Biochemistry 460 - Dr.

Tischler
LIPOLYSIS, BETA-OXIDATION, KETONES, LIPOGENESIS

Related Reading: Chapter 22: 619-644 in Stryer 6th edition

OBJECTIVES:

1. For the lipolytic pathway (lipolysis): describe the pathway, identify where it occurs, name the
principal enzyme involved, and explain the role of albumin and fatty acid binding protein in
the transport and metabolism of free fatty acids liberated by lipolysis
2. For the degradation of fatty acyl CoAs: describe the roles of acyl CoA synthetase, carnitine-
palmitoyl transferases (CPT-I and CPT-II), and carnitine acylcarnitine translocase (CAT) and
discuss the relationship of the products of the β-oxidation pathway to energy production.
3. For ketone body metabolism: identify where and when ketone body formation (ketogenesis)
occurs, state the role of ketogenesis, identify where ketone oxidation occurs and explain why
normally individuals do not develop ketoacidosis even when producing ketone bodies.
4. Describe the reactions catalyzed by malic enzyme and acetyl CoA carboxylase
5. For the fatty acid synthase reaction: list the substrates and key products, identify the sources of
NADPH for the reaction, and describe its general mechanism.
6. Describe how fatty acids are stored as a source of fuel during starvation or stress.

PHYSIOLOGICAL PREMISE

Would you believe that diabetics having a ketotic crisis have actually been arrested for DUIs even though
they have consumed no alcohol? Indeed a blood analysis would show no alcohol. Why would this occur?
During a ketotic crisis a byproduct of the excess ketone production is acetone. Having nowhere else to go,
it is expired through the lungs. It is the acetone that arresting officers have smelled on the breath of these
individuals and despite their protestations have innocently believed them to be consuming alcohol.

LIPOLYSIS

Lipolysis is a simple process whereby the fatty acids attached to glycerol in triacylglycerols are
hydrolytically removed yielding free fatty acids plus glycerol. Lipolysis largely occurs in adipose tissue
for the mobilization of fatty acids to serve as a fuel in the body, as well as a precursor for the synthesis of
ketone bodies. Additionally, lipolysis may also occur in muscle or liver where smaller amounts of fatty
acids are stored to produce energy for the use of the cell in which they are stored. Hormone-sensitive
(cyclic AMP-regulated) lipase initiates lipolysis by cleaving off the first fatty acid. Then this lipase and
other lipases remove the remaining two fatty acids from the glycerol backbone. The fatty acids and
glycerol are then released from the adipose tissue into the blood. Glycerol is water-soluble and therefore
can freely travel through the blood. However fatty acids are very hydrophobic because of their long
hydrocarbon tails. Consequently they must bind to albumin, a protein released from liver, to be carried
through the blood.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-1

DEGRADATION OF FATTY ACIDS

Overview of degradation of fatty acids

lipoproteins
(chylomicrons
or VLDL)
L FABP MITOCHONDRION
[2]
P FA
CAPILLARY L TCA
A acetyl-CoA [7]
[3] C cycle
[4] β-oxidation
FA FA S
[6]
albumin FA FA acyl-CoA acyl-CoA
FA FABP FABP
[5]
[1] CYTOPLASM carnitine
transporter
from fat cells
FA = fatty acid
LPL = lipoprotein lipase
cell membrane FABP = fatty acid binding protein

Figure 1. Overview of fatty acid degradation. Fatty acids are delivered bound to albumin or released
from lipoproteins. Fatty acid binding protein carries the fatty acids within the cytoplasm. Fatty acids are
transported as the carnitine derivative into the mitochondrion for subsequent oxidation.

Albumin delivers free fatty acids (FA) from fat cells following lipolysis (Fig. 1, [1]). Lipoproteins also
deliver fatty acids via chylomicrons or very low density lipoproteins (VLDL) by the action of lipoprotein
lipase that is located in the capillary cell wall (Fig. 1, [2]). Fatty acids are solubilized within the cell by
binding to fatty acid binding protein (FABP) (Fig. 1, [3]). Fats in the liver may also be synthesized
(lipogenesis) or released from triacylglycerols or phospholipids. Fatty acids are then activated to their
acyl CoA form in a reaction catalyzed by acyl CoA synthetase (ACS; fatty acid + CoA + ATP → fatty
acyl CoA + AMP + 2 Pi) (Fig 1, [4]; Fig 2, [1]) This reaction also activates fatty acids derived from
lipogenesis. Recall that CoA is also used for converting acetate to the chemically reactive acetyl CoA,
via pyruvate dehydrogenase, for use in the citric acid cycle.

Following activation of the fatty acids, they are transported into the mitochondria via the carnitine
transport system (Fig. 1, [5]) (see Fig. 2 for more detail). In the mitochondria the fatty acids are oxidized
via the beta-oxidation pathway (Fig. 1, [6]) (see Fig. 3 for more detail). Acetyl CoA produced by
beta-oxidation feeds into the citric acid cycle (TCA cycle) for energy production (Fig. 1, [7]) or may be
used by the liver in the synthesis of ketone bodies (see Fig. 5 for more detail).

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-2

Uptake of fatty acids into mitochondria

Palmitoyl CoA, like all acyl CoA molecules, cannot directly pass through the inner mitochondrial
membrane. Instead, fatty acids are transported across the membrane attached to carnitine. Palmitate is first
activated to palmitoyl CoA on the outside of the outer mitochondrial membrane (Fig. 2, [1]). Palmitoyl
CoA diffuses through the outer membrane. Then palmitoylcarnitine is formed by the reaction of
palmitoyl CoA with carnitine via carnitine-palmitoyl transferase I (CPT-I) (Fig. 2, [2]). CPT-I is
located in the outer mitochondrial membrane.

ATP + CoA AMP + PP i

palmitoyl-CoA

Cytoplasm

Outer
ACS Mitochondrial
CPT-I
[1] [2 Membrane

CoA
palmitoyl-CoA

Intermembrane palmitoyl-carnitine
carnitine
Space

Inner
Mitochondrial CAT [3
Membrane

Matrix CPT-
[4
carnitine palmitoyl-carnitine

palmitoyl-CoA CoA

LEGEND
ACS = acyl CoA synthetase
CPT = carnitine-palmitoyl transferase
CA = carnitine-acylcarnitine translocase
Figure 2. Activation of
palmitate to palmitoyl CoA and its mitochon-drial uptake via the carnitine-cylcarnitine translocase (steps
4 and 5 in Fig. 1).

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-3

The carnitine transporter, carnitine-acylcarnitine translocase (Fig. 1, [5]; Fig. 2, [3]) is an integral
protein of the mitochondrial inner membrane that exchanges palmitoyl-carnitine from the intermembrane
space for carnitine in the mitochondria matrix. Palmitoylcarnitine is the principal molecule transported
into the matrix on this translocase though other fatty acylcarnitines use it as well. The palmitoylcarnitine
is converted back to palmitoyl CoA in the mitochondria in a reaction catalyzed by carnitine palmitoyl
transferase II (CPT-II) that is attached to the matrix side of the inner mitochondrial membrane (Fig. 2,
[4]). Thus palmitoyl CoA is regenerated in the mitochondrial matrix and carnitine is liberated to be
transferred back to the intermembrane space. Other specific carnitine-fatty acyl transferases participate in
the conversion of less common fatty acyl molecules to their carnitine derivative. A specific transferase is
needed for palmitoyl CoA because this fatty acid is the most prevalent stored in triacylglycerols.
Carnitine-palmitoyl transferase defects lead to considerable muscle weakness, since fatty acids are a
major fuel during muscle utilization. Furthermore, because fatty acid oxidation is obligatory for
gluconeogenesis to occur in the liver, such defects can contribute to hypoglycemia in fasting.

Beta-oxidation of even-chain fatty acids

Saturated (no HC=CH bonds) fatty acyl CoA molecules of any chain length that enter the mitochondrial
matrix can be substrates for the β-oxidation pathway (Fig. 3). Palmitoyl CoA is given as a basic example.
It is first transported into the mitochondrial matrix as the carnitine derivative, and then reactivated to
palmitoyl CoA. The first reaction in the pathway is an oxidation reaction (acyl CoA dehydrogenase) that
uses FAD as a coenzyme. There are several different dehydrogenases that catalyze this reaction
depending on the length of the fatty acid hydrocarbon tail (short-, medium-, or long-chain). The
remaining reactions include a hydratase, another dehydrogenase step with NAD+ as the coenzyme, and
finally a thiolase that removes acetyl CoA from the end of the chain. The products of each of these steps
are acetyl CoA and a fatty acyl CoA molecule that is two carbons shorter than the one that initiated the 4-
step sequence. Thus in this example palmitoyl CoA is shortened after one cycle to a fatty acyl CoA with
14 carbons. The β-oxidation reactions recycle to consecutively remove 2-carbon units as acetyl CoA. On
the final cycle, which begins with the 4-carbon fatty acyl CoA intermediate, two acetyl CoA molecules
are formed as the product when this intermediate is cleaved. Thus, the 16-carbon palmitoyl CoA molecule
needs to cycle just 7 times to produce 8 molecules of acetyl CoA. During each cycle one molecule each of
FADH2 and NADH are produced. The overall reaction of β-oxidation of palmitoyl CoA is:

palmitoyl CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA → 8 acetyl CoA + 7 FADH2 + 7 NADH

Energy is produced indirectly from oxidation of fatty acids in several ways. The FADH2 produced is
oxidized subsequently by the respiratory chain to produce 2 ATP via oxidative phosphorylation per each
FADH2. Similarly each NADH is oxidized via the respiratory chain to produce 3 ATP via oxidative
phosphorylation. Thus the 7 FADH2 and the 7 NADH will ultimately yield a total of 35 molecules of
ATP. Additional ATP also can be produced when the acetyl CoA is oxidized via the citric acid cycle
which, as you should recall, produces 3 NADH, 1 FADH2 and 1 GTP for each acetyl CoA oxidized. In
liver, this acetyl CoA maybe used instead for the synthesis of ketone bodies (ketogenesis), as described
below.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-4

 Palmitoylcarnitine

carnitine
inner membrane translocase respiratory chain

matrix
Palmitoylcarnitine
2 ATP Figure 3. Processing and
3 ATP β-oxidation of palmitoyl
CoA, an even-chain fatty
Palmitoyl-CoA acid.
FAD
oxidation
FADH2

hydration H2O

recycle NAD+
6 times oxidation
NADH
thiolase CoA

CH3(CH2)12-C-S-CoA + Acetyl CoA


O
citric
acid
cycle
2CO2

Very long-chain fatty acids (20-carbon or longer) are processed via a modified β-oxidation pathway in
peroxisomes with acetyl CoA and peroxide as products. The process ends with an 8-carbon fatty acid that
is then converted to its carnitine form and further oxidized in the mitochondria. The acetyl CoA products
are converted to acetyl carnitine and oxidized via the citric acid cycle after transport and conversion to
acetyl CoA.

KETONE METABOLISM:

Ketogenesis:

Ketogenesis occurs only in liver mitochondria and only when the production of acetyl CoA from fatty
acids exceeds the capacity of the citric acid cycle to oxidize it (Fig. 4). The excess acetyl CoA then is
used to produce ketones. Hydroxymethylglutaryl CoA (HMG CoA), an intermediate in ketogenesis, is
formed via mitochondrial HMG CoA synthase. Hydroxymethylglutaryl CoA is also formed in the
cytoplasm as a precursor of cholesterol biosynthesis. In ketogenesis, HMG CoA is cleaved by HMG CoA
lyase to form acetoacetate with acetyl CoA as the other product. β-Hydroxybutyrate, the primary ketone
body in the blood, is formed from acetoacetate via β-hydroxybutyrate dehydrogenase, which requires
NADH as a coenzyme.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-5

 MITOCHONDRION
oxidation to CO2
Fatty acid
2 Acetyl CoA
β-oxidation Citric acid cycle
(excess amounts Thiolase
of acetyl CoA)
CoA
Acetoacetyl CoA
acetyl CoA
HMG-CoA synthase
Figure 4. Ketone body
CoA formation (ketogenesis) in
liver mitochondria from
excess acetyl CoA derived
Hydroxymethylglutaryl CoA
from the β-oxidation of
fatty acids
HMG-CoA-lyase
acetyl CoA

(non-enzymatic) Acetoacetate
NADH

Acetone β-Hydroxybutyrate
dehydrogenase
NAD+
β-Hydroxybutyrate

Ketone body oxidation:

Only under conditions of high rates of lipolysis (e.g., long-term starvation or in uncontrolled diabetes) are
there sufficient amounts of ketones in the blood to be effective as a fuel. If glucose, ketones and fatty
acids are all available in the blood, ketones are the preferred fuel; that is they will be used preferentially
in many tissues over glucose and/or fatty acids. The primary tissues using ketones, when they are
available, are brain, muscle, kidney and intestine, but not the liver. β-hydroxybutyrate is oxidized to
acetoacetate by β-hydroxybutyrate dehydrogenase in the mitochondria. This reaction is the reverse of the
one catalyzed by this enzyme in ketogenesis. Hence the reaction produces NADH. Acetoacetate is
converted to acetoacetyl-CoA, which is cleaved into two acetyl-CoA molecules that can be oxidized for
energy.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-6

Ketosis:

Both β-hydroxybutyrate and acetoacetate are acids. Acetoacetate is spontaneously (non-enzymatic)

cleaved to acetone (Fig. 5), which is non-acidic. When abnormal amounts of ketones are produced in the
body (i.e. ketosis), they may appear in the urine or be expired as acetone (see physiological premise).
When excessive build-up of ketone bodies leads to a fall in the pH of the blood due to the acidic ketone
bodies ketoacidosis results. In normal individuals, ketosis is prevented as follows:

1) excess ketone bodies promote the pancreatic release of insulin,

2) insulin subsequently inhibits lipolysis to decrease the release of fatty acids
3) the supply of fatty acids reaching the liver is decreased so that there are not sufficient amounts of
acetyl CoA for the synthesis of ketones (Fig 6).
A diabetic loses this important control because of their inability to secrete insulin (type I, juvenile\
diabetes) or because their adipose cells have lost their ability to respond normally to insulin (type II,
adult-onset diabetes).

Adipose
Free fatty LIVER
tissue
X acids Figure 5. Mechanism for prevention
of ketosis due to excess ketone body
production that can lead to
ketoacidosis.
Insulin Ketone
Bodies

Pancreas

BIOSYNTHESIS AND STORAGE OF FATTY ACIDS

Lipogenesis

Synthesis of fatty acids (lipogenesis) principally occurs in adipose tissue and liver. In adipose tissue the
fatty acids are stored immediately as triacylglycerols formed via esterification, as discussed later in this
lecture. Liver also produces triacylglycerol that is packaged into VLDL and exported into the blood (as
dsicussed in the preceding lecture). Any compound metabolized to acetyl CoA can serve as a precursor
for fat synthesis. However, glucose is the primary source of carbons for fat biosynthesis. Glucose is
converted to pyruvate via glycolysis, and pyruvate is then transported into the mitochondrial matrix.
However, lipogenesis occurs in the cytoplasm and requires acetyl CoA.

Because acetyl CoA cannot be directly transported across the mitochondrial membrane, its carbons must
be carried to the cytoplasm via a different mechanism. This is accomplished by incorporating the two
carbons from acetyl CoA into citrate. Citrate is then transported from the matrix to the cytoplasm as a
carrier of the carbons destined for fatty acids (Fig.6). The production of citrate requires equal amounts of
oxaloacetate and acetyl CoA for the citrate synthase reaction. Therefore pyruvate must be converted to
both oxaloacetate via pyruvate carboxylase and acetyl CoA via pyruvate dehydrogenase. Recall that
pyruvate carboxylase is also important in gluconeogenesis and requires biotin as a cofactor and ATP as
an energy source. The pyruvate dehydrogenase mechanism was described in an earlier lecture.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-7

pyruvate + CO2 + ATP → oxaloacetate + ADP + Pi pyruvate carboxylase

pyruvate + NAD + coenzyme A (CoA) → acetyl CoA + CO2 + NADH pyruvate dehydrogenase

Glucose CYTOPLASM MITOCHONDRIAL MATRIX

PPP Glycolysis NAD, CoA NADH, CO2

Fatty
Acid PDH
CO2 Pyruvate Pyruvate Acetyl CoA
ATP, CO2
NADPH ME
FAS PC
Malate NADP+
ADP, Pi
Malonyl CoA NAD+
ADP, Pi MDH Oxaloacetate
ACC NADH CS
ATP, CO2 Oxaloacetate
Acetyl CoA ADP+Pi

ATP, CoA
CL
Citrate Citrate

LEGEND:
ACC = acetyl CoA carboxylase
CL = citrate lyase
CS = citrate synthase
FAS = fatty acid synthase
MDH = malate dehydrogenase
= translocase
ME = malic enzyme
PC = pyruvate carboxylase
PDH = pyruvate dehydrogenase
PPP = pentose phosphate pathway

Figure 6. Export of acetyl CoA incorporated into citrate for fatty acid biosynthesis, generation of
NADPH and pathway of lipogenesis.

When there is excessive intake of dietary glucose then a lot of citrate is produced and is available to
participate in lipogenesis. In the cytoplasm, citrate is cleaved by citrate lyase (CL) to regenerate acetyl
CoA and oxaloacetate in an energy requiring reaction. Coenzyme A is required as a co-substrate for this
lyase reaction. Oxaloacetate is then reduced to malate via malate dehydrogenase (MDH) that uses
NADH as the co-substrate. Malate is oxidized to pyruvate by NADP+ via malic enzyme (ME). The
NADPH produced by malic enzyme is obligatory for fatty acid synthesis.

citrate + CoA + ATP → acetyl CoA + oxaloacetate + ADP + Pi citrate lyase

+
oxaloacetate + NADH → malate + NAD malate dehydrogenase
malate + NADP+ → pyruvate + NADPH malic enzyme

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-8

Acetyl CoA carboxylase (ACC) is the committed step for fat biosynthesis with malonyl CoA as its
product (Fig. 6). The mechanism for acetyl CoA carboxylase is identical to that of pyruvate carboxylase
in that biotin, a prosthetic group, is obligatory for the reaction as well as CO2 (bicarbonate).

acetyl CoA + CO2 + ATP → malonyl CoA + ADP + Pi acetyl CoA carboxylase

Fatty acid synthase is a dimeric enzyme consisting of 7 enzyme activities. Two of these activities are
responsible for attaching acetyl CoA and malonyl CoA to the complex to initiate the reaction and for
attaching malonyl CoA in subsequent steps to build the fatty acid molecule. A third activity, condensing
enzyme (CE), contains an acyl carrier protein (ACP) to which the growing fatty acid carbon chain is
attached (initially this is acetyl CoA) (Fig. 7). A second acyl carrier protein provides the site for
attachment of malonyl CoA, the source of 2-carbon units for the growing chain. The condensing enzyme
initially joins two carbons from the malonyl group with the two carbons of the acetyl group, This
condensation results in release of CO2 (the third malonyl carbon). The condensation product is a 4-carbon
intermediate (Fig. 7, row 1). Finally in a series of three reactions that use two molecules of NADPH
(reductase steps), a 4-carbon fatty acid is formed (Fig. 7, row 1).

The new 4-carbon unit now moves to the condensing enzyme site thus allowing a second molecule of
malonyl CoA to be attached for the next cycle (Fig. 7, row 2). The reaction then continues through five
more cycles that include attachment of five more molecules of malonyl CoA and condensation of the
growing acyl chain with these new malonyl groups.

The cycling ends when palmitate (16 carbons) is formed (Fig. 7, row 3). The palmitate is cleaved from the
condensing enzyme in a reaction catalyzed by a thioesterase (Fig. 7, row 3). A total of seven cycles are
required to form palmitate and thus oxidation of 14 molecules of NADPH (2 per cycle) occurs. Note that
the product of lipogenesis is the free fatty acid NOT the fatty acyl CoA form. The fatty acid must be
activated by acyl CoA synthetase, a reaction described before.

Overall reactions:

acetyl CoA + 7 malonyl CoA + 14 NADPH → palmitate + 7 CO2 + 8 CoA + 14 NADP

fatty acid synthase

palmitate + ATP + CoA → palmitoyl CoA + AMP + 2 Pi

acyl CoA synthetase

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-9

 reduction A
A condensation A CE C
C dehydration
CE CE C reduction acp P
acp P acp P
CO2
2 NADPH +
2 NADP
4-carbon
acetyl malonyl 4-carbon unit
CoA CoA unit
reduction A
A condensation A CE C
C C dehydration
CE CE reduction acp P
acp P acp P
CO2
2 NADPH 2 NADP+
6-carbon
4-carbon malonyl 6-carbon unit
unit CoA unit
A
5 more cycles thioesterase CE C
A A P
C adding 10 carbons C cleavage acp
CE CE
acp P acp P
palmitate

6-carbon malonyl 16-carbon unit

unit CoA (palmitate)

Figure 7. General mechanism for the fatty acid synthase reaction. CE is condensing enzyme. ACP is acyl
carrier protein. The first row is the initial steps for priming the reaction with acetyl CoA and the addition
of two carbons from malonyl CoA. The second row depicts a typical cycle of adding two more carbons to
the fatty acid chain. The final row shows the release of the finished product, palmitate, through cleavage
by thioesterase.

Sources of NADPH for Lipogenesis

Malic enzyme generates NADPH during lipogenesis when it processes the carbons from oxaloacetate, a
product of the citrate lyase reaction (Fig. 1). However malic enzyme provides only about half of the
NADPH required for lipogenesis. The remaining NADPH needed for the fatty acid synthase reaction
comes from the oxidative branch of the pentose phosphate pathway.

Esterification of Fatty Acids for Storage

The backbone of triacylglycerols and phospholipids (phosphoglycerides) is glycerol. Both glycerol 3-

phosphate and dihydroxyacetone phosphate are precursors for these pathways. The pathway in Fig. 8
depicts just the triacylglycerol pathway. Phosphatidic acid is an intermediate in the biosynthesis of both
molecules. The primary fatty acid esterified to triacylglycerols is palmitoyl CoA. These triacylglycerols
are then stored as a fuel for mobilization during starvation or stress. Unsaturated fatty acids are not stored
as triacylglycerols because their oxidation for energy is more complicated. In times of starvation or stress
the rapid mobilization of fats for fuels is essential for survival. Since palmitoyl CoA is the major product
of lipogenesis, it is the primary storage form of lipid fuel.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-10

In contrast phospholipids contain unsaturated (containing C=C double bonds) fatty acids. In the
membrane, phospholipids play roles other than for structural purposes. Phospholipases catalyze the
specific cleavage of side groups from phospholipids. We learned earlier that phospholipase C acts on
membrane phosphatidylinositol to generate the second messengers diacylglycerol and IP3. Additionally,
phospholipase A2 action releases arachidonic acid (see below). Snake venoms contain phospholipases and
destroy phospholipids in membrane of tissues they infiltrate, possibly causing tissue necrosis at the site of
the bite. When venoms attack inner mitochondrial membrane the large amounts of fatty acids released act
as natural uncouplers causing severe loss of the ability of the mitochondrion to produce energy.

Glycerol Dihydroxyacetone phosphate

ATP fatty acyl CoA

glycerol
kinase
ADP
Glycerol-3-P CoA
fatty acyl CoA Acyldihydroxyacetone phosphate
NADPH

CoA
Lysophosphatidic acid
NADP+
fatty acyl CoA

CoA
Phosphatidic acid Diacylglycerol
phosphatase fatty acyl CoA

CoA
Triacylglycerol

Figure 8. Formation of phosphatidic acid from glycerol-3-phosphate or dihydroxyacetone phosphate, and

its conversion to triacylglycerol.

Lipolysis, Beta Oxidation, Ketones & Lipogenesis-11