Download Lipids are biological molecules that are insoluble, or only sparingly

Metabolism of Triacylglycerol
John O. Thomas
Lipids are biological molecules that are insoluble or only sparingly soluble in water.
These include triacylglycerols (also known as triglycerides), phospholipids, cholesterol (often in
ester form), glycolipids, steroids, fat-soluble vitamins, and other compounds present in smaller
amounts. These lectures will deal primarily with the metabolism of
O
triacylglycerols. Cholesterol metabolism and steroid metabolism
||
O H2 C - O - C - R 1
will be presented more extensively in the third part of this course,
||
|
and phospholipids will be presented in the cell biology course.
R2 - C - O - CH
|
The body stores triacylglycerol for use as an energy source
H2 C - O - C - R 3
during times of starvation. A triacylglycerol is a glycerol esterified
||
O
with three fatty acids, the fatty acids being the energy-rich part of the
triacylglycerol. Triacylglycerols are highly insoluble in water, which
Triacylglycerol
is an advantage for storage since they can be stored without any
added weight from associated water.
Fatty acids consist of a hydrophobic hydrocarbon chain and a hydrophilic, terminal
carboxyl group (-COOH;), which is ionized (-COO-) at physiological pH. Fatty acids are
therefore amphipathic; that is, they have both hydrophobic and hydrophilic characteristics. Most
naturally occurring fatty acids have a hydrocarbon chain of 14 - 20 carbons (these are commonly
called long-chain fatty acids), and can be placed within one of three categories: saturated (no
double bonds), monounsaturated (one double bond), and polyunsaturated (more than one double
bond).
β-carbon
(carbon number 3)
ω-carbon
Palmitate
Carbon number 1
Palmitic acid
9
∆9 bond
Cis-palmitoleic acid (18:1∆ )
(an ω7 fatty acid)
ω7 carbon
Trans-palmitoleic acid
Figure 1 Fatty acid structures. The cis double bond of naturally occurring unsaturated fatty acids
places a kink in the molecule that lowers the melting point of triacylglycerols and increases the fluidity
of membranes. Trans-fatty acids have physical properties similar to saturated fatty acids.
1
Fatty acids have both chemical names and common names. The common names
are more frequently used in the medical literature and are listed in the following table.
Linoleic acid and linolenic acid are required compounds that can not be synthesized by the
body and must be taken in the diet. These are essential fatty acids. Arachidonic acid can be
synthesized from linoleic acid so it is not an essential fatty acid.
Number of carbons: Number
and positions of double bonds
Common name
Physiological importance
Saturated Fatty Acids
1:0
2:0
3:0
4:0
10:0
16:0
18:0
Formic
Acetic
Propionic
Butyric
Capric
Palmitic
Stearic
Important components of breast milk.
Major components of triacylglycerols and structural lipids.
Monounsatruated Fatty acids
16:1 ∆9 (ω7)
18:1 ∆9 (ω9)
Major components of triacylglycerols and structural lipids.
Palmitoleic
Oleic
Polyunsaturated Fatty acids
An omega-3
fatty acid
18:2 ∆9,12 (ω6)
18:3 ∆9,12,15 (ω3)
20:4 ∆5,8,11,14 (ω6)
Linoleic
Linolenic
Arachidonic
Essential fatty acids:
must be taken in diet.
Precursor of eicosinoids.
The symbols ∆ and ω refer to two different systems for denoting the positions of double
bonds in unsaturated fatty acids as illustrated in figure.1.
Nearly all naturally occurring unsaturated fatty acids have their double bonds in the cis
configuration. Chemically processed fats such as margarine or fats heated to high temperatures
contain a mixture of cis and trans double bonds. While the body is able to metabolize both cis
and trans double bonds, cis and trans fatty acids have different physical properties that may
affect their metabolism. Some epidemiological evidence correlates an increased intake of trans
as opposed to cis fatty acids with an increased risk of coronary artery disease. The following
table shows the fatty acid composition of some common foods.
Food:
% Saturated % Trans % Monounsat. %polyunsat.
Olive oil
16%
0.1%
74%
10%
Sunflower oil
13%
0.5%
17%
70%
Cheddar cheese
72%
2.6%
24%
4%
Margarine - tub
19%
16.9%
43%
38%
Margarine - stick
19%
31.8%
60%
20%
French fries (typical values)
29%
32.3%
62%
9%
2
Oxidative damage to polyunsaturated fatty acids (PUFA).
Lipids are easily oxidized. Oxidized lipids are dangerous. They are taken up by
macrophages, contributing to their transformation into foam cells. Foam cells are a major
factor in the development of atherosclerosis. Vitamins E and C are important anti oxidants
that, together with glutathione, protect against this damage.
I. A free radical (R·) can initiate a chain reaction that results in the formation of many molecules
of PUFA peroxides. The acronym PUFA refers to polyunsaturated fatty acid. PUFA
peroxides are highly reactive compounds that can cause further damage to lipids and cells.
R· is any free radical,
including a PUFA radical
R·
Chain reaction
RH
PUFA
O2
PUFA
PUFA radical
PUFA·
PUFA peroxide radical
PUFA preoxide
II. Vitamin E, Vitamin C, Glutathione and NADPH function together to limit oxidative damage
to PUFAs
2. Vitamin E, which is lipid soluble, reacts with
the PUFA radical. The vitamin E radical that is
formed is chemically stable
1. A dangerous PUFA
free radical is formed.
Dehydrovitamin C
PUFA·
Vitamin E
Vitamin C·
PUFA
Vitamin E·
Vitamin C
2 GSH
NADP+
G-S-S-G
NADPH
+
4. Glutathione reduces
the oxidized vitamin C.
3. Vitamin C picks up radicals
from two molecules of vitamin E.
Vitamin C is water soluble and
forms a stable radical.
5. Oxidized glutathione is reduced by NADPH obtained
from the pentose phosphate pathway.
III. Glutathione peroxidase detoxifies PUFA peroxides:
Glutathione
peroxidase
Glutathione
reductase
PUFA peroxide
2 GSH
NADP+
PUFA hydroxide
G-S-S-G
NADPH + H+
3
Overview of Tissues involved in Triacylglycerol Metabolism
FED STATE
Intestin
Glucose
Liver
Glycogen
Glucose
T.G. Synthesis
Monoacylglycerol
Triacylglycerol
(CM/VLDL)
Fatty acid
Muscle
Fatty acids
Use
Glycogen
T.G.(Small)
Adipose
Storage
STARVED STATE
Brain
Energy
Liver
Amino acids
(from muscle)
Glucose
Ketone Bodies
Energy
Muscle
Adipose
Energy
Fatty acids
Storage
OVERVIEW OF SUBSTRATE FLOW
Triacylglycerol metabolism in the liver
I. Fatty acid synthesis in the liver
Overview:
When a person is on a high carbohydrate diet, a major function of glycolysis in the liver
is to provide acetyl-CoA for fatty acid synthesis. Acetyl-CoA gives rise to malonyl-CoA,
which is the major source of carbons for fatty acid synthesis. Fatty acid synthesis also requires
a large amount of NADPH, some of which is derived from the pentose pathway, and some of
which is derived from the malic enzyme.
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A. Production of cytosolic acetyl CoA
Glycolysis, the pentose phosphate pathway and pyruvate dehydrogenase convert glucose
to acetyl-CoA that is located inside of mitochondria. Cytosolic acetyl-CoA is required for
fatty acid synthesis, but the mitochondrial acetyl-CoA can not pass through the mitochondrial
inner membrane and the inner mitochondrial membrane has no transporter for acetyl-CoA.
To obtain cytosolic acetyl-CoA, citrate leaves the mitochondria via the citrate-malate
transporter (Fig. 2), citrate gives rise to acetyl-CoA and malate in the cytosol, and malate then
re-enters the mitochondria via the citrate-malate transporter.
Glucose
Fatty acid
CoA
Pyruvate
ADP
+ Pi
Citrate
Acetyl-CoA
Citrate
Citrate synthase
CO2
ATP
CoA ATP
ADP + Pi
Acetyl-CoA
Citrate lyase
Citrate – malate
transporter
+
NAD+ NADH + H+
NADH + H+ NAD+
Oxaloacetate
Oxaloacetate
Malate
Malate dehydrogenase
(cytosolic)
Malate
Malate dehydrogenase
(mitochondrial)
Mitochondrial matrix
Cytosol
Inner membrane
Outer membrane
B. Formation of malonyl-CoA is the committed step of fatty acid synthesis.
Fatty acids are synthesized by the sequential addition of two-carbon units. These carbons are
derived from malonyl-CoA, a three carbon compound. In the process, one carbon of malonylCoA is lost as CO2, providing some of the energy required for fatty acid synthesis. The
concentration of malonyl-CoA in the cell limits the rate of fatty acid synthesis.
1. Malonyl-CoA is synthesized from cytosolic acetyl-CoA by acetyl-CoA carboxylase
(ACC). Like other carboxylases, acetyl-CoA carboxylase contains biotin as a cofactor.
2. Acetyl-CoA carboxylase is regulated such that fatty acid production is stimulated
when both blood glucose is high and the energy needs of the cell have been met.
a. Allosteric regulation:
i. Activated by citrate (a precursor of fatty acid synthesis
ii. Inhibited by palmitoyl-CoA (a product of fatty acid synthesis)
b. Phosphorylation inhibits the enzyme. Phosphorylation is by:
i. Protein kinase A (PKA; stimulated by cAMP via glucagon)
ii. AMP activated protein kinase (AMPK).
c. Transcriptional regulation: High carbohydrate diets increase the synthesis of
acyetyl-CoA carboxylase, snd other enzymes involved in fatty acid synthesis are
also increased (e.g. fatty acid synthase, ATP-citrate lyase, glucose-6-phosphate
5
dehydrogenase). The mechanism of this transcriptional regulation is described
below
Xylulose-5-phosphate
+
Insulin
H2O
P P
Phosphorylated
Acetyl CoA
carboxylase
(Inactive)
Acetyl-CoA
Transcription
Citrate
Palmitoyl-CoA
Pi
+
+
Protein phosphatase
PKA AMPK
+
ADP + Pi
Glucagon
+
ATP
Acetyl CoA
carboxylase
(Inactive)
AMP
─
CO2
Acetyl CoA
carboxylase
(Active)
ATP
ADP + Pi
Malonyl-CoA
Glucagon, AMP, Palmitoyl-CoA slow fatty acid synthesis
Insulin, citrate, xylulose-5-P speed up fatty acid synthesis
3. Malonyl CoA also functions as the primary regulator of fatty acid oxidation Thus,
fatty acid synthesis and fatty acid oxidation do not occur at the same time. There are two
isozymes of ACC.
a. ACC1 (also known as ACC-alpha) is located in cells that actively synthesize fatty
acids in the liver and lactating mammary gland.
b. ACC2 (also known as ACC-beta) is located in most cells. It synthesizes small
amounts of malonyl-CoA as a regulator of fatty acid oxidation.
C. Fatty acid synthesis requires reducing power in the form of NADPH + H+
1. Up to half of the required NADPH + H+ can be produced by the pentose phosphate
pathway. Because of the large amount of NADPH that is required, the pentose
phosphate pathway becomes a predominant pathway of glucose metabolism of the
liver when blood glucose is high. The amount of glucose-6-phosphate
dehydrogenase is increased in people who eat a diet rich in carbohydrate.
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2. The remainder of the needed NADPH is produced by the malic enzyme and a shuttle
system that transports malate out of the mitochondria and pyruvate back into the
mitochondria:
NADH + H+
NAD+
Oxaloacetate
Malate
H+ H2PO4
Phosphate
carrier
Pyruvate
Malate
dehydrogenase
HPO4
CO2 +
ATP
ADP + Pi
Pyruvate
carboxylase
Mitochondrial matrix
Dicarboxylate
carrier
H+
Pyruvate
carrier
Inner membrane
Outer membrane
CO2 +
H
+
H2PO4
HPO4
NADP
Malate
+
H+
NADPH + H+
Pyruvate
Malic enzyme
Malic enzyme
Cytosol
Net half reaction:
NADH + H+
(mitochondrion)
NADPH + H+
(cytosol)
ATP
ADP + Pi
D. Fatty acids are synthesized by a single multifunctional enzyme
Fatty acid synthesis is initiated using acetyl-CoA and proceeds by the sequential addition
of two carbon units derived from malonyl-CoA. Each two-carbon unit that is added is
reduced, dehydrated and reduced again. All of these reactions are catalyzed by a single
enzyme, fatty acid synthase. The fatty acid is released from the enzyme once it reaches a
length of 16 or 18 carbons, the primary product being the 16-carbon saturated fatty acid,
palmitic acid. The overall reaction is
Acetyl-CoA + 7 Malonyl-CoA + 14NADPH + 14H+
Fatty acid synthase
Palmitic acid + 7CO2 + 14 NADP+ + 8CoA + 6H2O
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E. Fatty acid activation, elongation and desaturation.
1. Activation of fatty acids: formation of acyl-CoAs
Metabolic reactions involving fatty acids require that the fatty acids be converted to
acyl-CoA. This is necessary for elongation and desaturation (described in this section),
for β-oxidation (described in section III), for triacylyglycerol synthesis and for
phospholipid and glycolipid synthesis. The thioester bond that joins the fatty acid and
the CoA is a high energy bond.
Fatty acid + CoA + ATP
Acyl-CoA + AMP + PPi
AcylCoA synthetase
O
||
R-C-OH
Fatty acid
O
||
R-C-S-CoA
acyl-CoA
A thioester
There are multiple isozymes of acyl-CoA synthase that differ in their subcellular location
and in their specificity for the chain length of the fatty acid.
a. The isozyme that is used for fatty acid biosynthetic reactions, including elongation
and desaturation. is located on the endoplasmic reticulum.
b. Other isozymes of acyl-CoA synthetase are involved in fatty acid oxidation (see
below) and are located on the mitochondrial outer membrane, and on the inside of the
mitochondrial inner membrane.
2. Elongation of fatty acids
a. Palmitic acid (16 carbons) produced by fatty acid synthase can be elongated in two
carbon units. Most tissues can elongate palmitate to produce 18 and 20 carbon fatty
acids.
b. Very long chain fatty acids (>20 carbons) are produced by neural tissues.for the
synthesis of phosphlipids and glycolipids.
3. Desaturation of fatty acids
Fatty acids can be desaturated by enzymes that insert double bonds between the
carbons 9 and 10 from the COOH end. Desaturation can also occur closer to the
COOH end, but not further away (hence linoleic acid and linolenic acid must be taken
in the diet).
F. Regulation of fatty acid synthesis
1. Pyruvate carboxylase is activated by acetyl-CoA thus providing an adequate supply
of oxaloacetate and hence citrate for transport to the cytosol.
2. The major point of regulation is acetyl-CoA carboxylase (see above).
3. The entire block of enzymes involved in fatty acid synthesis is regulated at the
transcriptional level, under control of a transcription factor termed carbohydrate
response element binding protein (ChREBP). ChREBP is inactivated through
phosphorylation by protein kinase A (via cAMP, which is elevated in response to
glucagon). ChREBP is activated by removal of the phosphate by protein
phosphatase2A (the same phosphatase that removes the phosphate from
phosphofructokinase 2). As discussed in the carbohydrate lectures, this phosphatase
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is stimulated by xyulose-5-phosphate, which is elevated under conditions of high
glucose availability (much of the glucose passes through the pentose phosphate
pathway in route to fatty acid synthesis).
II. Triacylglycerol production in the liver and export to the blood.
A. The synthesis of triacylglycerol is unregulated, and limited only by the availability of the
substrates: glycerol-3-phosphate and acyl-CoAs.
1. Glycerol-3-phosphate for triacylglycerol synthesis is obtained from dihydroxyacetone
phosphate, an intermediate of glycolysis.
NADH + H+
NAD+
Dihydroxyacetone phosphate
Glycerol-3-phosphate
Glycerol-3-phosphate dehydrogenase
H2C-OH
|
O=C O |
|
H2 C-O-P-O ||
O-
H2C-OH
|
HO-CH O |
|
H2 C-O-P-O ||
O-
Dihydroxyacetone phosphate
L-Glycerol 3-phosphate
2. In the liver, triacylglycerol is formed by the sequential addition of acyl-CoAs to
glycerol 3-phosphate (a different pathway is used in the intestine).
2 Acyl-CoA
Glycerol 3-phosphate
2 CoA
H2O
Phosphatidate
Pi
Acyl-CoA
Diacylglycerol
O
||
O H2C-O – C R1
||
|
R2 C –O -CH O |
|
H2 C-O- P-O ||
OPhosphatidate
O
||
O H2C-O – C R1
||
|
R2 C –O -CH
|
H2 C-OH
Diacylglycerol
CoA
Triacylglycerol
O
||
O H2C-O – C R1
||
|
R2 C –O -CH O
|
||
H2 C-O – C R3
Triacylglycerol
B. Export of triacylglycerol.
1. The liver exports lipids into the circulation in the form of Very Low Density
Lipoprotein (VLDL) particles. These particles contain triacylglycerol, cholesterol
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cholesterol ester, phospholipids, and one molecule of apolipoprotein B-100. Several
other proteins are also associated with the VLDL particle.
2. Apolipoprotein B-100 is a huge protein (4536 amino acids). As it is being
synthesized, the N-terminus forms a nucleus for the generation of the VLDL particle.
3. As the apolipoprotein B-100 protein continues to be synthesized, microsomal
triacylglycerol transfer protein (MTP) delivers triacylglycerol to be packaged by the
still growing peptide chain. MTP also assists in the further maturation of the VLDL.
4. The rate of synthesis of apolipoprotein B-100 limits the rate at which triacylglycerol
can be exported from the liver. If the rate of synthesis of triacylglycerol is faster
than apolipoprotien B-100 synthesis, triacylglycerol will accumulate, giving rise to a
fatty liver.
III. Fatty acid oxidation
A. The liver uses fatty acids as its primay energy source. Sources of the fatty acids that enter
the cytosol of the liver include:
Fasting State:
- free fatty acids released from adipose tissue
Fed State:
- free fatty acids released from chylomicrons by lipoprotein lipase located on
extrahepatic tissues (lipoprotein lipase is discussed below).
- fatty acids produced by the action of hepatic lipase on lipoproteins (primarily IDL
and LDL). Hepatic lipase is anchored to the outside of the cell. It is homologous in
structure and function to lipoprotein lipase, which is discussed below.
- chylomicron remnants and low density lipoproteins (LDL) that have been taken up
by the cell into lysosomes where triacylglycerol and phospholipids are hydrolyzed
by lysosomal enzymes.
The transport processes that get these molecules to the liver are discussed below.
Inside of the cell, fatty acids are associated with Fatty Acid Binding Protein (FABP),
which facilitates their transport into and within the cell.
B. Fatty acids that enter the cell are converted to their CoA thioesters by an acyl-CoA
synthetase located on the outer mitochondrial membrane.
Fatty acid + CoA + ATP
Acyl-CoA + AMP + PPi
AcylCoA synthetase
O
||
R-C-OH
Fatty acid
O
||
R-C-S-CoA
acyl-CoA
A thioester
C. Fatty acid oxidation takes place in the mitochondria. The rate of transport into the
mitochondria is the rate controlling step of fatty acid oxidation.
1. In order to be transported into the mitochondrion, acyl-CoA must first be converted to
acyl-carnitine. This occurs on the cytosolic side of the inner mitochondrial membrane,
and is catalyzed by carnitine acyltransferase I (CAT-I). CAT-I is the rate limiting step
in fatty acid oxidation. It is inhibited by malonyl CoA, the starting point of fatty acid
10
synthesis. This inhibition of CAT-I functions to prevent the oxidation of newly
synthesized fatty acids.
2. Acyl-carnitine is transported into the mitochondria.
3. Inside of the mitochondria acyl-carnitine is converted back to acyl-CoA. This is
catalyzed by carnitine acyl transferase II (CAT-II)
4. Carnitine is transported back to the cytosol. The carnitine carrier protein catalyzes an
exchange of acyl-carnitine and carnitine
Inhibited by malonyl-CoA
Outer mitochondrial membrane
CAT-I
Acyl-CoA
Acyl-carnitine
Carnitine
CoA
Mitochondrial membrane space
Carnitine carrier
protein
inner mitochondrial membrane
mitochondria
Carnitine
CoA
Acyl-CoA (mitochondria)
Acyl-carnitine (mitochondria)
CATII
D. The metabolic break down of acyl-CoA occurs in a series of steps that remove two
carbon units at a time. The two carbon units appear as acetyl-CoA. Important aspects
are summarized here. If you wish, you may refer to the text for the actual series of steps.
1. The first step in the removal of two carbons is catalyzed by acyl-CoA dehydrogenase.
This reaction is catalyzed by one of three enzymes, depending on the size of the acylCoA
- Long chain acyl-CoA dehydrogenase (LCAD)
- Medium chain acyl-CoA dehydrogenase (MCAD)
- Short chain acyl-CoA dehydrogenase (SCAD)
2. MCAD is of clinical importance since a deficiency of this enzyme can lead to sudden
infant death if left untreated, and mutations affecting this gene are relatively common.
The pattern of inheritance is autosomal recessive. Several states, including New York,
routinely screen newborns for this deficiency. The Baby Ian tutorial available from the
MGB web site focuses on this disorder. Like succinate dehydrogenase, this enzyme
utilizes FAD as a hydrogen acceptor
3. The overall oxidation of palmitoyl-CoA can be summarized as:
Lots of energy in
these hydrogens
Palmitoyl-CoA + 7 FAD + 7 NAD+ + 7CoA + 7H2O →
8 Acetyl-CoA + 7FADH2 + 7NADH + 7 H+
11
4. The FADH2 and NADH + H+ are oxidized by the electron transport chain to ultimately
yield ATP and water. The acetyl-CoA can have a number of fates including oxidation
via the TCA cycle. When all of these reactions proceed to completion, over 100
molecules of ATP are produced per palmitic acid oxidized.
E. Regulation
1. CAT-1 inhibition by malonyl-CoA is the major short term point of control for fatty acid
oxidation in the liver as well as in other tissues. Malonyl-CoA concentration is
determined by the availability of citrate and the activity of the highly regulated enzyme
acetyl-CoA carboxylase.
2. In the liver, the genes involved in fatty acid oxidation are regulated at the level of
transcription. A major regulator is PPARα (Peroxisome Proliferator-Activated
Receptor alpha). This is a member of the nuclear receptor family of transcription
factors, and works by the same mechanism as the estrogen receptor (described earlier in
the course). PPARα is stimulated by a number of lipids, and when stimulated it
increases the transcription of target genes including the fatty acid binding protein and
enzymes of fatty acid oxidation. Refer to the estrogen receptor for a model of the
mechanism of activation.
3. The fibrate class of lipid lowering drugs function by activating PPARα.
IV. Propionic acid metabolism
A. Propionyl-CoA is a product of branched-chain amino acid metabolism and the
metabolism of fatty acids that contain an odd number of carbons. The amount of
propionyl-CoA produced is significant and under some conditions medically important.
While we have elected to present propionyl-CoA metabolism at this point in the
Molecules to Cells module, it is important to note that the majority of the propionyl-CoA
that is produced is derived from amino acid metabolism rather than odd-chain-length fatty
acid metabolism.
B. Propionic acid is the end product of the metabolism of fatty acids that have an odd number
of carbons. When these compounds are metabolized by the beta-oxidation pathway
described above, propionyl-CoA is the last product rather than acetyl-CoA.
C. Propionyl-CoA is converted into succinyl-CoA in a series of reactions. In the first
reaction, carbon dioxide is added. This is catalyzed by a biotin-dependent enzyme
(propionyl-CoA carboxylase). The product, D-methylmalonyl-CoA is converted to the L
isomer, then an intramolecular rearrangement (catalyzed by methylmalonyl-CoA mutase)
produces succinyl-CoA.
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HCO3 - + ATP
ADP + Pi
Propionyl-CoA
D-methylmalonyl-CoA
Propionyl-CoA carboxylase
Requirement
for biotin
Propionyl-CoA
L-methylmalonyl-CoA
Methylmalonyl-CoA
mutase
Requirement for
vitamin B12
Succinyl-CoA
Methymalonyl-CoA
Succinyl-CoA
D. Methylmalonyl-CoA mutase requires vitamin B12. If not treated, a lack of vitamin B12
will cause irreversible neural damage due to the build up of toxic byproducts of
propionyl-CoA metabolism. Propionyl-CoA metabolism is one of two places where
vitamin B12 is required. The other, methionine synthase, will be presented in the amino
acid lectures.
E. Vitamin B12
1. The symptoms of vitamin B12 deficiency are related to the two reactions for which it is
required.
a. Megaloblastic anemia (pernicious anemia) results from a block of folate
metabolism and will be discussed in the amino acid metabolism lecture. These are
the first symptoms that appear and can be reversed by administering B12.
b. Methylmalonic acidosis and resulting irreversible neural damage are long term
consequences of blocking methylmalonyl-CoA mutase
2. Vitamin B12 is present in bacteria and animal products, but not vegetables.
Consequently, strict vegetarians are at risk for vitamin B12 deficiency, although the
deficiency requires about a decade to develop due to the normally large stores
maintained in the body.
3. Vitamin B12 deficiency is most often due to a problem with absorption of the vitamin
rather than a lack of dietary intake. The absorption of B12 is dependent on the presence
of “intrinsic factor”, a protein that is secreted by the parietal cells of the stomach. Loss
of the parietal cells by autoimmune mechanisms is the most common cause of B12
deficiency. This deficiency can be treated by periodic injections of B12.
V. Glycerol utilization
A. Glycerol is released into the blood by the hydrolysis of triacylglycerol (see below under
adipose tissue). The glycerol is taken up by the liver and converted to glycerol-3-
13
phosphate which is oxidized to give dihydroxyacetone phosphate, an intermediate of both
gluconeogenesis and glycolysis.
B. Adipose tissue can not use glycerol since it lacks glycerol kinase.
ATP
NAD+
ADP + Pi
Glycerol
Glycerol-3-phosphate
NADH + H+
Dihydroxyacetone phosphate
Glycerol kinase
Gluconeogenesis or Glycolysis
Not present in adipose
VI. Ketone body synthesis: the liver is the major producer of ketone bodies, but it can not
use them.
A. During the fasting state, the production of acetyl-CoA exceeds the capacity of the TCA
cycle to use it. This is because the liver derives most of its energy from the FADH2 and
NADH produced by fatty acid oxidation. The excess acetyl-CoA is converted to
acetoacetate and β-hydroxybuyrate which are exported to other tissues for use.
B. Synthesis of acetoacetate and β-hydroxybutyrate
CoA
2 Acetyl-CoA
H2O + Acetyl-CoA
Acetoacetyl-CoA
CoA
Acetyl-CoA
HMG-CoA
Acetoacetate
NADH + H+
Acetone is a by product:
NADH + H+
CO2
Acetoacetate
NAD+
Acetone
NAD+
(Not catalyzed)
β-hydroxybutyrate
O
||
CH3-C-S-CoA
Acetyl-CoA
O
O
||
||
CH3-C- CH2-C-S-CoA
Acetoacetyl-CoA
O
OH O
||
|
||
O-C-CH2-C-CH2-C-S-CoA
|
CH3
HMG-CoA
(3-hydroxy-3-methyl-glutaryl-CoA)
14
O
O
||
||
CH3-C- CH2-C-O –
Acetoacetate
OH
O
|
||
CH3-CH-CH2-C-O –
β-hydroxybutyrate
C. Synthesis of acetoacetate and β-hydroxybutyrate
D. HMG-CoA that is produced in this pathway is in the mitochondria. There is a distinct
pool of HMG-CoA that is present in the cytosol, which is used for the synthesis of
cholesterol.
E. The liver is the major source of ketone bodies Heart and renal cortex use large amounts
of ketone bodies, and the brain will adapt to the utilization of ketone bodies during prolonged
starvation. The liver is unable to utilize ketone bodies since it does not have the enzyme
required to convert acetoacetate back to acetoacetyl-CoA (see below under Muscle), the first
step in its utilization.
F. Acetoacetate and β-hydroxybutyrate are moderately strong acids. The release of large
quantities into the blood can cause a significant drop in blood pH. Under pathological
conditions, such as uncontrolled diabetes, this acidosis can be life threatening.
G. Acetoacetate is converted to acetone by a non-enzymatic chemical reaction. The small
amounts of acetone produced as not physiologically important, but are noticeable on the
breath of a diabetic in ketoacidosis.
Adipose Tissue
I. Triacylglycerol uptake and storage: Triacylglycerols are broken down outside of the
cell, fatty acids are taken up by the cell and then resynthesized into triacylglycerol
A. Lipoprotein lipase
1. Reaction: hydrolysis of the glycerol – fatty acid ester bonds of triacylglycerols found
in VLDL and chylomicron particles.
2. Located on the surface of capillaries. Lipoprotein lipase is synthesized by adipose
cells (as well as muscle cells and other fatty acid-utilizing tissues). It is exported
from the cell and binds non-covalently to heparin sulfate located on the outside of
endothelial cells.
3. The Km of the adipose isoform of lipoprotein lipase is higher than the lipoprotein lipase
that is present in other tissues. Thus, when triacylglycerol is abundant it is stored in
adipose tissue; when it is scarce, it is used by other tissues
4. Regulation: The synthesis of adipose lipoprotein lipase is increased by the action of
insulin.
Triacylglycerol
Lipoprotein lipase
adipocyte
Gycerol
LL
Fatty acids
Capillary
Capillary wall
15
B. Resynthesis of triacylglycerol for storage in the adipose cell
1. Triacylglycerol is formed from acyl-CoA and glycerol-3-phosphate by the same series
of reactions as in the liver (see above).
2. Adipose does not contain glycerol kinase. Hence, adipose can not reuse glycerol; it
must manufacture new glycerol-3-phosphate
3. Availability of glycerol-3-phosphate is limiting. Glycerol-3-phosphate is derived from
dihydroxyacetone, an intermediate of glycolysis. In adipose tissue, the rate of
glycolysis, and hence availability of dihydroxyacetone phosphate, is limited by
GLUT-4. As with the muscle, insulin causes intrecellualar membrane stores of
GLUT-4, to move to the plasma membrane. Hence, insulin stimulates triacylglycerol
synthesis and storage by the adipocyte.
4. Fatty acid acids are activated to acyl-CoAs by acyl-CoA syntetase as described above.
Fatty acid + CoA + ATP
Acyl-CoA + AMP + PPi
Acyl-CoA synthetase
II. Triacylglycerol mobilization from adipose tissue.
A. The first, and most highly regulated enzyme is hormone sensitive lipase.
Triacylglycerol + H2O
2,3-Diacylglycerol + fatty acid
Hormone sensitive lipase
Highly regulated
B. Hormone sensitive lipase is inactivated by the action of insulin and activated by
phosphorylation by protein kinase A.
1. Inactivation by insulin is probably the most important level of control.
2. Activation by protein kinase A (PKA) catalyzed phosphorylation is also important.
The primary activators of PKA in adipose are catacholamines (epinephrine and
norepinepherine) acting through β-adrenergic receptors.
3. The 2,3-diacylglycerol formed by hormone sensitive lipase is further hydrolyzed to
fatty acids and glycerol by other unregulated lipases.
4. The net products of triacylglycerol mobilization are free fatty acids and glycerol, both
of which are released into the blood.
5. In the blood, free fatty acids are transported as a complex with serum albumin. Other
tissues (but not brain or red blood cells) use the free fatty acids for the production of
energy. The liver uses these for both energy production as well as ketone body
production.
C. Regulation
1. The primary regulator of triacylglycerol metabolism in adipose is insulin.
a. Insulin increases storage of triacylglycerol by glucose entry into adipocytes and in
the long term by increasing lipoprotein lipase levels.
b. Insulin inactivates triacylglycerol mobilization by hormone sensitive lipase.
16
2. The development of adipocytes and the regulation of their enzymes is controlled to a
large extent by the activity of PPARγ, which is analogous to PPARα, (discussed
above in the section on liver. Activation of PPARγ by fatty acids stimulates the
synthesis of enzymes involved in glucose uptake, fatty acid uptake and storage. It
also stimulates the production of several hormones that are released by adipocytes.
3. The thiazolidinedione class of insulin-sensitizing drugs used for treating type II
diabetes function by stimulating PPARγ.
Insulin
Noepinepherine
Epinepherine
+
-
H2O
PPi
ATP
cAMP
Adenylyl cyclase
+
Pi
AMP
phosphodiesterase
ATP
+
ADP
Triacylglycerol
Protein kinase A
P
Hormone sensitive lipase
Hormone sensitive lipase
(inactive)
(active)
Lipase phosphatase
H2O
Pi
Insulin
17
Fatty
acid
Diacylglycerol
Other
lipases
+
Adipocyte
H2O
2 Fatty acid
+
Glycerol
A
L
B
U
M
I
N
Muscle
I. Muscle can utilize fatty acids as fuel.
A. Albumin-bound fatty acids mobilized from adipose tissue can be taken up from the
circulation.
B. Fatty acids in chylomicrons and VLDL are released by hydrolysis of the triacylglycerol
This hydrolysis is catalyzed by lipoprotein lipase bound to the outside of capillary
endothelial cells (see above section on adipose)
C. Once taken up, the fatty acids are imported into the mitochondria and metabolized by
beta-oxidation as described for the liver (see above section on liver III.A).
D. Regulation
1. The amount of fatty acid that can be utilized is regulated by the rate of entry of fatty
acids into the mitochondria. As in the liver, CAT-I is inhibited by malonyl-CoA (see
above section on liver III.C). Muscle has a small amount of acetyl-CoA carboxylase
that functions to synthesize regulatory amounts of malonyl-CoA. Muscle does not use
the malonyl-CoA for fatty acid synthesis. As in liver, muscle acetyl-CoA carboxylase
is stimulated by citrate. Therefore, when glucose is abundant, citrate increases,
malonyl-CoA increases and oxidation of fatty acids is inhibited.
2. PPARα, (see Liver section V.E.3) stimulates the synthesis of the muscle enzymes
required for lipid metabolism in response to increased concentration of a number of
lipids.
E. Triacylglycerol. Muscle contains a small amount of triacylglycerol that can be used as an
immediate energy source. The regulation of muscle triacylglycerol storage and used is
poorly understood, but of potential importance for the field of sports medicine.
II. Muscle imports and metabolizes ketone bodies
A. The reactions:
NAD+
β-hydroxybutyrate
NADH + H+
Succinyl-CoA
Acetoacetate
This enzyme is missing in liver.
Hence liver produces, but does not
use ketone bodies
18
Succinate
Acetoacetyl-CoA
CoA transferase
CoA
2 Acetyl-CoA
Brain and Red Blood Cells
I. Red blood cells lack mitochondria and therefore can not metabolize either fatty acids or
ketone bodies. They are dependent on glucose for their energy needs.
II. The brain normally uses glucose, but during prolonged starvation it can use ketone bodies
for a considerable amount (but not all) of its energy needs. Neither triacylglycerols nor
fatty acids are used by the brain for the production of energy.
Intestine
+
2-Monoacylglycerol
Triacylglycerol
Fatty acids
1,2,3
Other lipids
4
Bile Salts
(from liver)
Jejunum
Bile Salts
(to ileum)
Mixed micelles
5
Enterocyte
Other lipids
6
2-Monoacylglycerol
Fatty acyl CoA
AMP + PPi
Enzymes:
1. acid-stable lipase (mouth)
2. pancreatic lipase (pancreas)
3. colipase
4. phospholipase A2/other lipases
5. intestinal fatty acid binding protein
(required for transport to the
endoplasmic reticulum)
6. acyl-CoA synthase
7. acyltransferase
8. microsomal transfer protein
Fatty acid
ATP
7
CoA-SH
Triacylglycerol
Apolipoprotein B48
phospholipid, cholesterol
8
Apolipoprotein A1
Chylomicron
Apolipoproteins C, E
Lymph
To tissues via lymph
19
I. Digestion
A. Digestion begins in the mouth with the action of lingual lipase. The free fatty acids,
together with phospholipids present in food emulsify the fat into small droplets.
B. When the gastric contents are emptied into the duodenum, the presence of lipids and
proteins stimulates the release of the peptide hormone cholecystokinin from the lower
duodenum and jejunum. This hormone stimulates the gall bladder to contract and release
gall (micelles of bile salts, phospholipids and cholesterol) into the duodenum. The gall
acts on the partially emulsified lipids from the stomach to form smaller mixed micelles.
Cholecystokinin also stimulates the release of digestive enzymes from the exocrine cells
of the pancreas. These enzymes include several that work at the surfaces of the mixed
micells to remove fatty acids from various lipids.
C. Pancreatic lipase (not to be confused with lipoprotein lipase or hormone sensitive lipase)
catalyzes the hydrolysis of fatty acids from positions 1 and 3 of triacylglycerols.
1. Pancreatic lipase requires the protein colipase which is also released from the pancreas.
Colpiase anchors the lipase to the surfaces of the miced micelles where digestion takes
place the protein.
2. Pancreatic lipase is inhibited by the weight loss drug, orlistat At the recommended
doses, Orlistat blocks about a third of dietary triacylglycerol from being digested
D. The resulting fatty acids and 2-monoacylglycerol are taken up by the enterocytes.
II. Resynthesis: Fatty acids and 2-monoacylglycerol enter the intestinal mucosal cells
(entereocytes) where they are resynthesized into triacylglycerols.
A. Fatty acids are activated by conversion to acyl-CoA by acyl-CoA synthetase. This is the
same reaction described above for the liver.
Fatty acid + CoA + ATP
Acyl-CoA + AMP + PPi
Acyl-CoA synthetase
B. The acyl-CoA combines with the 2-monoacylglyceride to reform triacylglycerol:
O CH2-OH
||
|
R-C-O-CH
2 Acyl-CoA + 2-Monoacylglycerol
|
CH2-OH
2-monoaycl-CoA
20
Triacylglycerol + 2 CoA
C. Triacylglycerol is exported from the intestine in combination with apolipoprotein B-48,
phospholipids, cholesterol, cholesterol ester, and other apolipoproteins and lipids in the
form of chylomicrons.
1. Apolipoprotien B-48 is a truncated form of apolipoprotein B-100 (B-100 is produced
in the liver and found in VLDL).
2. The modification that gives rise to B-48 as opposed to B-100 occurs at the level of
RNA editing. In intestinal cells, the mRNA sequence is modified so that an amino
acid codon is converted to a stop codon.
3. The process of chylomicron formation is analogous to the formation of VLDL in the
liver (see above; Liver I.E.3)
4. Chylomicrons enter the lymphatic system for distribution to the tissues After
depletion of lipids, the chylomicron remnants are taken up by the liver (see below).
D. Chylomicrons that have been depleted of their triacylglycerol (chylomicron remnants) are
taken up by the liver.
Lipid transport
I. Overview. Lipids are transported between tissues as complexes with carrier proteins or as
components of lipoprotein complexes.
A. Free fatty acids are transported in the blood bound to serum albumin, the major protein
present in the serum.
B. There are three major lipid transport pathways
1. Chylomicrons are released from the intestine and carry one molecule of apolipoprotein
B-48, dietary triacylglycerol, cholesterol ester, phospholipids and other water
insoluble compounds. They mature in the blood through the addition of other
apolipoproteins, notably apolipoproteins C and E. As a chylomicron passes through
the tissues, the triacylglycerol is depleted through the action of lipoprotein lipase,
resulting in a Chylomicron remnant. This is taken up by the liver.
2. Very Low Density Lipoproteins (VLDL) are released from the liver, and carry one
molecule of apolipoprotein B-100, newly synthesized triacylglycerol, cholesrterol,
cholesterol ester and phospholipid. They mature in the blood through the addition of
other apolipoproteins, notably apolipoproteins C and E. In the blood the VLDL loses
triacylglycerol to the tissues and gains cholesterol ester, progressing to Intermediate
Density Lipoprotein (IDL) and then Low Density Lipoprotein (LDL). The LDL is
taken up predominantly by the liver although all tissues are capable of taking up some
of it.
3. High Density Lipoproteins (HDL) are released from the liver and intestine, and
mature in the blood through the addition of other proteins and cholesterol which is
picked up from the tissues. HDL functions primarily as a carrier of cholesterol, which
it picks up from the tissues, and converts to cholesterol ester. The cholesterol ester is
then donated to IDL and LDL.
21
C. Characteristics of the lipoproteins
1. Physical properties and composition (TG - triacylglycerol; PL - phospholipid; ChE cholesterol ester; the predominant compound(s) transported are in bold)
Class
Diameter (nm)
% Protein % TG %PL %ChE
Chylomicron 100-1000
1-2
8
3
86
VLDL
30-80
6-10
18
13
55
IDL
25-30
15-20
21
25
28
LDL
20-25
22
9
20
40
HDL
5-10
35-50
5
30
15
2. Functional properties (TG - triacylglycerol; ChE - cholesterol ester)
Class
Source
Apolipoproteins Transports:
Fate
Chylomicron intestine
B-48, C, E, A
Dietary TG
TG to tissues, remnant to liver
VLDL
liver
B-100, C, E
New TG from liver TG to tissues/ becomes IDL
IDL
VLDL
B-100, E
New TG/
TG to tissues/ ChE from HDL/
ChE from HDL
becomes LDL
LDL
IDL
B-100
ChE to liver
uptake by liver
HDL
liver/
A-I, A-II, C, E
Cholesterol from
ChE to IDL/eventual
intestine
tissues/ ChE to IDL uptake by liver
3. Functional properties of some of the apolipoproteins
Apolipoprotein Source
Lipoproteins
Function
A-I, A-II
liver, intestine HDL, chylomicrons Structural protein of HDL
B-48
intestine
Chylomicrons
Structural protein of chylomicron
B-100
liver
VLDL, IDL, LDL
Structural protein of VLDL, IDL, LDL
C-I, C-II, C-III
liver
VLDL, IDL HDL,
Transferred between different classes
CM
C-II activates lipoprotein lipase
E
liver
HDL., VLDL, IDL, Mediates uptake of chylomicron remnants
CM remnants
and IDL by liver
II. Transport by chylomicrons
A. Chylomicrons are formed in the intestine with apolipoprotein B-48 as the key structural
protein. Apolipoproteins C and E are transferred to chylomicrons in the blood by HDL.
B. In addition to triacylglycerol, chylomicrons transport other dietary lipids and fat soluble
vitamins.
C. Triacylglycerol is depleted from chylomicrons by the action of lipoprotein lipase lipase in
adipose tissue, skeletal muscle, heart, and other tissues. Lipoprotiein CII stimulates this
process. Apolipoproteins C and A are transferred to HDL leaving the chylomicron
remnant, which contains aploliproteins B-48 and E.
D. Chylomicron remnants are taken up by the liver chylomicron remnant receptor (also
known as LRP for LDL-receptor Related Protein). Processing by the liver is as for LDL
described below.
22
Intestine
B-48
TG
PL, Ch, ChE
A
C
HDL
E
A
Chylomicron
B-48
TG
PL, Ch, CE
E
B-48
B-48
Liver
cell
TG
PL, Ch, ChE
TG
PL, Ch, ChE
E
E
LRP
C
A
C
A
Chylomicron
remnant
C
A
MG + FA
Lipoprotein
Lipase
E
n
d
o
t
h
e
l
i
a
l
C
e
l
l
HDL
III. Transport by VLDL, IDL, LDL
A. VLDL is formed in the liver with apolipoprotein B-100 as the key structural protein.
Apolipoproteins C and E are transferred to VLDL from HDL in the blood.
B. Triacylglycerol is depleted from VLDL by the action of lipoprotein lipase in adipose
tissue, skeletal muscle, heart, and other tissues. Lipoprotein CII stimulates this process..
C. Considerable remodeling of the VLDL takes place to produce IDL and LDL. This
includes the transfer of cholesterol ester from HDL (see below), the transfer of
apolipoproteins C to HDL, and hydrolysis of excess triacylglycerol and phospholipid by
hepatic lipase. Hepatic lipase is analogous to the lipoprotein lipase found in other tissues,
but it does not hydrolyze triacylglycerol present in chylomicrons or VLDL.
D. LDL is taken up by liver via the LDL receptor.. After binding to the receptor, the LDL is
taken into the cell by endocytosis where it is digested by lysosomal enzymes (this process
will be covered in detail in the cell biology course). In the lysosome, cholesterol esterase
hydrolyses cholesterol ester to cholesterol which then enters the cholesterol pool of the
liver cell. Phospholipids are also hydrolyzed and the B-100 protein is digested to amino
acids.
23
Liver
B-100
TG
C
PL, Ch, ChE
E
FA
HDL +
glycerol
VLDL
Liver
cell
B-100
E
TG
B-100
IDL
V
PL, Ch, ChE
TG
PL, Ch, ChE
E
C
E
Liver
B-100
ChE
TG
PL, Ch, ChE
B-100
ChE
E
TG
C
PL, Ch, ChE
LDL
E
Bile
S Salts
MG + FA
C
Ch
E
n
d
o
t
h
e
l
i
a
l
C
e
l
l
HDL
LDL receptor
FA
+
glcerol
V
Fatty Acids
ChE
LDL receptor
V
Ch
Tissues
E. Most of the cholesterol of the liver is exported to other cells in the form of VLDL (largely
after being converted to cholesterol ester) or is used for the synthesis of bile, which
consists of cholesterol, phospholipids and bile salts. Bile salts are derived from
cholesterol. The synthesis and entero-hepatic circulation of bile will be discussed later in
the course.
IV. Overview of cholesterol metabolism
A. Cells obtain cholesterol from a combination of endocytosis mediated by the LDL receptor
and de novo synthesis. Although nearly all cells can synthesize cholesrterol, most of the
body’s cholesterol is produced by the liver; on a typical western diet about a third comes
from food. The synthesis of cholesterol will be discussed later in the course.
Cholesterol is stored within liver cells and some other cells that are active in cholesterol
metabolism (such as the adrenal cortex) as cholesterol ester. This is formed by the action
of ACAT (acyl-CoA-cholesterol acyl transferase). Free cholesterol induces the enzyme.
24
Acyl-CoA
CoA
ACAT
Cholesterol
Cholesterol ester
B. Cholesterol is lost from the body through the death of intestinal cells and through the
inadvertent loss of bile.
C. Regulation of intracellular cholesterol levels:
1. Free cholesterol represses the de novo synthesis pathway by inhibiting the rate limiting
step catalyzed by HMG-CoA reductase (this enzyme will be discussed later in the
course).
2. Free cholesterol represses cholesterol uptake from the blood by repressing the
synthesis of the LDL receptor and promoting the degradation of the LDL receptor.
D. Familial hypercholesterolemia is due to a severe mutation in the LDL receptor gene.
Heterozygotes have half the normal ability to synthesize LDL receptors. The decreased
amount of receptors leads to an increase in the amount of cholesterol in the blood by two
mechanisms.
1. Low number of LDL receptors leads to low uptake of cholesterol into cells..
2. This leads to low intracellualar cholesterol.
3. This stimulates cholesterol synthesis.
4. Additional cholesterol is exported from the liver
5. Blood cholesterol is increased.
E. Macrophages remove LDLs that contain oxidized lipids via lipoprotein receptors that are
only are only marginally affected by cholesterol concentrations. Since these LDLs also
contain cholesterol ester, macrophages can accumulate large amounts of cholesterol when
the blood cholesterol is high. This accumulation leads to the progression of macrophages
to lipid rich foam cells; an important event in atheroschlerosis.
F. Excess cholesterol is eliminated from cells by an ATP dependent transport pathway
catalyzed by the ATP Binding Cassette A1 (ABC-A1) transporter. Homozygotes who
lack this protein have Tangier Disease.
V. Transport by HDL
A. HDL functions primarily in the transport of cholesterol between tissues, the intestine and
the liver.
B. Apolipoprotein A, which forms the nascent HDL particle, is released by the liver and
apolipoprotiens C and E quickly join. Aplolipoprotein A is also released by the intestine.
In the blood the nascent HDL acquires two key enzymes: Cholesterol Ester Transfer
Protein (CETP) and lecithin-cholesterol acyl transferase (LCAT).
C. The HDL particles complete their maturation by picking up cholesterol exported from
cells by the ABC-A1 transporter. The cholesterol is rapidly converted to cholesterol ester
by LCAT.
25
D. The HDL particle then donates the cholesterol ester to LDL particles in exchange for
lecithin. The process is mediated by CETP.
E. The LDL particles that are now enriched with cholesterol ester, are taken up by liver cells.
F. Triacylglycerol and phospholipid acquired by HDL is removed by hepatic lipoprotein
lipase.
G. HDL particles are also metabolized by the liver through the Scavenger Receptor B1
(SRB-1). This receptor binds the HDL, removes most of the cholesterol ester then
releases a greatly depleted HDL. The cholesterol ester is taken up by the liver.
A
PL, Ch
Intestine
C
E
A
PL, Ch
Tissues
Liver
hepatic lipase
E
A
TG
C
PL, ChE
LCAT
ABC-A-1
transporter
Ch
Ch
CETP
ChE
HDL
Ch
Bile
S Salts
SR-B-I receptor
Liver
cell
ChE ChE
TG
ChE
LDL
LDL
IDL
LDL receptor
26
LDL
receptor