybutyrate
from the circulating blood into the cytosol, &
into the mitochondrial matrix
47
-hydroxybutyrate is oxidized back to acetoacetate
by
-hydroxybutyrate dehydrogenase
This rxn produces NADH
Subsequent steps convert
acetoacetate to acetyl CoA
In mitochondria,
acetoacetate is activated to acetoacetyl CoA;
by succinyl CoA:acetoacetate CoA transferase
/thiotransferase/thiophorase
As the name suggests, CoA is transferred from
succinyl CoA to acetoacetate
48
Although the liver produces ketone bodies, it does not use
them,
b/c this thiotransferase (thiophorase) enzyme is not
present in sufficient quantity

Acetoacetyl CoA is cleaved to 2 molecules of acetyl CoA

by acetoacetyl CoA thiolase
The principal fate of this acetyl CoA is
oxidation in the TCA cycle

49
50
 When the rate of formation of ketone bodies is greater than the
rate of their use, their levels begin to rise in the blood (ketonemia)
and eventually in the urine (ketonuria).
This state is known as ketoacidosis or Ketosis.
Extreme acidosis can lead to coma & in some cases death
During Ketosis acetone is eliminate by the breath.

There are three principal causes of ketosis:

1. Diabetes mellitus
2. Starvation periods
3. A rich fat and free carbohydrate diet.

51