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BIOLOGICAL CHEMISTRY
Chapter 6: LIPID METABOLISM
(April 2010)
Dr. T.K. Bose
Department of Zoology,
Miranda House,
University of Delhi,
Delhi-110007, India
CONTENTS
INTRODUCTION
SECTIONS:
6.1
SOURCES OF LIPIDS AND THEIR TRANSPORT
6.2
OXIDATION OF FATTY ACIDS
6.3
KETOGENESIS
6.4
BIOSYNTHESIS OF FATTY ACIDS
6.5
METABOLISM OF TRIGLYCERIDES
6.6
METABOLISM OF COMPLEX LIPIDS
6.7
METABOLISM OF CHOLESTEROL
2
LIPID METABOLISM
INTRODUCTION
The metabolism of lipids provides a host of benefits to our body. Catabolism of
triacylglycerols and phospholipids provides energy fuel, synthesis of triacylglycerols creates
energy reserves while de novo synthesis, breakdown and re-modelling of lipids builds
membranes. Several lipids including the sterols, are the source of a host of important
hormones, intercellular messengers and carrier molecules. Lipid oxidation is even a
critical source of metabolic water in desert animals like the camel and gerbil, and in sea
animals like the killer whale.
GERBIL
CAMELS
KILLER WHALE
Fig. 6.A. Animals which are critically dependent on lipid oxidation for provision of water for their body
functions: Gerbils and camels do not have access to water for long periods of time while the killer whale does
not drink sea water. (Source: Grisham and Grisham 1999, p 790 fig 24.18)
3
Tissues depend on three main types of metabolic fuels:

Glucose – available from the diet, and from glycogenolysis and gluconeogenesis in
the liver

Free fatty acids – available from the diet, and from hydrolysis of triacylglycerols in
adipose tissue

Ketone bodies – derived from fatty acids by ketogenesis in the liver
In terms of calorie yield per unit, lipids are superior to carbohydrates as energy fuels. The
energy yield from lipids is estimated to be 37 kJ/gm while that from carbohydrates is 16
kJ/gm. In absence of dietary intake, glycogen reserves can supply energy for approximately
12 hours while lipid reserves can provide energy for 12 weeks! No wonder then that
migratory birds can bank on their lipid reserves to supply energy during long flights!
RUBY-THROATED HUMMINGBIRD
AMERICAN GOLDEN PLOVER
Fig. 6.B. Migratory birds which oxidize lipids to obtain energy for their long, non-stop flights: The
hummingbird
flies across the Gulf of Mexico while the golden plover flies from Alaska to Hawaii.
(Source: Grisham and Grisham 1999, p 790 fig 24.18)
The organs in the body co-operate with each other to utilize available energy sources in an
efficient and physiologically tenable manner. Extra-hepatic tissues like the muscle, show a
preference for metabolic fuels in the order ketone bodies > fatty acids > glucose, even though
glucose is the most readily available fuel from the blood. The use of lipids as energy fuels in
such tissues has a “sparing” effect on glucose, a compound which is indispensable to the
brain and RBC (refer Chapter 5 Section 6).
4
Fig. 6.C. Metabolic co-operation between organs in the supply and use of major metabolic fuels: Fatty acids are
obtained from the diet and from adipose tissue; glucose is obtained from the diet and from liver, while ketone
bodies are synthesized only in the liver. (FFA = free fatty acids; LPL = lipoprotein lipase; TG =
triacylglycerols; VLDL = very low density lipoproteins). (Source: Murray et al, 2003, p 233 fig 27-1)
5
Lipid molecules differ from carbohydrates and protein molecules in being essentially
hydrophobic in nature. Before delving in the details of lipid metabolism it is worthwhile to
appreciate the ingenuous manner in which the body deals with a major problem viz. the
supply of large hydrophobic lipid molecules continuously to tissues using a hydrophilic
medium (blood) as vehicle.
SECTION 1: SOURCES OF LIPIDS AND THEIR TRANSPORT
Lipids are obtained from the diet or synthesized actively in adipose tissue, liver and a few
other sites in the body. The blood distributes lipids to different organs where they are
metabolized. Plasma lipids are constituted by triglycerides (16%), phospholipids (30%),
cholesterol (14%), cholesteryl esters (36%) and free fatty acids (4%).
TABLE 6.1.1. SYNTHESIS AND TRANSPORT OF MAJOR LIPIDS
LIPID CLASS
Fatty acids
Phospholipids
Plasmalogens
Sphingolipids
Cholesterol
MAIN SITES OF
SYNTHESIS
 Cytosol: Upto
16-C chain
length
 MC and ER:
Elongation of
chain
 ER:
Desaturation
 ER: main site
 MC: to some
extent
 Peroxisomes
 ER and Golgi
complex
 Cytosol + ER
MODE OF
INTERCELLULAR
TRANSPORT
Bound to plasma
proteins like albumin
OR
as constituent of
other lipids in
lipoproteins
As lipoproteins
As lipoproteins
As lipoproteins
Lipids released from the adipose tissue, liver and other sites in the body are carried by the
blood as free fatty acids bound to albumin, or as soluble ketone bodies, or as amphipathic
lipoprotein complexes. Some of the smaller lipid molecules are transported by specific
carrier proteins e.g. cortisol is transported by corticosteroid-binding globulin (CBG).
Lymph also circulates lipids but in a more restricted manner.
6.1.1 PROCESSING OF DIETARY LIPIDS
Most dietary lipids are ingested as triacylglycerols (TGs) which are hydrophobic molecules.
Two strategies are employed to facilitate their digestion and absorption in the aqueous
intestinal milieu:
6
1. Emulsification of large lipid droplets by coating them with amphipathic bile salts
and phospholipids
2. Enclosure of the hydrophobic lipid digestion products within micelles
Small emulsion droplets (1mm) formed in the intestinal lumen are then hydrolyzed by
pancreatic lipase into monoacylglycerols and fatty acids. The process is aided by another
amphipathic protein secreted by the pancreas and called co-lipase.
i) Bile salt: glycocholate
ii) Orientation of polar and
non-polar groups
in a bile salt
(iii) An emulsion droplet
Fig. 6.1.1. The constitution of an emulsion droplet in the intestinal lumen: (i) and (ii) show the chemical
structure and amphipathic nature (polar groups are in red) of a bile salt; (iii) is an emulsion droplet formed with
central lipid (yellow), coated with the amphipathic bile salts (green and red) and phospholipids (pink and blue).
(Adapted from: Vander et al, 2001, p 563 figs 17-9 and 17-10)
2 FATTY ACIDS
2 H2 O
TRIACYLGLYCEROL
MONOACYLGLYCEROL
pancreatic lipase
Fig. 6.1.2. Hydrolysis of triacylglycerol by pancreatic lipase
Bile salts now help to form small (4-7nm), soluble aggregates called micelles which enable
the lipid digestion products to be “ferried” to the surface of the intestinal epithelium. Micelles
are clusters containing bile salts, fatty acids, monoglycerides, phospholipids, cholesterol and
fat-soluble vitamins. The central core contains the hydrophobic portions of the molecules and
the polar, hydrophobic regions project towards the aqueous intestinal medium.
7
Fig. 6.1.3. The use of bile salts (green and red) to form micelles from the products of lipid digestion.
(Adapted from Vander et al, 1999, p 564 fig 17-11)
The products of lipid digestion undergo re-synthesis and re-modelling before passage into
the blood stream (ct. carbohydrate and proteins products). Following release from the
micelles, the digested lipids diffuse into cells of the intestinal epithelium. Here
triacylglycerols are re-synthesized from fatty acids and monoglycerides. This time they are
packaged and released as chylomicrons into lymph. Lacteals carry lymph out of the intestine,
join the general lymphatic circulation and subsequently link up with the systemic blood
supply.
8
Fig. 6.1.4. Summary of the process of lipid digestion and absorption in the intestine (refer to text for details)
(Source: Vander et al, 1999, p 565 fig 17-12)
9
6.1.2. LIPOPROTEIN COMPLEXES AND THEIR CIRCULATION
In order to transport the hydrophobic lipids in the aqueous medium of the blood, the liver and
intestine sequester them in soluble lipoprotein complexes.
Fig 6.1.5. Structure of a low density lipoprotein (LDL) showing the hydrophobic core (yellow and red) and the
hydrophilic external coat (in blue). (Source: Nelson and Cox, 2005, p 821 fig 21-39a)
The design of the lipoprotein complexes follows a basic and logical pattern. Non-polar lipids
like triglycerides and cholesteryl esters are held in a central core, surrounded by a single
layer of amphipathic phospholipids and free cholesterol. The amphipathic molecules are so
oriented that their hydrophilic “heads” are directed outwards while their hydrophobic “tails’
project inwards. The surface layer of the lipoprotein complex is hydrophilic, and constituted
by special proteins called apoproteins/apolipoproteins.
Lipoprotein complexes are differentiated into four major classes on the basis of their
densities:

Chylomicrons – formed in cells of intestinal epithelium for transport of absorbed
lipids

VLDL (very low density lipoproteins; pre-β-lipoproteins) – synthesized in the liver
from excess endogenous lipids for export to different organs

LDL (low density lipoproteins; β lipoproteins) – formed on catabolism of VLDL

HDL (high density lipoproteins; α-lipoproteins) – originate in the liver and intestine
but attain “mature” form in the plasma
10
Fig.6.1.6. Electron micrographs of plasma lipoproteins complexes (Source: Nelson and Cox, 2005, p 821 fig 21-39b)
TABLE 6.1.2. PROPERTIES OF THE MAJOR CLASSES OF PLASMA LIPOPROTEINS
(Source: Lodish et al, 2003, p 752 Table 18-3)
Chylomicrons and VLDL contain high proportions of triglycerides while LDL and HDL have
high content of cholesterol and its ester. The difference in densities is due to the proportion of
lipid/non-lipid in the complexes (recall that lipids are less dense than water) and this allows
separation of the complexes by ultracentrifugation.
11
The lipoprotein complexes are assigned α / β symbols on the basis of their electrophoretic
properties.
Apoproteins are synthesized by the liver. Their proportion in lipoprotein complexes varies
from 1% (in chylomicron) to 70% (in HDL). There are several types of apoproteins viz ApoA, -B, -C, -D and -E, which, in turn, have many sub-types. They are differentiated on the
basis of their size, antigenic properties, and distribution. Apoproteins not only make lipid
transport possible in plasma but also function as activators of enzymes that work on
lipoproteins, and as signals/ligands that direct lipoproteins to specific tissues.
Lipoprotein complexes enable transport and exchange of lipids between different organs.
Chylomicrons and VLDL transport triacylglycerols (TGs) to sites like the adipose tissue,
heart, skeletal muscle and mammary glands. Free fatty acids are made available to the tissues
when the TGs are hydrolyzed by an enzyme, lipoprotein lipase, located in the capillary walls.
LDL delivers cholesterol and its ester to extra-hepatic tissues while HDL delivers them to the
liver.
Fig.6.1.7. Overview of the circulation of plasma lipoproteins HDL, LDL, VLDL and chylomicrons in the
body. (Source: Nelson and Cox, 2005, p 822 fig 21-40)
12
6.1.3. INTRACELLULAR SITES OF LIPID METABOLISM
The figure below demarcates the major sites of lipid metabolism within an animal cell like
the hepatocyte: The reactions of lipid synthesis and catabolism occur simultaneously within
the cell but are segregated in different areas so that futile cycles are prevented. Thus lipid
synthesis takes place in the cytosol while catabolism takes place in the mitochondrion, in
close proximity to the enzymes of the citric acid cycle and electron transport chains.
Sometimes the pathways of anabolism and catabolism may be very similar (e.g. for fatty acid
synthesis and breakdown) even though they proceed in opposite directions. Differences in
location, use of different co-enzymes (e.g. NADPH is the donor of high-energy electrons in
lipid synthesis while FAD and NAD+ are electron acceptors in lipid catabolism) and
attachment to different activating ligands (e.g. Coenzyme A in catabolism and acyl carrier
protein in anabolism) ensures that the anabolic and catabolic pathways proceeds to
completion.
Fig 6.1.8. Sites of lipid metabolism in an animal cell. ( Adapted from Nelson and Cox, 2005, p 795, fig 21-8)
In the ensuing pages we will primarily focus on the metabolism of the simple lipids. We will
take you through the pathways of their oxidation and synthesis in detail, but restrict ourselves
to only a glance at the metabolism of the complex lipids.
13
SECTION 2: OXIDATION OF FATTY ACIDS
The oxidation of long-chain fatty acids is a major energy-yielding process in many tissues.
The liver and muscle meet almost 50% of their energy needs from this source. Additionally,
products of fatty acid oxidation like. acetyl-CoA and propionyl-CoA, are precursors in lipid
biosynthesis. Interestingly, water released during fatty acid oxidation fulfills physiological
needs in desert animals.
6.2.1. PROVISION OF FATTY ACIDS TO CELLS
Cells oxidize non-esterified or “free fatty acids” (NEFA/FFA) which are made available to
them from four sources:
 Dietary lipids
 Stored lipids (mainly triacylglycerols) in adipose tissue
 Plasma lipoprotein complexes
 Intracellular structural lipids (mainly phospholipids)
Short chain fatty acids are generally water-soluble and carried free in the plasma in the
neutral or anionic state. Longer chain fatty acids, bound by serum albumin or by cytosolic
fatty acid-binding protein, are unesterified and considered as “free”. Such fatty acids can be
oxidized readily.
Fig 6.2.1.
Ribbon diagram of the structure of a cytosolic fatty acid-binding protein from an intestinal cell.
The fatty acid chain is depicted yellow. (Source: Voet and Voet, 1995, p 666 fig 23-4)
However, the majority of fatty acids in the plasma or in storage tissues are in the esterified
form; they are released by suitable lipases prior to undergoing oxidation.
14
TG
GPC
GLYCEROL
H2 O
lipases
OXIDATION
FFA
H2 O
phospholipases
PC
or RE-USE IN
SYNTHESIS
Fig.6.2.2. Release of free fatty acids from triacylglycerols (TG) and phospholipids like phospatidylcholine
(PC), by
enzymatic action. (GPC = glycerylphosphoylcholine)
6.2.2. STAGES IN CATABOLISM OF FATTY ACIDS
A fatty acid is completely oxidized within a tissue when the primary purpose is to obtain
energy. There are three stages in this catabolic process:
Stage 1: oxidation of a long-chain fatty acid to yield acetyl-CoA residues, NADH and
FADH2
Stage 2: oxidation of the acetyl groups in the TCA cycle to yield CO2, NADH, FADH2 and
GTP
Stage 3: transfer of electrons from Stages 1 and 2 to molecular O2 in the electron transport
chain for ATP synthesis
In this section we will take you through Stage 1; for details of Stages 2 and 3 we refer you to
chapters 5 and 9 respectively. Stage I has the primary objective of producing C-2 units, viz.
acetyl-CoA. Subsequently the acetyl-CoA is processed by the TCA cycle and the electron
transport chain to ensure complete oxidation to CO2 and H2O and concomitant production of
ATP. If required, acetyl-CoA can also be diverted to biosynthesis.
When fatty acid oxidation exceeds normal levels ketogenesis takes place, a process on which
we will elaborate in section 3 of this chapter.
15
Fig.6.2.3. Stages in oxidation of fatty acids: Stage 1 yields acetyl-CoA which is then processed through the
citric acid cycle (Stage 2) and the electron transport chain (Stage 3) for complete oxidation and yield of ATP.
(Source: Nelson and Cox, 2005, p 637 fig 17-7)
6.2.3. SITES OF OXIDATION ON THE ACYL CHAIN
Oxidation of fatty acids can occur at α, β or ω carbon atoms of their ‘R’ chains (refer
chapter 2 section 2). We will deal briefly with the two less common modes of fatty acid
oxidation viz. α- and ω-oxidations, before taking up the most prevalent mode of oxidation i.e.
β-oxidation, in more detail.
Oxidation of the α-C atom (i.e. C-2) occurs in the peroxisome if the fatty acid is already
branched at the β-carbon atom as in phytanic acid (notice the majenta methyl group at C-3 in
phytanic acid (Fig 6.2.4a.i). The sequence of reactions in its oxidation requires molecular
oxygen and removes the -CH2 group in the α–position (i.e. C-2). Pristanic acid so formed is
now branched at the α-C atom and its ‘R’ chain can undergo β-oxidation. A genetic failure to
carry out α-oxidation results in Refsum’s disease, which is associated with severe
neurological problems.
C H3
C H3
(
CH
C H3
C H2 C H2 C H2 ) CH
3
-
C H2 C O O
(i) Phytanic acid
C H3
CH3
(
CH
C H3
C H2 C H2 C H2 ) CH
3
-
COO
(ii) Pristanic acid
Fig.6.2.4a. Chemical structures of: (i) phytanic acid, a branched-chain fatty acid which has a methyl group on its
16
β-C atom (shaded majenta), and (ii) pristanic acid, with the methyl group now on its α- C atom.
C H3
CH3
(
CH
bond broken
C H2
C H3 O
C H2 C H2 ) CH
3
C
_
S Co A
(iii) Acyl-CoA form of pristanic acid
C H3 O
CH3
C H C S Co A
isobutyryl-CoA
O
O
_
+ 3
C H3
C
acetyl-CoA
_
S Co A
+ 3 C H3
CH 2
C
_
S Co A
propionyl-CoA
(iv) Final products of oxidation of phytanic acid
Fig.6.2.4b. Chemical structures of an intermediate, acyl-CoA (iii), and all final products (iv), in the α-oxidation
of phytanic acid in the peroxisome. Details of reactions have been excluded.
Oxidation of the ω-terminal C atom occurs in the endoplasmic reticulum (liver and kidney
cells); a dicarboxylic acid is produced which subsequently enters either β-oxidation or the
citric acid cycle routes of degradation.
dicarboxylic intermediate in ω-oxidation
of lauric acid
lauric acid
succinic acid
adipic acid
Fig.6.2.5. The products of ω-oxidation of a medium-chain fatty acid, lauric acid, in the endoplasmic reticulum: a
dicarboxylic intermediate is obtained which then undergoes β-oxidation to finally yield two dicarboxylic
acids, succinic and adipic acids. (Adapted from Nelson and Cox , 2005, p 649 fig 17-16)
6.2.4. β-OXIDATION OF FATTY ACIDS (Knoop’s β-oxidation of fatty acids)
The preferred method of fatty acid oxidation in animal cells is β-oxidation, which involves
the β-C atom (i.e. C-3). It occurs mainly in the mitochondrion and to some extent, in the
peroxisome. Its characteristic feature is the oxidation of the β-C atom to a keto form,
followed by cleavage of the acyl chain between α- (C-2) and β- (C-3) Carbon atoms. Units of
2-C are removed one at-a-time as acetyl-CoA from the carboxylic end of the molecule.
17
The process is essentially similar for all fatty acids but shows some differences in detail
between ‘R’ chains that are saturated/unsaturated, linear/branched and with an even/odd
number of C atoms.
Fig.6.2.6. Overview of β-oxidation of palmitic acid, a common long-chain fatty acid in animal cells.
(Source: Murray et al, 2003, p 181 fig 22-2)
A single pass of an acyl-CoA molecule (like palmitoyl/stearoyl-CoA) through a sequence of
four reactions in the pathway produces one molecule of acetyl-CoA and an acyl chain
shortened by a 2-C unit. Repeated passes of the progressively shortened acyl chain are
required for complete conversion to acetyl-CoA.
PREPARATORY REACTIONS
β-oxidation occurs in the mitochondrial matrix. In order to undergo this process, fatty acids in
the cytosol (that have entered from the blood or have been released from intracytosolic lipids)
need two kinds of help:

esterification with CoA to “activate” the relatively stable C-C bonds in the ‘R’ chain

a “shuttle” to take cytosolic acyl-CoA across the barrier of the mitochondrial
membrane
Activation of fatty acid to acyl-CoA:
Cytosolic fatty acids are acted on by acyl-CoA synthetase to produce the corresponding fatty
acyl-CoA. There are at least 3 isozymes of synthetase, specific for fatty acids with short,
intermediate and long chains. The isozymes are associated either with the endoplasmic
reticulum or with the outer mitochondrial membrane (OMM).
A high-energy thioester bond links the acyl group with CoA. Energy released from the
hydrolysis of two phosphoanhydride bonds of ATP is used. Formation of the acyl-ester
expends energy from one phosphoanhydride bond with an initial release of AMP and PPi.
The highly exergonic hydrolysis of the phosphoanhydride bond in PPi, drives the whole
reaction to completion. Note that this is the only step in β-oxidation where energy is
consumed.
18
Fig 6.2.7. The activation of fatty acid by conversion to acyl-CoA (Source: Nelson and Cox, 2005, p 635 fig 17-5)
Overall reaction catalyzed by acyl-CoA synthetase
FATTY ACID + CoA + ATP  ACYL-CoA + AMP + 2 Pi
ΔG’o = -34 kJ/mole
The acyl-CoA so produced can now be transported into the mitochondrion for β-oxidation or
it can remain in the cytosol for synthesis of membrane lipids.
Transport of acyl-CoA to mitochondrial matrix
Fatty acids with chain lengths of 12-C or less, enter the mitochondrion freely. However, most
dietary or endogenous fatty acids are longer and require the carnitine shuttle to cross the
mitochondrial membrane.
19
Fig 6.2.8. Representation of the carnitine shuttle: the four enzymes of the shuttle are located differently in the
mitochondrial membrane. The acyl group is transferred to carnitine in the intermembrane space and
carried across the inner membrane as acyl-carnitine (Source: Murray et al, 2003, p 180 fig 22-1)
C H3
CH3
N
+ CH
2
C H3
CH
CH2
-
COO
OH
Fig 6.2.9. Chemical structure of carnitine (β-hydroxy-γ-trimethylammonium butyrate)
The steps in the operation of the carnitine shuttle are:
1. Formation of acyl-carnitine: The acyl group is transferred from CoA to carnitine by
the enzyme, carnitine acyltransferase I. The enzyme is located in the OMM; hence the
reaction is essentially cytosolic and the CoA released remains in the cytosolic pool.
Esterification with carnitine commits the acyl group to oxidation.
2. Passage of acyl-carnitine into the intermembrane space through large pores in the
OMM
3. Facilitated diffusion of acyl-carnitine into mitochondrial matrix: enabled by
carnitine/acyl-carnitine translocase, a transporter in the inner mitochondrial
membrane (IMM)
20
4. Transfer of acyl group from acyl-carnitine to mitochondrial CoA: catalyzed by
carnitine acyltransferase II, located on the inner face of the IMM. Thus acyl-CoA is
regenerated but this time it is within the mitochondrial matrix.
5. Free carnitine released in Step 4 returns to the intermembrane space via the same
translocase. Thus, inward transport of acyl-carnitine is coupled to the outward
transport of carnitine.
The carnitine acyltransferases that are used frequently are specific for acyl groups with 14 to
18 carbon atoms and hence often called carnitine palmitoyltransferase (CPT-I and CPT-II).
Notice that two separate pools of CoA are connected by the carnitine shuttle. Cytosolic CoA
participates in the synthesis of fatty acids, while mitochondrial CoA is required for the
oxidative degradation of pyruvate, fatty acids and some amino acids.
Mitochondrial ‘fatty acid oxidase’ system
The reactions in the pathway of β-oxidation are catalyzed by enzymes collectively referred to
as “fatty acid oxidase” and located in the mitochondrial matrix or IMM, near the respiratory
chain. To oxidize fatty acyl chain lengths of 12 or more C atoms, 3 of the 4 enzymes needed
are associated together in a membrane-bound, multi-enzyme complex called trifunctional
protein (TFP) in the IMM. TFP has 2 enzyme activities (viz. enoyl-CoA hydratase and βhydroxyacyl-CoA dehydrogenase) in its α-subunit and another enzymatic function (viz.
thiolase) in its β-subunit. The tight association of these enzymes enables substrate-channeling
and lends efficiency as we saw earlier in the PDH complex (chapter 5 section 2). When chain
lengths are of 12 or less C atoms or longer chain-lengths have been reduced to this size,
further oxidations are carried out by four soluble enzymes in the matrix.
Fig 6.2.10. Schematic representation of the four membrane-bound enzymes for β-oxidation of long-chain fatty
acids. (Source: Nelson and Cox, 2005, p 648 fig 17-15)
We shall first take up in detail the steps in the β-oxidation of fatty acids that have linear,
saturated chains with an even number of C atoms, and deal later with those that show
variations. Saturated fatty acids with an even number of carbon atoms are common in our
body and we will use palmitic acid to understand the pathway.
21
SEQUENCE OF REACTIONS IN β-OXIDATION
β-oxidation of saturated fatty acids.
There are four reactions in the pathway of mitochondrial β-oxidation:
Reaction 1: Dehydrogenation of acyl-CoA to trans-Δ2 -enoyl-CoA
Dehydrogenation/oxidation of acyl-CoA creates a trans double bond between its α
and β carbon atoms (C-2 and C-3), and the products are trans-Δ2 -enoyl-CoA and
FADH2. The reaction is catalyzed by acyl-CoA dehydrogenase, a flavoprotein bound
to the IMM, and carrying FAD as co-factor. An electron transferring protein (ETF)
links FADH2 for re-oxidation in the ETS.
Specific isozymes of acyl-CoA dehydrogenase (AD) exist for fatty acids with very
long chains (VLCAD: C12 to C18), medium chains (MCAD: C4-C14) and short chains
(SCAD: C4-C8).
Reaction 1 is analogous to the SDH reaction of the TCA cycle.
Reaction 2: Hydration of trans-Δ2-enoyl-CoA to L-β-hydroxyacyl-CoA
The addition of –H and –OH across the α-β double bond produces L-β-hydroxyacylCoA (= 3-hydroxyacyl-CoA). The reaction is catalyzed by enoyl-CoA hydratase and
is similar to the fumarase reaction of the TCA cycle.
Reaction 3: Dehydrogenation of L-β-hydroxyacyl-CoA to β-ketoacyl-CoA
Removal of 2 H atoms by NAD+ from the β-C atom of L-β-hydroxyacyl-CoA creates
a keto group in this position; the products are β-ketoacyl-CoA and NADH.H+. The
enzyme catalyzing the reaction is β-hydroxyacyl-CoA dehydrogenase which functions
in a manner similar to the MDH reaction of the TCA cycle.
The linkage of the α-C atom with two carbonyl groups in β-ketoacyl-CoA makes the
bond between β-C and γ-C less stable and prone to nucleophilic attack in the next
reaction.
Reaction 4: Thiolysis of β-ketoacyl-CoA with release of acetyl-CoA
The enzyme thiolase (= acyl-CoA acetyl transferase) uses a molecule of free CoA to
cleave the Cα–Cβ bond and splits β-ketoacyl-CoA. into one molecule of acetyl-CoA
and an acyl-CoA which is shorter by 2 C atoms.
The shortened acyl-CoA now goes through the reactions 1-4 again, while acetyl-CoA
enters the TCA cycle.
Notice that the mechanisms of the first three reactions of β-oxidation are very similar
to reactions of the citric acid cycle from succinate to OAA.
22
Fig.6.2.11. The pathway of β-oxidation of fatty acids in the mitochondrion: A single pass of the acyl group
through the oxidative system involves four consecutive reactions viz. oxidation, hydration, a 2 nd oxidation and
thiolysis. Re-cycling of the ultimate product enables complete cleavage of the fatty acid to units of
acetyl-CoA. (Source: Hames and Hooper, 2000, p 318 fig 3)
23
Fig 6.2.12. Ribbon diagram of the active site of acyl-CoA dehydrogenase: The substrate is octanoyl-CoA (blue
and white) and the prosthetic group is FADH 2 (green) (Source: Voet and Voet, 1995, p 670 fig 23-11)
As mentioned above, repeated passes from reaction 1 to 4 are required to cleave off one C 2
unit at every passage till the original acyl-CoA is completely demolished into acetyl-CoA
units. Thus 8 acetyl-CoA are obtained from the C16 fatty acyl ester, palmitoyl-CoA, after 7
passes down the β-oxidation pathway.
7
6
5
4
3
2
1
O
C
_
S Co A
Fig.6.2.13. β-oxidation of palmitic acid: the red lines indicate bonds cleaved in seven successive cycles of βoxidation and the numerals indicate the order of cleavage. The green lines represent the final yield of eight C 2
units of acetyl-CoA.
The β-oxidation pathway is closely associated with enzymes of the citric acid cycle and is
coupled to oxidative phosphorylation in the ETS. One molecule each of NADH and FADH2
are generated at every pass through the pathway; they are re-oxidised in the ETS to yield
ATP.
Complete oxidation of the fatty acid is achieved when all the acetyl-CoA produced is
oxidized to CO2 and H2O by the TCA cycle and ETS. Notice how these three systems viz.
“fatty acid oxidase”, citric acid cycle and the electron transport system, are located close to
each other and work in tandem to ensure complete oxidation as well as coupling with ATP
production.
OVERALL REACTION for β-oxidation of Palmitoyl-CoA
PALMITOYL-CoA + 7 CoA + 7 FAD + 7 NAD+ + 7 H2O 
8 ACETYL-CoA + 7 FADH2 + 7 NADH + 7
H+
24
OVERALL REACTION for complete oxidation of Palmitoyl-CoA
PALMITOYL-CoA + 23 O2 + 108 Pi + 108 ADP  CoA + 108 ATP + 16 CO2 + 23 H2O
The mitochondrial β-oxidation sequence can be used only for acyl chain lengths upto C18. For
very long-chain fatty acids (C20 and above) a modified form of β-oxidation occurs in the
peroxisomes. The end products here are acetyl CoA and H2O2 and the system is not directly
coupled to oxidative phosphorylation. H2O2 is broken down by the enzyme catalase.
Energetics of β-oxidation
Let us now calculate the energy yield from β-oxidation, using palmitic acid (C16) as an
example:
Number of C2 units produced by β-oxidation
= 8 Acetyl-CoA
Total number of passes through pathway
= 07
Each pass produces:
1 FADH2 + 1 NADH.H+ equivalent to (1.5 + 2.5) ATP
=
Total yield for 7 passes = 7 x 4
=
4 ATP
28 ATP
Since oxidation of 1 Acetyl-CoA (mitochondrial) via TCA cycle and ETS produces 10 ATP
Therefore oxidation of 8 Acetyl-CoA produce 8 x 10
Therefore the total yield for oxidation of Palmitoyl CoA
ATP
=
80 ATP
=
80 + 28 = 108
Since the equivalent of 2~P had been used in activating palmitic acid to palmitoyl-CoA
Hence:
NET YIELD = 108 - 2 = 106 ATP/MOLECULE OF PALMITIC ACID
Since energy conserved by synthesis of 1 molecule of ATP
=
51.6 kJ/mol
Hence synthesis of 106 ATP conserves
=
5469.6 kJ/mol
This represents conservation of 68% of the free energy of combustion of palmitic acid
(Compare the yield of ATP per molecule of palmitic acid to that obtained per molecule of
glucose.)
β-oxidation of unsaturated fatty acids.
Most fatty acids in triglycerides and phospholipids are unsaturated, with 1-more double
bonds in cis configuration.
Double bonds at odd positions or with cis configuration pose two problems which are
overcome by deploying two additional enzymes in the β-oxidation pathway. We take the
example of a polyunsaturated fatty acid, linoleic acid (18:2), to study this situation.
25
The 1st problem arises after 3 rounds of β-oxidation when the system encounters a cis double
bond between C-3 and C-4. Since enoyl-CoA hydratase (Reaction 2) can add water only
across a trans double bond and the desired location is between C-2 and C-3, an additional
enzyme called Δ3, Δ2 -enoyl-CoA isomerase, catalyzes isomerization of cis Δ3-enoyl-CoA to
trans Δ2-enoyl CoA.
The 2nd problem is in the 5th round of the pathway when the presence of a pre-existing double
bond at an even-numbered C atom (Δ4) results in the formation of 2, 4-dienoyl-CoA. This
intermediate has two double bonds very close together and is an unsuitable substrate for the
next enzyme enoyl-CoA hydratase. An NADPH-dependent enzyme, dienoyl-CoA reductase,
now intervenes to reduce the Δ4 double bond. In mammals, the product of this reaction, trans3-enoyl-CoA is then isomerized to trans-2-enoyl-CoA by enoyl-CoA isomerase. β-oxidation
can now proceed normally.
β-oxidation of a fatty acid with three double bonds (e.g.linolenic acid, 18:3) also uses the
same two additional enzymes, isomerase and reductase, while a monounsaturated fatty acid
(e.g. oleic acid, 18:1) needs only the isomerase.
Oxidation of unsaturated fatty acids generates less ATP than a saturated fatty acid of same
chain-length. This is because the acyl-CoA dehydrogenase step is skipped and hence less
FADH2 is produced.
26
Fig.6.2.14. The pathway of β-oxidation of a polyunsaturated fatty acid, linoleic acid, which finally yields acetylCoA (refer to text below for details). (Source: Garret and Grisham, 1999, p 795 fig 24..24 )
27
β-oxidation of a fatty acid with an odd number of C atoms
The process is identical to that for fatty acids with even number of C atoms until the last
cleavage when a propionyl-CoA moiety is also formed in addition to acetyl-CoA.
O
O
C H3
( C H2 ) n C _ S Co A
(
n -1 ) CH
3
2
_
C
O
_
S Co A
acetyl - CoA
acyl - CoA
+
CH3
_
C H2
_
C
_
S Co A
propionyl - CoA
Three additional reactions viz carboxylation, epimerization and mutation, convert propionyl
CoA to succinyl-CoA, an intermediate of the TCA cycle. Alternately the succinyl-CoA can
also be converted to acetyl-CoA.
28
Fig. 6.2.15. Conversion of propionyl-CoA to succinyl-CoA using three additional enzymes viz. a carboxylase,
an epimerase and a mutase (Source: Garret and Grisham, 1999, p 791 fig 24.19)
Regulation of β-oxidation
β-oxidation of fatty acids is co-ordinated with fatty acid synthesis and glucose oxidation. The
carnitine-mediated passage of acyl groups into the mitochondrion is the rate-limiting step of
β-oxidation and hence the activity of carnitine acyltransferase-I (CPT-I) is a suitable point for
regulation. CPT-I is inhibited by malonyl-CoA, the first intermediate in the fatty acid
synthetic pathway, so that simultaneous degradation and synthesis of fatty acids is prevented.
The supply of NAD+ and CoA also regulate β-oxidation. High [NADH]/[NAD+] ratio inhibits
β-hydroxyacyl-CoA dehydrogenase, while high [acetyl-CoA] inhibits thiolase.
29
Fig.6.2.16. Regulation of fatty acid oxidation in the liver (Source: Murray et al, 2003, p 188 fig 22-10)
In the well-fed state (i.e. high carbohydrate intake), the hepatocyte receives large quantities
of glucose but low quantities of FFA. Activation of acetyl-CoA carboxylase, diverts excess
glucose into the synthesis of fatty acids. The inhibitory action of malonyl-CoA on CPT-I
ensures that the oxidation of fatty acids is concommittantly suspended. Thus fatty acids are
not oxidized when adequate glucose is available as energy fuel.
Conversely, the [fatty acids] is high in starvation/high-fat diet/on increased lipolysis (as in
diabetes mellitus). Increased entry of FFA into the cell increases intracellular [acyl-CoA]
which directly inhibits acetyl-CoA carboxylase. Since [malonyl-CoA] is now low, CPT-I is
not inhibited and acyl-CoA enters the mitochondrion for β-oxidation.
In the same way, the rate of fatty oxidation increases in the period between meals when the
levels of blood glucose are low. Oxidation of FFA released from lipid stores in the body,
provides sustained energy in hibernators, polar animals and in migratory birds.
30
SECTION 3: KETOGENESIS
Excess oxidation of fatty acids results in ketogenesis, a metabolic condition when the
products of β-oxidation are processed differently to produce acetoacetate, D-βhydroxybutyrate and acetone. These compounds are collectively referred to as ketone bodies.
They are produced mainly in the liver and replace fatty acids and glucose as energy fuel in
many extra-hepatic tissues.
O
C H3
CO2
C
_
C H3
Acetone
O
C H3
C
_
CH 2
Acetoacetate
spontaneous
decarboxylation
-
COO
reduction by
N A D H. H
dehydrogenase
+
NAD
OH
+
C H3
CH
CH 2
-
COO
D- 3 - Hydroxybutyrate
Fig.6.3.1. Chemical structures and inter-relationships of the three ketone bodies viz. acetoacetate, acetone and
D-3- hydroxybutyrate (i.e. D-β-hydroxybutyrate).
Free fatty acids in the plasma exceed their normal concentration (4%) under certain metabolic
or nutritional conditions. Consumption of a high-fat diet raises [FFA] absorbed from the GIT,
starvation or disorders like diabetes mellitus deprive cells of their glucose supply and our
body tries to compensate for this deficiency of energy fuel by releasing more fatty acids from
its triglyceride stores.
However, it is not desirable to have high concentrations of FFA in the blood because:

they have limited solubility in an aqueous medium

they are toxic at high concentrations

they saturate the carrying capacity of plasma even at low concentrations
When plasma [FFA] exceeds normal levels the liver once again comes to the rescue. It
converts the products of β-oxidation of FFA to ketone bodies and releases them into the
plasma as alternate substrates to FFA and glucose for energy. Ketone bodies are preferable to
FFA since:

they are highly soluble in plasma
31

they have low toxicity even at high concentrations

they diffuse rapidly through membranes

they are rapidly metabolized to yield CO2, H2O and energy in extra-hepatic tissues
Fig.6.3.2. Summary of the production, distribution and utilization of ketone bodies: ketogenesis is in the liver,
oxidation for energy yield is in the extra-hepatic tissues and excretion is by the kidneys and lungs.
(Source: Murray et al, 2003, p 184 fig 22-6)
Blood is the medium for a net transfer of ketone bodies from their almost exclusive site of
production in the liver to their sites of utilization in extra-hepatic tissues like skeletal muscle
and heart. The brain, which normally uses glucose as a respiratory substrate, can adapt to the
use of ketone bodies under conditions of acute hypoglycemia. The liver itself cannot oxidize
ketone bodies for energy. The kidneys and lungs excrete ketone bodies.
6.3.1. SYNTHESIS OF KETONE BODIES
Ketogenesis occurs in the mitochondrial matrix of hepatic cells. The steps in the pathway are:
Step 1: Formation of acetoacetyl-CoA
Two molecules of acetyl-CoA condense to form acetoacetyl-CoA by reversing the thiolase
reaction of β-oxidation. Acetoacetyl-CoA is also the terminal 4-C ketoacyl-CoA formed as
an intermediate in the β-oxidation pathway.
Step 2: Condensation of acetoacetyl-CoA with acetyl-CoA
This crucial reaction is catalyzed by HMG-CoA synthase and the products are β-hydroxy-βmethyl glutaryl-CoA (HMG-CoA) and one molecule of free CoA.
32
Fig.6.3.3. The pathway of ketogenesis in the mitochondria of hepatic cells. Refer to the text for details.
(Source: Nelson and Cox, 2005, p 651 fig 17-18)
Step 3: Production of acetoacetate and release of acetyl-CoA
HMG-CoA is split in a reaction catalyzed by another crucial enzyme of ketogenesis, HMGCoA lyase. The products are the 1st ketone body, acetoacetate, and a molecule of acetyl-CoA
is released.
Step 4: Acetoacetate is the source of the other two ketone bodies. It undergoes reversible
reduction in the liver to yield D-β-hydroxybutyrate using an NADH-dependent enzyme, D-βhydroxybutyrate dehydrogenase. Acetoacetate and D-β-hydroxybutyrate are the main ketone
bodies released by the liver into the blood. The predominant ketone body in the blood is D-βhydroxybutyrate.
33
Spontaneous or enzymatic decarboxylation of acetoacetate (by acetoacetate decarboxylase)
produces acetone. The formation of acetone is nominal in healthy people but high in those
with untreated diabetes mellitus.s
The liver is unique in its ability to produce ketone bodies because hepatocytes are the only
major cell types in the body to possess both synthase and lyase in their mitochondria. The
rumen epithelium can carry out ketogenesis to some extent.
6.3.2. UTILIZATION OF KETONE BODIES
Following their release into the blood, acetoacetate and D-β-hydroxybutyrate are taken up by
extra-hepatic tissues like the muscle, while acetone is excreted in urine or exhaled. D-βhydroxybutyrate is re-oxidized to acetoacetate by β-hydroxybutyrate dehydrogenase.
Acetoacetate is then activated to acetoacetyl-CoA by a rather unusual enzyme CoA
transferase (= thiophorase), which uses succinyl-CoA to donate CoA.
Subsequently, acetoacetyl-CoA is split by thiolase into 2 molecules of acetyl-CoA that are
oxidized to yield CO2, H2O and ATP via the TCA cycle and ETS.
Ketone bodies are oxidized for energy yield in preference to FFA and glucose. Even though
the liver is the bulk producer of ketone bodies, it cannot use them as energy fuel since
hepatocytes lack CoA transferase.
Several amino acids like Phe, Leu and Tyr, are “ketogenic” i.e. they can also be converted to
acetoacetate at times of metabolic need. Lactating cattle use ketone bodies as a source of
energy..
Fig.6.3.4. Schematic representation of the production and release of ketone bodies by the liver and their
oxidation in the muscle. (Source: Murray et al, 2003, p 186 fig 22-8)
34
6.3.3. REGULATION OF KETOGENESIS
Regulation of ketogenesis is excercised at the sites 1, 2 and 3 as shown in figure 6.3.5.
Site 1 – The mobilization of FFA from adipose tissue determines the [FFA] in the blood.
The extent of mobilization of FFA from the triglyceride stores depends on the activity of the
enzyme Hormone-Sensitive-Lipase (HSL). In turn, HSL is regulated by hormones and
metabolic factors (refer section 5).
Site 2 - Control of esterification vs degradation of FFA in the liver determines the
availability of substrate for ketogenesis. The liver extracts almost 30% of FFA passing
through its tissue. Hence when [FFA] in the blood is high, substantial quantities of FFA enter
the cytosol of the hepatic cells. The crucial point of regulation is the activity of carnitine
palmitoyl transferase I (CPT-I) as explained earlier (Fig 6.2.16). When high levels of glucose
are available as respiratory substrate, the pathway of triglyceride synthesis from FFA is
preferred over β-oxidation and ketogenesis.
Fig.6.3.5. The three sites of regulation of ketogenesis ( Source: Murray et al, 2003, p187 fig 22-9)
Site 3 - Control on the partitioning of mitochondrial acetyl-CoA between ketogenesis and
the TCA cycle in the hepatocytes determines the quantity of ketone bodies produced. When
[acetyl-CoA] is high, intrinsic factors in the mitochondrion determine whether it will undergo
ketogenesis or be oxidized by the TCA cycle. Production of OAA (from malate) and βhydroxybutyrate (from acetoacetate) both depend on the redox state of the tissue in terms of
the ratio [NADH]/[NAD+]. Oxidation of acetyl-CoA in the TCA cycle depends on the supply
of OAA and NAD+, while its metabolism by ketogenesis depends on NADH. This difference
35
in requirement of the two pathways is important when [acetyl-CoA] is high, for the rate of
regeneration of NAD+ from NADH depends on the rate of oxidative phosphorylation. The
oxidation of acetyl-CoA molecules produced from palmitic acid yields 129 ATP via TCA
cycle but only 54 ATP via ketogenesis. Hence when [FFA] is high, more acetyl-CoA is
metabolized by ketogenesis than by TCA cycle, thereby preventing over-loading of the
tightly coupled electron transport system.
6.3.4. PHYSIOLOGICAL IMPLICATIONS OF KETOGENESIS
The normal level of ketone bodies in the blood is approximately 0.2 mmol/L. An increase
beyond this level is associated with their increased oxidation in extra-hepatic tissues.
However, since the tissues cannot handle levels above 12mmol/L, further increase results in a
metabolic disorder called ketosis. Starvation and untreated diabetes mellitus are common
causes of pathologic ketosis.
Ketonemia and ketonuria refer to excess levels of ketone bodies in the blood and urine
respectively. Since ketone bodies are acidic, high levels deplete alkali reserves in the body
and cause metabolic acidosis / ketoacidosis. The degree of ketosis is assessed by the
measurement of [ketone bodies] in the blood. A fruity smell of acetone in the breath is an
indicator of untreated diabetes mellitus. Weight-watchers on low calorie diets should be wary
of ketosis.
36
SECTION 4: BIOSYNTHESIS OF FATTY ACIDS
Fatty acids are essential components of major cellular lipids like the triacylglycerols and
phospholipids. Even though dietary sources do supply fatty acids, synthesis of fatty acids is
required in the cells of many tissues to meet their requirement for structural components (like
membrane constituents), or to convert excess glucose into reserve lipid. Active fatty acid
synthesis takes place in cells of the liver, kidney, adipose tissue, brain, lungs and mammary
glands.
Fatty acid synthesis occurs primarily in the cytosol and is an endergonic, reductive process.
The nature of the reactions, the enzymes used and the cytosolic location….all ensure a
complete segregation of their synthetic pathway from their degradation.
We take up first the de novo synthesis of fatty acids which can produce acyl chain-lengths of
upto C16 and for which the sequence of reactions are exclusively in the cytosol. Later, we take
a look at the processes of “chain-elongation” and desaturation which occur in the
microsomes.
We shall focus mainly on the synthesis of fatty acids which are saturated and which have an
even number of C atoms.
6.4.1. DE NOVO SYNTHESIS OF FATTY ACIDS
Fatty acid synthesis basically requires glucose. However, the immediate precursors are:

Acetyl-CoA – the fundamental C2 unit required to constitute fatty acids

NADPH – which serves as a reductant
In addition, the pathway needs ATP as a source of energy and HCO3- as a donor of COO-.
Sources of acetyl-CoA
Fatty acid synthesis takes place in the cytosol which is not a significant site for production of
acetyl-CoA. The system, however, is smart enough to outsource acetyl-CoA production to the
mitochondrion and arrange for its transport to the cytosol.
The major contributors of mitochondrial acetyl-CoA are pyruvate (oxidation) and several
glucogenic amino acids like Ala, Phe, Leu, and Tyr. Contribution of acetyl-CoA from βoxidation is not significant since the pathways of fatty acid oxidation and synthesis are
reciprocally regulated.
As we have seen earlier, the mitochondrial membrane debars free passage of acetyl-CoA and
hence an indirect shuttle is used to transfer mitochondrial acetyl-CoA to the cytosol when its
mitochondrial concentration is high.
Acetyl-CoA is camouflaged by condensation with OAA to form citrate (citrate synthase
reaction), which then crosses the inner mitochondrial membrane on a citrate/ tricarboxylate
transporter. Once in the cytosol, citrate is cleaved by an ATP-dependent enzyme citrate
lyase to release acetyl-CoA and OAA. This acetyl-CoA is used in fatty acid synthesis.
37
Fig.6.4.1. The transfer of acetyl-CoA from the mitochondrion to the cytosol by use of a tricarboxylate
transporter
(blue oval) (Source: Berg et al, 2002, p 928 fig 22.25)
OVERALL REACTION catalyzed by citrate lyase:
A DP
ATP
CITRATE
+
Co A
+
Pi
ACETYL
citrate lyase
- Co A + O A A
Following its release, OAA is converted to malate by cytosolic MDH:
+
N AD H. H
OXALOACETATE
NAD
ma la te
d e h y d r ogena s e
+
MALATE
Cytosolic malate can be processed in two ways. The action of “malic enzyme” (NADPmalate dehydrogenase) converts malate to pyruvate and generates NADPH for fatty acid
synthesis.
NA DP+
MALATE
+
N AD P H. H
malic enzyme
PYR UVATE
+
C O2
Alternately, malate can be transported back into the mitochondrion by the malate-αketoglutarate shuttle and then reduced to OAA. Condensation of OAA with acetyl-CoA
now produces more citrate for outward transport.
38
Fig.6.4.2. Composite diagram of the routes by which acetyl-CoA and NADPH are provided for fatty acid
synthesis
(Source: Murray et al, 2003, p 176 fig 21-4)
Sources of NADPH
The ratio of [NADPH]/[NAD+] is very high in the cytosol of hepatocytes. This is ideally
suited for fatty acid synthesis. As shown in Fig 6.4.2 above, NADPH generators in the
cytosol are the HMP pathway and the action of “malic enzyme”. Cytosolic IDH can also
produce NADPH by oxidation of cytosolic isocitrate.
PATHWAY OF FATTY ACID SYNTHESIS
Fatty acid synthesis is a cyclic pathway and involves repeated addition of C2 units till the
desired chain-length is obtained. However, the actual step-wise input is not of a C2 unit but of
a C3 unit, malonyl-CoA, followed by its decarboxylation to a C2 unit and reduction.
Preparatory reaction
The preparatory reaction is the attachment of a carboxylic group to acetyl-CoA (C2 unit)
converting it to malonyl-CoA (C3 unit). It is catalyzed by acetyl-CoA carboxylase, an
enzyme that resembles other biotin-dependent carboxylases (like pyruvate carboxylase).
39
O
CH 3
_
C
_
S
_
- OO C
Co A
ENZYME
ENZYME
C
_
S
_
Co A
- biotin
AT P
+
CH 2
_
Malonyl - CoA
- biotin - C OO -
A DP
O
_
+
H C O 3-
Pi
Fig.6.4.3. Summary of the conversion of acetyl-CoA to malonyl-CoA
Acetyl-CoA carboxylase is a multi-enzyme protein, constituted by a variable number of
identical subunits. Each subunit carries biotin, biotin carboxylase, biotin carboxyl carrier
protein and transcarboxylase, and also has a binding site for an allosteric modulator.
Fig.6.4.4. Schematic diagram of acetyl-CoA carboxylase with carboxylic group attached to a flexible biotin arm.
The white unlabelled portion is the transcarboxylase domain. (Source: Nelson and Cox, 2003, p 788 fig 21-1)
Energy from the hydrolysis of ATP is used to first attach the carboxylic group from HCO 3- to
the long, flexible biotin arm of the carrier protein. The arm now moves the COO- group from
biotin carboxylase to transcarboxylase which then transfers it to acetyl-CoA. The reaction is
irreversible and a potential site for regulation of fatty acid synthesis.
Malonyl-CoA is the direct donor of the C2 unit which is fed repeatedly into the pathway of
fatty acid synthesis.
Nature of Fatty Acid Synthase
A unique feature of the pathway is the use of a multifunctional “fatty acid synthase complex”.
This is an extraordinary polypeptide with seven enzymatic activities. It is a dimer of two
identical polypeptide chains/sub-units oriented in opposite directions to each other (Fig
6.4.5). Each sub-unit has a 3-domain organization (Fig 6.4.6), constituted by a total of seven
enzymes and an acyl carrier protein, ACP. The growing fatty acid chain is attached to ACP
40
which is used as a “swinging arm” to move the chain “back and forth” between the different
enzyme activities. The complex synthesizes two identical acyl-CoA chains simultaneously
and releases them as free fatty acids.
“Fatty acid synthase” can synthesize only saturated fatty acyl chains of upto 16-C chain
length.
Fig.6.4.5. Schematic representation of the dimeric multi-enzyme complex, fatty acid synthase: The two subunits
(shown in different colors) are aligned in opposite directions. Each has seven enzyme activities and an acyl
carrier protein, ACP. (Source: Murray et al, 2003, p 174 fig 21-2)
41
Fig.6.4.6. Schematic representation of the 3-domain organization of each sub-unit of fatty acid synthase: domain
1(blue) has acetyl transferase (AT), malony transferase (MT) and condensing enzyme (CE i.e ketoacyl
synthase); domain 2 (yellow) has dehydratase (DH /hydratase), enoyl reductase (ER), β-ketoacyl reductase
(KR) and ACP; domain 3 (red) contains thioesterase. The flexible phospho-pantetheinyl arm (green) of ACP
can carry an acyl group. (Source: Berg et al, 2002, p 927 fig 22.3)
Even though each monomer has all the activities needed to synthesize a fatty acid chain, it is
probable that the actual functional unit is constituted by “one half of one monomer interacting
with the complementary half” of the second monomer (refer fig 6.4.5). We thus have a
unique enzyme complex in which the constitution of structural and functional units is
different.
Notice that in each polypeptide chain ACP at one terminal end and the enzyme ketoacyl
synthase at the other terminal, separately carry a projecting thiol (-SH) group. We will refer
to them as the “Pan-arm” of ACP and the “Cys-arm” of ketoacyl synthase. Both form
thioesters with acyl groups. All the reactions of fatty acid synthesis are carried out with the
substrates attached to the enzyme-complex and these two arms are carriers of acyl
intermediates. As we have seen earlier, the hydrolysis of thioesters is highly exergonic and
can drive reactions to completion. This is useful in the relatively long, cyclic course of the
fatty acid synthetic pathway.
42
Fig.6.4.7. The phosphopantheine group on ACP and CoA which enables both to serve as acyl group carriers.
(Source: Berg et al, 2002, p 925 fig 22.21)
SEQUENCE OF REACTIONS IN FATTY ACID SYNTHESIS
Reaction 1: Priming reaction (reactions 1a and 1b in fig 6.4.8):
The fatty acid synthase complex is initially “primed” by the attachment of:

an acetyl group to the Cys-arm of β-ketoacyl synthase, catalyzed by acetyltransacylase

a malonyl group to the Pan-arm of ACP, catalyzed by malonyl-transacylase
The enzyme-substrate complex is acetyl-malonyl enzyme in which the two activated acyl
moieties are held in close proximity to each other.
Reaction 2: Condensation
A condensation reaction between the acetyl group and the methylene group of malonyl
residue, removes one C atom from the latter as CO2. This frees the –SH group on the Cysarm of the enzyme-substrate complex. A 4-C acyl intermediate, acetoacetyl-S-enzyme, is
obtained. The CO2 released is taken up by acetyl-CoA carboxylase to synthesize a fresh
molecule of malonyl-CoA.
The condensation reaction is catalyzed by 3-ketoacyl synthase. Coupling of condensation to
decarboxylation makes the overall process highly exergonic and thermodynamically
favorable.
Reaction 3: Reduction 1
The keto group on C-3 of the acetoacetyl moiety is reduced, using NADPH and the enzyme
3-ketoacyl reductase. The product is D-3-hydroxybutyryl-S-enzyme.
43
Fig.6.4.8. The sequence of reactions in fatty acid (palmitic acid) synthesis: “fatty acid synthase” has been shown
schematically with its acyl-carrying arms –Cys-SH and –Pan-SH attached to sub-units 1 and 2 respectively
(see Fig 6.4.5). (Adapted from Murray et al, 2003, p 198 fig 23-3)
Reaction 4: Dehydration
A double bond is created between C-2 and C-3 of the acyl residue by hydratase (=
dehydrase), which removes –H and –OH as water. The product is trans-Δ2 butenoyl-Senzyme (= 2,3 unsaturated acyl enzyme in Fig 6.4.6).
44
Reaction 5: Reduction 2
Enoyl reductase uses NADPH again to reduce the double bond in the acyl group in reaction
4. The product is a saturated 4-C acyl ester, butyryl-S-enzyme i.e. a C2 acyl chain (acetyl
CoA) has been lengthened by addition of another 2-C unit.
Translocation and re-cycling
The freshly synthesized C4 acyl unit, so far attached to the Pan-arm, is displaced to the Cysarm by a new molecule of malonyl CoA. The acyl (butyryl)-malonyl enzyme obtained is now
re-cycled through the series of reactions from 2 to 5 to extend the chain by another C2 unit.
This process of translocation and re-cycling continues repeatedly until a saturated C16 acyl
chain viz. palmityl-S-enzyme is produced.
Deacylation
Free palmitic acid is finally liberated from the enzyme-complex by the action of thioesterase.
As mentioned earlier, the complex releases two molecules of palmitic acid simultaneously.
In the mammary glands, a different thioesterase specifically releases acyl residues of C8, C10
and C12 for constitution of the milk lipids.
OVERALL REACTION:
8 ACETYL-CoA + 14 NADPH + 14 H+ + 7ATP 
PAMITATE + 14 NADP+ + 8 CoA + 6
H2O + 7 ADP + 7 Pi
Synthesis of saturated fatty acids with an odd number of C atoms takes place in the same way
as described above, except that propionyl-CoA is used instead of acetyl-CoA in the
“priming” reaction.
6.4.2. ELONGATION OF THE SATURATED FATTY ACID CHAIN
Palmitic acid is the principal product of the fatty acid synthase system in animal cells. Longer
chain-lengths (C18 or more) of both saturated and unsaturated fatty acids are synthesized from
palmitic acid by a fatty acid elongase system. This system is particularly important in the
brain where C22 and C24 fatty acids are needed for the synthesis of sphingolipids.
Fatty acid elongase systems are located in the endoplasmic reticulum and mitochondrion
and do not involve ACP-bound intermediates. The ER (“microsomal”) system adds only a C2
unit to give stearoyl-CoA. The system is very active and similar to the fatty acid synthase
system; the acyl moiety is bound to CoA, malonyl-CoA is the acetyl group donor and
NADPH is the reductant. The mitochondrial system elongates the acyl chain by a pathway
which is the reverse of fatty acid oxidation; units of acetyl-CoA (and not malonyl-CoA) are
used successively.
45
ACYL - C o A + MALONYL- C o A
ACYL - C o A + ACETYL - C o A
( Cn)
( Cn)
CoA + CO2
synthase
CoA
thiolase
3 - KETOACYL - C o A
3 - KETOACYL - C o A
N A D P H. H +
N A D H. H+
reductase
dehydrogenase
N A D P+
NAD
3 - HYDROXYACYL - C o A
dehydrase
+
3 - HYDROXYACYL - C o A
hydratase
H2O
2 - trans - ENOYL- C o A
H2O
2 - trans - ENOYL- C o A
N A D P H. H +
N A D P H. H +
reductase
reductase
N A D P+
N A D P+
ACYL - C o A
ACYL - C o A
( C n +2 )
( C n +2 )
A.
B.
Fig.6.4.9. Comparison of the fatty acid elongase systems in the endoplasmic reticulum (A) and mitochondrion
(B). Significantly dissimilar components in the pathways have been colored and generic names of enzymes
have been used.
6.4.3. SYNTHESIS OF UNSATURATED FATTY ACIDS
Introduction of double bonds in the fatty acyl chain requires reactions mainly on the
endoplasmic reticulum of many tissues including the liver. Monounsaturated fatty acids like
palmitoleate (16:1) and oleate (18:1) can be synthesized from the corresponding saturated
acyl chains (palmitate and stearate respectively). Insertion of the double bond is catalyzed by
a mixed-function oxygenase system, specifically referred to as fatty acyl-CoA desaturase and
located on the luminal face of the smooth ER. The 1st double bond is inserted at Δ9 by Δ9desaturase. The reaction involves Cyt b5 and a flavoprotein, Cyt b5 reductase, and requires
molecular O2 and NADH/NADPH.
Fig.6.4.10. Fatty acyl-CoA desaturase system for converting saturated acyl-CoA (stearoyl-CoA) to
monounsaturated acyl-CoA (oleoyl-CoA). (Source: Berg et al, 2002, p 933 fig 22.29)
46
Synthesis of polyunsaturated fatty acids requires both desaturase and elongase systems:
Δ6desaturase
elongase
Δ5 desaturase
elongase
Δ4desaturase
18:1------18:2--------20:2------------ 20:3--------- 22:3 ---------22:4
(oleic acid)
Mammalian desaturases can readily synthesize the ω9 family of fatty acids but they cannot
introduce a double bond beyond Δ9 of the fatty acyl chain. Hence linoleic and α-linolenic
acids which have unsaturation beyond C-9 cannot be synthesized in our body. We need to
ingest them from plant sources, following which the elongase and desaturase systems can
synthesize the ω6 and ω3 family of fatty acids.
The C20 polyunsaturated fatty acid, arachidonic acid, is synthesized mainly from linoleic acid.
It is the essential precursor of the eicosanoids viz. prostaglandins, thromboxanes and
leukotrienes.
Fig.6.4.11. Outline of the synthesis of longer chains in unsaturated fatty acids. Linoleate and linolenate can be
synthesized in plants but not in animals; hence they are essential fatty acids for animals.
(Source: Nelson and Cox, 2005, p 797 fig 21-12)
6.4.4. SYNTHESIS OF PROSTAGLANDINS AND OTHER EICOSANOIDS
Prostaglandins (PGs) are local, short-lived hormones derived from C20 fatty acids like
arachidonic acid. Arachidonate is obtained from the diet, synthesized from essential
47
unsaturated fatty acids like linoleate (Fig 6.4.11), or released by hydrolysis of membrane
phospholipids in response to hormonal and other signals. Prostaglandin synthesis from
arachidonate occurs on the smooth endoplasmic reticulum.
Prostaglandins and thromboxanes are synthesized by the “cyclic” pathway. A bifunctional
enzyme, prostaglandin H2 synthase (PGH2 synthase) or COX, enables the formation of
prostaglandin H2 (PGH2) from arachidonate. The reaction occurs in two steps aided by the
two catalytic activities of PGH2 synthase:
i.
Cyclooxygenease activity – which adds molecular O2 to arachidonate and
produces the cyclopentane-ring needed for prostaglandins. The product is PGG2
ii.
Peroxidase activity – which converts PGG2 to PGH2
Prostaglandin H2 (PGH2) is the immediate precursor of the other prostaglandins
and thromboxanes.
Fig.6.4.12. Outline of the synthesis of eicosenoids viz. prostaglandins, prostacyclins, thromboxanes and
leukotrienes from arachidonate (PLA2 = Phospholipase A2) (Source: Berg et al, 2002, p 933 fig 22.30)
Leukotrienes are formed by a “linear” pathway from arachidonate in which several
lipooxygenases catalyze the insertion of molecular O2 into the linear chain of arachidonate.
These enzymes are mixed-function oxidases which use cytochrome P-450.
PGH2-synthase is also referred to as COX owing to its cyclooxygenase activity. Mammals
have two isozymes of PGH2-synthase viz. COX-1 and COX-2. COX-1is expressed in most
tissues and supports levels of PG synthesis necessary for homeostasis. COX-2 is expressed in
certain tissues only in response to inflammatory stimuli and is the cause of inflammation,
pain and fever. .
48
Fig.6.4.13. Site of action of non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 inhibitors on the
cyclooxygenase isozymes COX-1 and COX-2 in prostaglandin synthesis.
(Source: Elliot and Elliot, 2005, p 243 fig 14-9)
Regulation of fatty acid synthesis
The enzymes of fatty acid synthesis are subject to two kinds of regulation:

short-term-regulation – which controls their activity

long-term regulation – which controls the genes that regulate their synthesis
The main target of short-term regulation is acetyl-CoA carboxylase (ACC), the first enzyme
in fatty acid synthesis.
The enzyme is activated allosterically by citrate, and is subject to product feedback
inhibition by long-chain acyl molecules (e.g. palmitoyl-CoA). Citrate levels are high when
[acetyl-CoA] is high and such a situation is conducive to fatty acid synthesis. Citrate has a
major role in shifting the metabolism of fuel compounds from oxidation to the synthesis of
fatty acids and their storage as triglycerides.
Fig 6.2.12. Allosteric regulation of acetyl-CoA carboxylase (Source: Nelson and Cox, 2005 p 797 fig 21-11)
Long-chain acyl-CoA inhibits fatty acid synthesis in many ways. It has a direct negative
feedback effect on ACC. Its inhibitory effect may be enhanced indirectly by suppression of
the tricarboxylate transporter so that citrate efflux from the mitochondrion is prevented.
High [Acyl-CoA] inhibits the ATP-ADP exchange transporter in the IMM and also stimulates
β-oxidation. This raises the mitochondrial ratio of [ATP]/[ADP] and [NADH]/[NAD+].
Consequently the PDH complex is inhibited and the production of acetyl-CoA falls.
Covalent modification of ACC is by reversible phosphorylation/dephosphorylation. The
phosphorylated form of the enzyme exists as protomers and is inactive. The
dephosphorylated form is polymerized and active.
49
Phosphorylation of ACC is caused by an AMP-activated protein kinase (AMPK) which is in
turn is activated by another kinase, AMPKK. The activity of AMPKK is increased by cAMP
via PKA, and also directly by acyl-CoA. The net result of the activation of AMPKK is
phosphorylation and hence inactivation of ACC and consequent fall in fatty acid synthesis.
On the other hand a phosphatase which dephosphorylates ACC increases fatty acid synthesis.
Fig 6.2.13. Covalent regulation of the activity of acetyl-CoA carboxylase (Source: Murray et al, 2003 p178 fig
21-6)
The hormones glucagon and insulin affect the activity of ACC. The inhibitory effect of
glucagon is via cAMP-dependent PKA. Thus when blood glucose is low, metabolism of the
fatty acids can switch from synthesis to oxidation. Insulin probably acts through an “activator
protein” and an insulin-dependent protein kinase to stimulate ACC and increase fatty acid
synthesis.
Long-term regulation of fatty acid synthesis is by alteration of the activity of the genes that
control synthesis of ACC and fatty acid synthase. The total amount of these enzymes is
determined by sustained nutritional states. Thus, prolonged fasting, high-fat diet and diabetes
result in decrease of the enzymes, while a well-fed state boosts their quantity. Insulin induces
enzyme synthesis but glucagon suppresses this action.
Non-steroidal anti-inflammatory drugs (NSAIDs) like aspirin and ibuprofen block the active
site of PGH2-synthase and prevent synthesis of prostaglandins and thromboxanes. They are
used widely for relief from inflammation, pain and fever. Low doses of aspirin are prescribed
for high-risk patients to decrease the production of thromboxanes and hence reduce the
chances of heart attacks and strokes. However these drugs have several undesirable sideeffects. Cox-2 inhibitors like celecoxib are more specific in action and have lesser sideeffects. Anti-inflammatory corticosteroids also inhibit transcription of COX-2 but not COX1.
50
SECTION 5: METABOLISM OF TRIACYLGLYCEROLS
The metabolism of triacylglycerols/triglycerides (TG) primarily involves:
(i)
esterification of excess fatty acids (derived from fats, carbohydrates and proteins) to
form TG that are stored in adipose tissue, or transported by the blood as lipoproteins
(ii)
hydrolysis of TG to release free fatty acids (FFA) for meeting the energy needs of
the body
The balance between esterification of fatty acids to TG and hydrolysis of triglycerides to
release FFA, is a prime determinant of the concentration of FFA in the blood. The state of
activity of the liver, adipose tissue and intestine influence FFA levels in the blood. The
nutritional state of the body acts via the hormones insulin, epinephrine, glucagon and
glucocorticoids to balance synthesis vs breakdown of the triglycerides.
The main sites of TG metabolism are the adipose tissue and liver. TG stores in adipocytes
are major energy reserves of the body. The liver (Fig 6.5.2) and intestine (Fig 6.1.4)
synthesize triacylglycerols from FFA, enclose them in lipoproteins and release them into the
blood. TGs in the lipoprotein complexes are hydrolyzed to release FFA to the tissues by
lipoprotein lipase, an enzyme located in the walls of the blood capillaries.
A
B
Fig.6.5.1. Transmission (A) and scanning (B) electron micrographs of adipocytes: The large dark mass within
the adipocyte in fig.A is a bulky lipid droplet which reduces the cytoplasmic content to a crescent- shaped
peripheral mass. (Source: A. Berg et al, 2002, p 898 fig 22.1; B. Garrett and Grisham, 1999, p 776 fig 24.1)
51
Fig.6.5.2. Diagram of a hepatic cell, showing the formation of very low density lipoproteins (VLDL) and their
release into blood sinusoids (RER = rough endoplasmic reticulum, SER = smooth endoplasmic reticulum, SD =
Space of Disse) (Source: Murray et al, 2003, p 208 fig 25-2)
An overall idea of biochemical pathways involved in the metabolism of triacylglycerols in
adipose tissue is given below in Fig 6.5.3. The synthesis of TGs takes place in the well-fed
state or when the intake of fatty acids is high. The major source of precursors in TG synthesis
is blood glucose and blood lipoprotein complexes. Their availability at the site of synthesis is
linked to pathways of carbohydrate metabolism. Mobilization of fatty acids from the TG
stores occurs when the demand for energy fuel in the body is high. Hydrolysis of
triacylglycerols releases free fatty acids (FFA) into the blood and the process is regulated
through the enzyme hormone- sensitive lipase. Insulin plays a major role in promoting
lipogenesis and inhibiting lipolysis.
52
Fig.6.5.3. Outline of the pathways in metabolism of storage triglycerides in adipose tissue. Refer to text for
details. (FFA = free fatty acids; TG = triglyceride; VLDL = very low density lipoproteins; PPP = pentose
phosphate pathway) (Source: Murray et al, 2003, p 214 fig 25-7)
6.5.1. SYNTHESIS OF TRIGLYCERIDES
Surplus carbohydrate, fat, or even proteins can be metabolized for storage in our body as
triacylglycerols. It is estimated that stored TGs account for 15kg in a 70kg man! The main
sites for TG synthesis and storage are the adipocytes, found in subdermal and retroperitoneal
“fat”. Lipid droplets are stored in cells of the adipose tissue and, to a lesser extent, in steroidsynthesizing cells of the gonads and adrenal cortex. As stated above, the liver and intestine
also synthesize triglycerides but, unlike adipose tissue, they manufacture lipoprotein
complexes within which the hydrophobic TGs can be released into the blood. The kidney,
adrenals and gonads have a relatively minor role in TG synthesis.
53
Fig.6.5.4. Schematic diagram of storage of lipid in an adipocyte: The nucleus has been pushed to the side and
the cytoplasm has been reduced by the large lipid droplet. (Source: Bettelheim et al, 2001, p 379 fig 18.1)
A lipid droplet in an adipocyte has an outer, protective coating of proteins called perilipins.
Inert, neutral lipids, like triglycerides and cholesterol esters, constitute the main, central core
in the droplet, which is surrounded by a layer of amphipathic phospholipids. (Note that
hydrophobic lipid molecules need the crutch of hydrophilic proteins for both storage and
transport).
PATHWAY OF TG SYNTHESIS
Triacylglycerols (and phospholipids) are synthesized by acylation of triosephosphates derived
from glycolytic pathways. TG synthesis takes place mainly in the endoplasmic reticulum of
adpocytes and hepatocytes.
54
D I H YD R O XYAC ET O N E
G L YC ER O L
3 - PH O SPH AT E
PH O SPH AT E
PHOSPHATIDATE
PAF
PLASMALOGENS
DIGLYCERIDE
CARDIOLIPIN
PHOSPHATIDYL
CHOLINE
PHOSPHATIDYL
INOSITOL
T R I G L YC ER ID E
PHOSPHATIDYL
ETHANOLAMINE
Fig.6.5.5. Overview of synthesis of triglycerides and phospholipids from triose phosphates (red)
The precursors required for TG synthesis are fatty acyl-CoA and glycerol 3-phosphate
(glycerol 3-P).
Acyl-CoA is formed from FFA in a reaction catalyzed by acyl-CoA synthetase:
O
R CH 2 CH 2 C
_
O- +
acyl-CoA
synthetase
Co A S H
Mg
AT P
O
R CH 2 CH 2 C
2+
A MP
_
S
_
Co A
+ PPi
Glycerol 3-P is obtained in two ways:
(i)
by the irreversible, ATP-linked activation of free glycerol by glycerol kinase.
Tissues like the liver and kidney obtain glycerol 3-P by this method but the
enzyme is absent in adipose tissue and muscle
CH 2
HO
_
_
OH
CH
CH 2
_
AT P
A DP
glycerol kinase
HO
_
_
OH
_
O
CH
CH 2
OH
_
P
Glycerol 3-phosphate
Glycerol
(ii)
CH 2
by glyceroneogenesis, a process which diverts dihydroxyacetone phosphate from
the glycolytic pathway. An NAD+-linked enzyme, glycerol-3-phosphate
dehydrogenase, catalyzes the reversible conversion of DHAP to glycerol 3-P
55
Fig.6.5.6. Outline of the pathway of glyceroneogenesis: Observe its similarity to gluconeogenesis. (DHAP =
dihydroxyacetone phosphate)
The synthesis of triacylglycerol from acyl-CoA and glycerol 3-P requires four simple steps:
Step 1: Formation of 1-acyl glycerol phosphate:
Glycerol 3-phosphate acyltransferase acylates the hydroxyl group at C-1 of glycerol 3-P,
producing 1-acylglycerol 3-phosphate (lysophosphatidic acid); and releasing CoA. The first
acyl group transferred is generally from a saturated fatty acid.
Step 2: Formation of 1,2-diacyl glycerol phosphate:
An identical acyl group transfer esterifies C-2 of the alcohol. The enzyme used is 1acylglycerol 3-phosphate acyltransferase, and the acyl group is usually from an unsaturated
fatty acid. The products are 1,2-diacyl glycerol phosphate (do you recognize phosphatidic
acid here ?) and CoA.
Step 3: Dephosphorylation
The phosphoryl group at C-3 of glycerol 3-P is removed before further acylation. Hydrolysis
by a phosphatase/hydrolase releases diglyceride/diacylglycerol (DG) and Pi
Step 4: Formation of triacylglycerol/triglyceride
A final transfer of an acyl group to DG by DG acyltransferase produces the
triglyceride/triacylglycerol and releases CoA.
56
CH 2
C
_
OH
O
CH 2
_
OP
DHAP
CH 2
glycerol 3-P
dehydrogenase
N A DH. H +
_
HO
N A D+
OH
_
OP
CH
CH 2
Glycerol 3-phosphate
O
R' C
_
_
S Co A
acyl transferase
_
Co A S H
O
_
CH 2 _ O _ C
HO
_
CH
CH 2
O
R'' C
_
_
R'
Lysophosphatidic
acid
OP
S Co A
acyl transferase
_
Co A S H
R''_ C
CH 2
_
O
_
H2O
CH 2 _ O _ C
O
R'' C
_
_
O
_
CH
_
O
_
CH2 O
_
C
_
R'
R'' '
Co A S H
R'' '
O
_
_
_
S Co A
acyl transferase
Phosphatidic
acid
OP
O
_
_
O
_
CH
CH 2
4
3
O
CH 2
_
R'
phosphatase
O
R''_ C
_
C
Pi
O
C
_
CH
CH 2
_
2
O
O
O
1
C
_
R'
Diglyceride
_
OH
Triglyceride
Fig.6.5.7. Sequence of reactions in triglyceride synthesis from acyl-CoA and DHAP in adipose tissue. The
active forms of most of the enzymes (generic names used here) are present in the endoplasmic reticulum.
REGULATION OF TG SYNTHESIS
The rate of triacylglycerol synthesis depends on dietary, metabolic and hormonal factors.
Caloric intake vs caloric utilization determines how much carbohydrate/fat/protein in the
blood (or tissues) is “surplus” and can be routed through the pathways of fatty acid or TG
synthesis. The major lipogenic hormone is insulin which stimulates synthesis of fatty acids
while simultaneously inhibiting lipolysis.
57
Fig.6.5.8. Stimulation of triglyceride synthesis by insulin ( Source: Nelson and Cox, 2005, p 806 fig 21-19)
Glyceroneogenesis is controlled by glucocorticoid hormones secreted by the adrenals under
stressful conditions like hypoglycemia. They stimulate glyceroneogenesis in the liver but
suppress it in adipose tissue.
Fig.6.5.9. Organ-specific regulation of glyceroneogenesis by hormone: Glucocorticoids activate the gene for
PEPCK (phosphoenolpyruvate-carboxykinase) synthesis in the liver but represses it in adipose tissue.
(Source: Nelson and Cox, 2005, p 808 fig 21-22)
Glucocorticoids act through diametrically opposite effects on the gene that controls synthesis
of PEP-carboxykinase (gluconeogenic enzyme) in the liver and adipose tissue. As shown in
fig 6.5.9 glycerol 3-phosphate synthesis is promoted in the liver but suppressed in adipose
tissue. This apparently puzzling action is beneficial to the body under stressful conditions like
hypoglycemia. Lack of glycerol 3-P reduces lipogenesis in adipose tissue and free glycerol is
preferentially released into the blood; this becomes available for gluconeogenesis in the liver.
Additionally, FFA is also released from adipose tissue and becomes available as energy fuel.
58
6.5.2. MOBILIZATION OF STORED TRIGLYCERIDES
Hydrolysis of stored triglycerides and release of FFA into the blood is referred to as
“mobilization”.
O
CH 2
O
R
_
C
_
O
_
_
O
_
CH
C
_
R
+
O
_
CH2 O
_
C
_
3 H2 O
lipases
R
CH 2 OH
H C OH
CH 2 O H
O
+
3 RC
_
OH
FREE FATTY ACIDS
GLYCEROL
TRIGLYCERIDE
Fig.6.5.10. Generalized reaction for the hydrolysis of triglycerides by lipases.
Mobilization of TG from adipose tissue is a function of a critical enzyme Hormone-Sensitive
Lipase (HSL). As the name implies, the activity of this lipase is regulated by hormones.
Epinephrine/glucagon act through a cAMP-mediated pathway to activate protein kinase A
(PKA). Subsequently PKA carries out two phosphorylations that promote hydrolysis of TGs:

phosphorylation of HSL: causing a 2-3 fold increase in its catalytic activity

phosphorylation of perilipin A: enables HSL to move to the surface of the lipid
droplet, stimulating its lipolytic activity by about 50 times
59
Fig.6.5.11. Schematic diagram of the mobilization of free fatty acids (FFA) from adipose tissue to myocytes:
Hormones like glucagon trigger a cAMP-dependent enzyme pathway (1-4) causing phosophorylation of
hormone-sensitive lipase and surface perilipin molecules on the lipid droplet. Hydrolysis of triacyl- glycerol
follows (5) and FFA is released into the blood. Bound FFAs (6) in blood enter myocytes with help of a
transporter (7) and undergo β-oxidation. (Source: Nelson and Cox 2005, p 634 fig 17-3)
The products of TG hydrolysis are fatty acids and glycerol. Adipose tissue releases a part of
the FFA produced into the blood while the rest is converted to acyl-CoA for re-use in TG
synthesis. Blood FFA is bound by serum albumin, and transported to the muscle, liver, renal
cortex and other tissues. At the level of the cells, the fatty acid dissociates from albumin and
is transported across the plasma membrane into the cytosol by a specific fatty acid
transporter.
Glycerol produced in lipolysis cannot be metabolized by adipocytes but is released into the
blood for use by cells of other tissues. Utilization of glycerol is possible only when it is
converted to glycerol 3-phosphate. This requires the action of glycerol kinase, an enzyme
which is absent in adipose tissue and muscle but present in other tissues like the liver and
kidney. For the same reason, glycerol that is released from its bound form in lipoprotein
complexes by lipoprotein lipase, cannot be used by adipose tissue.
60
C H 2 OH
ATP
A DP
H C OH
CH2 OH
N A D+
CH 2 OH
HO C H
glycerol kinase
Glycerol
N A DH. H +
glycerol 3-P
dehydrogenase
CH 2 O P
CH 2 OH
C
O
CH 2 O P
DHA P
Glycerol
3 - phosphate
GLUCO NEOGENESIS
GLYCOLYSIS
TRIGLYCERIDE
SYNTHESIS
Fig.6.5.12. Conversion of glycerol to glycerol 3-phosphate; subsequently the product can be utilized in
glycolysis, gluconeogenesis or in the synthesis of glycerides.
Regulation of HSL
The lipolytic activity of HSL is regulated by its reversible phosphorylation/dephosphorylation. Phosphorylated HSL is catalytically active while dephosphorylated HSL is
inactive. Activation of HSL is mainly by a cAMP-mediated cascade mechanism (as for
glycogenolysis, refer chapter 5 section 5.A), triggered by hormones like epinephrine. Other
lipolytic hormones include glucagon, the thyroid hormones, growth hormone and
vasopressin. Glucocoticoids promote lipolysis through a different pathway which involves
synthesis of a new lipase and activation of suitable genes, but does not involve cAMP.
61
G L U C AG O N
EPIN EPH R IN E
N O R EPI N EPH R IN E
+
+
+
IN SU LI N
_
THYROID HORMONES
GH
TSH
MSH
ACTH
_
Adenylyl cyclase
FFA
+
+
AT P
cAMP
Phospho diesterase
INSULIN
5 ' AMP
+
PKA
M
+
FFA
g2+
ATP
HSL
inactive
GLUCOCORTICOIDS
_
A DP
HSL
Pi
active
P
H2O
Lipase
phosphatase
DG
+ FFA
TG
HSL
+
2-MG
INSULIN
+ FFA
2-MG Lipase
FFA
+ GLYCEROL
Fig.6.5.12. Regulation of hormone-sensitive lipase (HSL): Continuous arrows show significant steps in
activation and catalytic action of HSL. Broken arrows indicate sites of action of major hormones viz
epinephrine, norepinephrine, glucagon and insulin. Thyroid and pituitary hormones have a facilitatory role.
Glucocorticoids activate HSL by a cAMP-independent route. Stimulation and inhibition are shown by symbols.
(PKA = protein kinase A; TG/DG/MG = Tri-/di-/mono-acylglycerols respectively)
Insulin is a major inhibitor of lipolysis in adipose tissue. It inhibits HSL indirectly in three
ways:

Inhibition of cAMP formation by adenylyl cyclase

Stimulation of the breakdown of cAMP to 5’AMP by phosphodiesterase

Stimulation of lipase phosphatase, which in turn, dephosphorylates and inactivates
HSL
62
Since insulin secretion is stimulated by high blood levels of glucose, hence the nutritional
state of the body has a major role in controlling the rate of lipolysis vs the rate of lipogenesis.
Insulin promotes glucose uptake and its increased conversion to TGs in adipose tissue,
concommittantly decreasing the release of FFA from TG stores.
It is believed that a hormone, leptin, which is secreted by the adipose tissue and signals
energy sufficiency, may also be important in regulation.
Re-cycling of TGs
Fig.6.5.13. Schematic representation of short- and long-range re-cycling of triacylglycerol between adipose
tissue and liver. (Source: Nelson and Cox, 2005, p 806 fig 21-20)
It is remarkable that the adipose tissue, generally regarded as rather “low-profile”, carries out
a continuous cycle of lipolysis and re-esterification in its cells and still maintains fairly
constant levels of stored lipids over long periods of time. 70% of all fatty acids that are
released by lipolysis are re-esterified to form triglycerides, either by short-re-cycling within
the adipose tissue, or by a systemic long-range re-cycling between the liver and adipose
tissue. Net release of FFA from the adipose tissue into the blood occurs only when hormones
stimulate high activity of HSL to the extent that [FFA] exceeds the ability of the adipocyte to
re-synthesize triacylglycerols. The liver re-synthesizes TGs and releases them into the blood
within VLDL. Breakdown of VLDL by lipoprotein lipase in the capillary walls again makes
FFA available to the adipose tissue.
63
TO SUM UP SECTIONS 2, 3, 4 AND 5:
We end this discourse on the metabolism of the simple lipids with a composite diagram to
clarify your concepts:
Fig.6.5.14. Summary of the metabolism of triacylglycerols and fatty acids in adipose tissue and liver. Red and
green circles are points of inhibition and stimulation respectively.
(Source: Voet and Voet, 1995, p 691 fig 23-35)
64
SECTION 6: METABOLISM OF COMPLEX LIPIDS
Phospholipids are major constituents of all cell membranes. They also have a significant role
as signal molecules. Phospholipid metabolism is generally intracellular and tissue-specific,
with many of them undergoing partial degradation followed by re-synthesis and re-modeling.
Details of phospholipid synthesis and degradation are complex and we will confine ourselves
to some significant features only.
6.6.1. SYNTHESIS OF PHOSPHOLIPIDS
The main site for phospholipid synthesis is the membrane of the smooth endoplasmic
reticulum. The pathway of synthesis resembles that of triacylglycerols. Phosphatidic acid,
obtained from glycerol 3-phosphate and acyl-CoA (Fig 6.5.7), is a central intermediate and
cytidine nucleotides play a crucial role.
The routes of syntheses of important glycerophospholipids are different in mammals from
those in other eukaryotes and bacteria. The immediate precursor here is diacylglycerol (DG),
obtained in the same way as shown in Fig.6.5.7.
Pathways in mammals for the synthesis of major glycerophospholipids are outlined below:
1. Phosphatidylcholine /Phosphatidylethanolamine:
The synthesis of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) depends on
the availability of FFA. Surplus FFA, which exceeds energy needs, is used preferentially for
phospholipid synthesis. When this need is satisfied, FFA is used for TG synthesis.
Choline
OR
Ethanolamine
A DP
ATP
Phospho choline
OR
kinase
Phospho ethanolamine
CT P
P Pi
transferase
CDP- Choline
OR
CDP- Ethanolamine
DG
transferase
CMP
PC / PE
Polar “head groups” like the nitrogenous bases choline or ethanolamine, are activated to their
phosphorylated CDP-derivative by CTP and then attached to diacylglycerol (DG) to produce
PC or PE respectively.
The above pathway takes place in most tissues. In addition the liver can convert PE to PC by
methylations, using S-adenosylmethionine as donor of methyl groups.
PE
3 - methylations
transferase
65
PC
2. Synthesis of Phosphatidylserine:
Phosphatidylserine (PS) is synthesized by exchange of polar “head groups” and CTP is not
involved. Re-use of such
“head groups” by suitable exchange is a “salvage” operation.
Ethanolamine
Serine
transferase
PE
PS
decarboxylase
C O2
The transfer of serine to PE occurs in the endoplasmic reticulum. Decarboxylation of PS in
the mitochondrion can give back PE.
3. Synthesis of Phosphatidylinositol and Cardiolipin:
For synthesis of phosphatidyl inositol (PI) and cardiolipin, CDP has to be attached to the
hydrophobic “tail” (DG) to give the activated intermediate CDP-DG. The phosphorylated
derivatives of PI are important signal molecules.
Inositol
CDP - DG
CMP
synthase
PI
kinases
phosphorylated
derivatives of
PI
Two molecules of CDP-DG are required for cardiolipin synthesis:
Glycerol
3 - phosphate
CDP - DG
synthase
CMP
Phosphatidyl glycerol
3 - phosphate
H2 O
Pi
phosphatase
Phosphatidyl
- glycerol
CDP - DG
synthase
CMP
Cardiolipin
66
4. Synthesis of Plasmalogens
The primary site for synthesis of plasmalogens is the peroxisome. An ether
glycerophospholipid is synthesized by replacing the acyl group at C-1 in 1-acyl DHAP by an
alkyl group from a saturated alcohol. The intermediate so obtained undergoes a series of
reactions to replace the keto- group at C-2 with an acyl group. A mixed-function oxidase now
introduces a double bond at C-1 in the alkyl-acyl derivative to give the plasmalogen.
Platelet-activating factor (PAF)
phosphocholine derivative.
is
synthesized
67
from
1-alkyl-2-acetylglycerol
3-
R' CH 2 CH 2 OH
O
1 C H2
2 C
_
R C OH
_
_
O C R
O
3 C H2
O
2 C
synthase
_
1 C H2
O_ P
_
O
3 C H2
1 - ACYL DHAP
O CH 2 C H 2 R'
_
_
O_ P
1 - ALKYL DHAP
REDUCTION
R''
O
C H 2 _ O _ CH 2 C H 2 R'
C O
CH
ACYLATION
DEPHOS PHORYLATION
C H2 _ O _ H
1- ALKYL , 2 - ACYL GLYCEROL
CDP Ethanolamine
transferase
CMP
O
R'' C O
_
_
C H 2 O CH 2 C H 2
R'
N A DH. H +
CH
C H2 _ O _ P C H2
+
C H2 N H3
N A D+
O2
2 H2 O
mixed - function oxidase
1- ALKYL , 2 - ACYL GLYCEROL
3 - PHOSPHOETHANOLAMINE
O
R'' C O
C H2 _ O _ C H
R'
CH
CH
C H2 _ O _ P C H2
+
C H2 N H3
1- ALKYL , 2 - ACYL GLYCEROL
3 - PHOSPHOETHANOLAMINE
PL AM AL O G EN
Fig.6.6.1. Outline of steps in the synthesis of a plasmalogen: Follow the change at C-1 (majenta) from saturated
acyl to saturated alkyl and then to vinyl alkyl group. The dotted arrow represents reactions not shown in detail.
(DHAP = dihydroxyacetone phosphate)
68
Incorporation of freshly synthesized phospholipids into membranes
Freshly synthesized phospholipids travel to their intracellular destinations via transport
vesicles or with specific cytosolic proteins. They are inserted into the outer leaflet of the ER
membrane as shown in the figure below. Their transfer to the inner leaflet of the ER
membrane and to other membranes in the cell may involve flippases and special
phospholipid-transfer proteins.
Fig.6.6.2. Incorporation of newly synthesized phospholipids into a membrane bilayer.
(Source: Elliot and Elliot, 2005, p 241 fig 14.7)
6.6.2. SYNTHESIS OF SPHINGOLIPIDS (Ceramide, Cerebroside, Ganglioside,
Sphingomyelin)
The base unit in synthesis of sphingolipids is a ceramide. Ceramides are synthesized from
palmitoyl-CoA and serine in four steps:
69
Palmitoyl Co A
Serine
synthase
CO 2 + Co A SH
3 - Keto sphinganine
N A D PH. H +
reductase
N A D P+
Sphinganine
Acyl
Co A
transferase
Co A SH
N - acyl sphinganine
FA D
desaturase
FA D H 2
OH H
O
H CH C
R C O N
C
H
CH
(
C H2
)12
C H3
CH 2 OH
CERAMIDE
Fig.6.6.3. Outline of the synthesis of ceramide from palmitol-CoA and serine.
Most sphingolipids require the attachment of a carbohydrate moiety at C-1 of ceramide.
Galactosyl/glucosyl moieties are transferred to ceramide from their UDP-Galactose/UDPGlucose form to produce the cerebrosides and gangliosides. The process occurs in the Golgi
apparatus. Sphingomyelins are formed from ceramide and phophatidylcholine.
70
U DP- Gal
OR
U DP- Glc
CERAMIDE
Oligosaccharide
U DP
PC
+
Sialic acid
DG
CEREBROSIDE
GANGLIOSIDE
SPHINGOMYELIN
PAPS
SULFOLIPIDS
Fig.6.6.4. Outline of the major routes of conversion of ceramide to other sphingolipids.
6.6.3. DEGRADATION OF COMPLEX LIPIDS
Degradation, replacement and re-modelling of polar lipid components of membranes occur
continuously in cells. The rate of synthesis generally balances the rate of breakdown.
Phospholipids and sphingolipids are degraded by specific hydrolases in the lysosomes.
Continuous exchange of fatty acids in phospholipids, in particular the polyunsaturated fatty
acids at C-2 of lecithins, yields new phospholipid molecules
Degradation of Phosphoglycerides
Specific ester links in a phosphoglyceride molecule are hydrolyzed by specific esterases
called phospholipases. Thus phospholipases A1 and A2 each release one molecule of free fatty
acid and reduce a phosphoglyceride to its corresponding lyso-compound. The lyso-compound
is cleaved of its 2nd fatty acyl group by specific lysolipases. Phospholipase B (which is
phospholipase A1 + A2) removes both fatty acyl groups simultaneously. Phospholipase C and
D free DG and phosphatidic acid respectively. Phospholipases are present in snake venom
and the lipid products of their hydrolytic action cause lysis of RBC, preventing clot formation
in the victims.
71
Fig 6.6.5. Sites of action of phospholipases on a glycerophospholipid.
(Source: Murray et al, 2003, p 201 fig 24-6)
(Since phospholipases are esterases they do not cleave the ether bond of plasmalogens).
As an example of the relatively simple way in which phospholipids are degraded, we follow
the three steps in the catabolism of a major membrane constituent phosphatidylcholine:
FATTY ACID
H2 O
PHOSPHATIDYLCHOLINE
phospholipase A 2
LYSOPHOHPHATIDYLCHOLINE
STEP I
FATTY ACID
H2 O
LYSOPHOHPHATIDYLCHOLINE
lysophospholipase
GLYCERYLPHOSPHORYLCHOLINE
STEP II
GLYCEROL 3 - PHOSPHATE
H2 O
GLYCERYLPHOSPHORYLCHOLINE
hydrolase
CHOLINE
STEP III
Fig.6.6.6. Steps in the enzymatic catabolism of phosphatidylcholine
72
The fatty acids released from phospholipids can be used for new phospholipid molecules or
for synthesis of cholesteryl esters. Lecithin/lysolecithin can undergo transacylation with
cholesteryl ester to generate a remodelled phosphatidylcholine.
Degradation of Sphingolipids
Gangliosides and other sphingolipids are degraded by a set of lysosomal enxymes that
catalyze the step-wise removal of sugar units to finally yield a ceramide. Genetic defects in
these enzymes lead to lipid accumulation have serious clinical consequences. Thus TaySach’s disease is caused by absence in hexosaminidase A which results in ganglioside
accumulation in the lysosomes and leads to mental retardation and death. Lack of
sphingomyelinase causes Niemann-Pick disease in which sphingolipid accumulation in brain
resulting in developmental abnormalities, paralysis and early death. Genetic counseling can
avert these dire consequences of inheritable gene defects.
SECTION 7: METABOLISM OF CHOLESTEROL
Cholesterol and cholesteryl esters are membrane lipids and precursors of steroid hormones,
bile acids and Vitamin D. Approximately half of the total cholesterol in our body is obtained
by de novo synthesis while the remainder comes from the diet.
6.7.1. SYNTHESIS OF CHOLESTEROL
Cholesterol synthesis takes place mainly in the liver but can occur in almost all tissues. The
reactions occur in the smooth endoplasmic reticulum and cytosol. Acetyl-CoA is the
ultimate source of all carbon atoms in cholesterol and its derivatives: 12 carbon atoms are
derived from the carbonyl group while remaining 15 are derived from the methyl group of
acetyl-CoA molecules.
The long synthetic pathway starts from acetyl-CoA (C2) and proceeds till HMG-CoA (C6) in
steps similar to ketogenesis (refer section 3). However, the two pathways are completely
separate in location since cholesterol synthesis is extramitochondrial while ketogenesis
occurs in the mitochondrion. Cytosolic isozymes of thiolase and HMG-CoA synthase produce
HMG-CoA for cholesterol synthesis.
HMG-CoA is reduced to mevalonate (C6) by HMG-CoA reductase, a characteristic and
crucial enzyme of cholesterol synthesis. Mevalonate is the first metabolite committed solely
to cholesterol synthesis.
73
Fig.6.7.1. Synthesis of mevalonate from HMG-CoA (Adapted from Garrett and Grisham 1999, p 833 fig 25.31)
Three successive reactions, each requiring ATP, convert mevalonate to the active isoprenoid
intermediate, isopentenyl pyrophosphate (IPP, C5). The involvement of isoprene unit is a key
point in the biosynthesis of steroids
and terpenes. Condensation of isoprenoid units gives farnesyl pyrophosphate, a C15
compound which is also the precursor of ubiquinone.
74
Fig.6.7.2. Conversion of mevalonate to squalene (Source: Garrett and Grisham, 1999, p 837 fig 25.34)
75
Two units of farnesyl pyrophosphate condense to form a linear, C30 molecule, squalene,
which then undergoes cyclization to form the parent C30 steroid, lanosterol. Lanosterol is
finally converted to cholesterol (C27) by a 20–step process, using enzymes embedded in the
membranes of the endoplasmic reticulum.
Fig. 6.7.3. Conversion of squalene to lanosterol (Adapted from Garret and Grisham, 1999, p 839 fig 25-3)
Smooth ER is the site for both cholesterol synthesis and for conversion to other steroids. Hence cells carrying
out steroidogenesis have enlarged smooth ER.
Fates of synthesized cholesterol
A small percentage of cholesterol synthesized in the liver is retained in hepatocytes for
incorporation into membranes. The remaining is exported in one of three forms

Biliary cholesterol – which is carried away by bile

Bile acids – approximately 0.5gms/day of cholesterol is disposed off in humans
through bile salts

Cholesteryl esters – which are either incorporated into lipoprotein complexes by
CETP (cholesteryl ester transfer protein) or stored in the liver.
In the liver cholesterol is converted into an activated intermediate cholyl-CoA which then
reacts with glycine/choline to form the bile salts glycocholate/taurocholate respectively.
These are excreted via bile.
76
6.7.2. SYNTHESIS OF CHOLESTERYL ESTER
Cholesteryl ester is the storage form of cholesterol in cells and the main form present in
lipoproteins. The replacement of its polar hydroxyl group at C-3 by a hydrophobic acyl group
makes cholesterol more suitable for storage as well as for packing into VLDL and other
lipoproteins.
Cholesterol acyl transferases catalyze the esterification of cholesterol; the specific enzyme
differs with the site of esterification:
i.
Within cells – the enzyme used is ACAT i.e. acyl-CoA: cholesterol acyl
transferase located on the ER
Fig.6.7.4. Conversion of cholesterol to cholesteryl ester using ACAT.
(Adapted from Garrett and Grisham, 1999, p 838 fig 25.35)
ii.
Within HDL – the enzyme used is LCAT i.e. lecithin: cholesterol acyl transferase
77
Fig.6.7.5. Conversion of cholesterol to cholesteryl ester using LCAT. ( Source: Nelson and Cox, 2005, p 823 fig 2141)
LCAT activity is associated with HDL containing apo A-1. Esterification of cholesterol,
creates a gradient for transfer of cholesterol from tissues and other lipoproteins to HDL, thus
enabling HDL to function in reverse cholesterol transport. Much of the cholesteryl ester
formed by LCAT is transferred to VLDL and chylomicron remnants and ultimately reaches
the liver.
6.7.3. REGULATION OF CHOLESTEROL SYNTHESIS
The maintenance of cholesterol homeostasis in the body is important for our well-being
Cholesterol levels depend on the diet and on metabolism. A high intake of saturated fatty
acids causes high levels of cholesterol in the liver while high intake of unsaturated fatty acids
(particularly ω-3 and ω-6) does not do so.
78
Cholesterol synthesis is controlled by regulation of the quantity or the activity of HMG-CoA
reductase. Short-term control of the enzyme is by product feedback inhibition by both
mevalonate and cholesterol. Covalent modification of the enzyme is by reversible
phosphorylation. In presence of high levels of cholesterol, phosphorylation is carried out by
AMP-dependent protein kinase. The phosphorylated form of the enzyme is poorly active and
is rapidly destroyed. Insulin and thyroid hormones stimulate HMG-CoA reductase while
glucagon/glucocorticoids inhibit it. Hormonal action is probably through reversible
phosphorylation/ dephosphorylation of the enzyme.
Fig.6.7.6. Major factors in the regulation of cholesterol synthesis (Source: Nelson and Cox, 2005, p 826 fig 2144)
Long-term regulation of the quantity of available HMG-CoA reductase is the primary
regulatory mechanism in cholesterol synthesis. Cholesterol itself regulates the expression of
several genes associated with its synthesis and uptake. Low levels of cholesterol in the cell
activate more than 20 genes coding for enzymes involved in its synthesis and uptake,
significant among them being the genes coding for HMG-CoA reductase and for LDL
receptor protein. The result is that cholesterol levels can be increased 200-fold.
The activation of the genes is by a remarkable mechanism. All the genes possess a specific
regulatory sequence called SRE (sterol regulatory element). When cholesterol levels are
high, a specific protein called SREBP (sterol regulatory element binding protein) resides in
the ER in association with SCAP (SREBP cleavage activating protein) as an SREBP-SCAP
complex. When cholesterol levels are high, SCAP transfers SREBP to Golgi where the latter
is cleaved to release its N-terminal domain that enters the nucleus, binds to SRE target genes
(CHECK) and induces their transcription.
6.7.4. CIRCULATION OF CHOLESTEROL
Cholesterol and cholesteryl esters are transported between tissues by the lipoprotein
complexes. The normal range for total plasma cholesterol in humans is < 5.2 mmol/L.
Animal cells acquire cholesterol either by synthesizing it or by taking it up from cholesterolrich particles like the LDL complexes. The highest proportion of cholesterol is in the LDLs.
Cholesterol efflux from tissues is mainly by its transfer to HDL.
79
Lipoprotein complexes are carried by the blood and lymph to all tissues of the body. VLDL
and chylomicrons (refer section 1) give up much of their TG-bound fatty acids to the tissues
but the VLDL and chylomicron remnants now left still contain cholesterol and cholesteryl
esters. Chylomicron remnants deliver 95% of their cholesterol to the liver.
With further loss of TG, circulating VLDL remnants are delipidated to LDL and IDL; these
now carry a higher proportion of cholesterol and its esters. LDL and IDL are prime movers of
cholesterol to the liver and extra-hepatic tissues. The uptake of degraded VLDLs and
chylomicron remnants by the liver is important since the liver is the only organ which can
remove significant quantities of cholesterol from the body.
Hepatocytes and many extra-hepatic cells posses a specific plasma membrane glycoprotein,
the LDL receptor, which recognizes apoB-100 on the LDL particle. These cells pick up
LDLs intact by receptor-mediated endocytosis. The LDLs are degraded in the lysosomes
and their load of cholesterol and fatty acids is released into the cell. The influx of this
cholesterol regulates endogenous cholesterol synthesis by the cells.
Fig.6.7.7. Structure of the LDL receptor showing domains of amino acid and oligosaccharide residues
(Source: Garrett and Grisham 1999, p 840 fig 25.40)
80
Fig.6.7.8. Receptor-mediated endocytosis of LDL and its subsequent processing within a cell
(Source: Berg et al, 2002, p 519 fig 12.40)
The LDL receptor can also bind ApoE of VLDL and chylomicron remnant for subsequent
ease in pick-up by hepatocytes. Probably there are other back-up receptors too.
HDL is the prime vehicle for a process called reverse transport of cholesterol. Disc-shaped,
nascent particles of HDL originate in the liver and intestine and are secreted into the blood.
They contain low quantities of cholesterol besides apoproteins and other lipids. They pick up
excess free cholesterol from tissues (and from VLDL and chylomicron remnants) and convert
it to cholesteryl ester by their own LCAT. As a consequence they are transformed into
spherical, mature HDL particles that transport cholesterol back to the liver. The liver
excretes excess cholesterol into bile as free cholesterol or as bile acids/bile salts.
Depleted HDL dissociates from the hepatocytes and re-circulates in the plasma to pick up
more cholesterol.
Reverse transport of cholesterol is aided by SR-B1, a cell-surface HDL receptor with a dual
role in HDL metabolism. In liver and steroidogenic tissues, it binds HDL via apo A-1 and
delivers cholesteryl ester selectively to the hepatic and steroidogenic cells. In other
cholesterol-rich tissues, SR-B1 mediates passive movement of cholesterol from cells into
HDL.
Reverse transport of cholesterol is also aided by a large membrane glycoprotein transporter in
cholesterol-rich cells called ABCA1 protein, which interacts with apoA-1 in depleted HDL.
It helps to preferentially transfer cholesterol from cells to poorly lipidated HDL particles (e.g.
pre –HDL), inducing efflux from tissues. ABCA1 protein belongs to a family of ATP-binding
cassettes.
81
6.7.5. PRODUCTION OF STEROID HORMONES
Fig.6.7.9. Cleavage of the side-chain of cholesterol by a mitochondrial mixed function oxidase system and
desmolase.
(Source: Nelson and Cox 2005, p 827 fig 21-47)
Cholesterol is the precursor for all steroid hormones synthesized by the body. Steroidogenic
tissues obtain most of their cholesterol supplies from the plasma and maintain adequate
reserves of cholesteryl esters in their cytoplasm.
82
The pathway of steroid synthesis is triggered by specific hormonal stimuli, like ACTH in the
adrenal and LH in the testis. This activates esterase to hydrolyze cholesteryl esters and free
cholesterol. A special transporter called steroidogenic acute regulatory protein (StAR),
conveys free cholesterol to the mitochondrion where a mixed function oxidase system
hydroxylates cholesterol at C-20 and C-22, using cytochrome P-450, O2 and NADPH. This is
followed by desmolase action which removes a 6-C unit, isocaproaldehyde. The C21
derivative now obtained is pregenenolone.
Pregnenolone is conveyed from the mitochondrion to the ER for conversion to progesterone
and other steroid hormones. It is the basic intermediate for the synthesis of steroid hormones
in the adrenals and gonads.
Fig. 6.7.10. Derivation of major groups of steroid hormones from pregnenolone
(Source: Berg et al, 2002, p 1091 fig 26.24)
Physiological implications of serum [cholesterol]
Serum cholesterol levels in humans depend on diet, lifestyle and hereditary factors. There is
actually no “good” or “bad” cholesterol; the term refers to the form in which it is packaged in
the blood. LDL is atherogenic and its high concentration in plasma means that high levels of
cholesterol are supplied to the tissues. On the other hand, high [HDL] means that liver is able
to metabolize/excrete excess cholesterol. High levels of cholesterol cause down-regulation of
LDL receptors in hepatic cells. This reduces uptake of LDL and IDL by the liver, so that
blood LDL cholesterol becomes high. Consequently, hydrophobic plaques of cholesterol can
form within walls of blood vessels, increasing the risks of strokes/heart attacks. High
[cholesterol] in bile can lead to formation of gallstones.
Physical exercise, substitution of saturated fats by PUFA in the diet, reduction in intake of
carbohydrates (particularly those containing sucrose and fructose), control of stress, and “NO
SMOKING!” are some of the measures that help to control LDL cholesterol. Hypolipidemic
drugs viz. the statins are administered to lower blood cholesterol levels in high-risk patients if
83
a change in diet and lifestyle does not do so. The statins inhibit HMG-CoA reductase and
prevent cholesterol biosynthesis.
SUMMARY
Lipid metabolism provides cells with energy fuels, energy stores, membrane constituents and
signaling molecules.
Lipids are transported in the blood either in the free form as fatty acids and ketone bodies, or
in the bound form within hydrophilic, lipoprotein complexes. Lipoproteins are synthesized by
the liver and intestine. Lipoprotein lipase in capillary walls releases free fatty acids from the
lipoprotein complexes to the tissues.
Catabolism and anabolism of lipids takes place at specific intracellular locations.
Mitochondrial β-oxidation of free fatty acids is a major generator of ATP in most cells. The
acyl chain is cleaved into acetyl-CoA units which are then processed through the citric acid
cycle and electron transport chain to obtain ATP.
Fatty acids upto chain lengths of C16 are synthesized in the cytosol from acetyl-CoA by a
fatty acid synthase system having multi-enzyme functions. The chain is further lengthened in
the ER/mitochondrion, and acquires double bonds in the ER. Eicosanoids are synthesized
from linoleic acid.
Starvation, high-fat diets and untreated diabetes mellitus results in excessive oxidation of
fatty acids causing ketogenesis. The liver produces ketone bodies which are used by nonhepatic tissues as alternate energy fuels to fatty acids and glucose
Triacylglycerols are synthesized in the adipose tissue and liver by esterification of glycerol 3phosphate with acyl-CoA derivatives. They are stored as large lipid droplets. The action of
hormone-sensitive lipase mobilizes fatty acids from stored triacylglycerols in times of need.
Insulin stimulates lipogenesis but inhibits lipolysis; epinephrine and glucagon stimulate
lipolysis.
Glycerophospholipids are synthesized from intermediates of triacylglycerol synthesis, with
cytidine nucleotides activating either the hydrophilic “head” group or the lipid “tail”.
Ceramide synthesis requires palmitoyl-CoA and serine as precursors. All sphingolipids are
built from ceramide. Synthesis of sphingomyelins requires phosphatidylcholine while
ganglioside formation requires attachment of sugar residues and sialic acid in the Golgi
complex.
Phospholipids are catabolised by phospholipases. Extensive remodeling of phospholipids is
carried out in cells, using fatty acyl groups released from cholesteryl esters and triacylycerols.
Sphingolipids are degraded in the lysosomes by a series of enzymatic hydrolyses and defects
in any of these enzymes results in lipid storage disease.
Cholesterol is synthesized from acetyl-CoA in hepatic cells as well as in other steroidogenic
cells. The process is long, occurring in the ER and cytosol. HMG-CoA is a crucial
intermediate and HMG-CoA reductase activity is controlled to regulate cholesterol synthesis.
Cholesterol is conveyed to tissues by LDL and taken up receptor-mediated endocytosis. HDL
is responsible for reverse transport of cholesterol from the tissues to the liver. The liver
excretes cholesterol mainly as bile salts. All steroid hormones are synthesized from
cholesterol.
84
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