Biochemistry 460 - Dr.

Tischler
LIPID TRANSPORT

Related Reading: Chapter 22: 619-620; Chapter 26: 745-748 in Stryer 6th edition

OBJECTIVES:

1. Describe the five steps of digestion and absorption of lipids including the important enzymes
and location where each step occurs.
2. Describe the general structure of a lipoprotein
3. Name the 4 main classes of lipoproteins and the major lipid component(s) and apoproteins of
each including the function of the apoprotein..
4. Describe the lipid-transport functions of each lipoprotein.
5. Discuss the function of lipoprotein lipase.
6. Compare and contrast the steps in the processing of chylomicrons and of VLDL.
7. Describe how defects in the LDL receptor are associated with familial hypercholesterolemia
and atherosclerosis.

PHYSIOLOGICAL PREMISE:
Atherosclerosis is a major cause of death in this country. Excess cholesterol in the blood accumulates on
the walls of blood vessels which provide oxygen to the heart. This accumulation causes the formation of
plaques. As the plaque increases in size, it occludes the blood vessel reducing oxygen delivery to the
affected part of the heart. Eventually no oxygen is delivered to the cells making them ischemic. The
ischemia causes cells to die resulting potentially in a myocardial infarction. In bypass surgery, blood
vessels, obtained from extremities, are attached to "bypass" the area of occluded blood vessel.

LIPID DIGESTION AND ABSORPTION:

Dietary Lipids: saturated fatty acid: CH3-(CH2)n-COOH

unsaturated fatty acid: CH3-CH=CH-(CH2)n-COOH
polyunsaturated fatty acid: CH3-CH=CH-CH2-CH=CH-(CH2)n-COOH

CH2----OOC-R1 CH2OH HOOC-R1
 | |
R2-COO----CH Î CHOH HOOC-R2
|  |
CH2----OOC-R3 CH2OH HOOC-R3
Triacylglycerol Glycerol Fatty acids

Figure 1. General structures of fatty acids and triacylglycerol. Lipolysis of stored triacylglycerol by
lipases produces fatty acids plus glycerol can occur in intestinal lumen, fat cells, muscle, liver, and
kidney.

Lipid Transport -1
On average, fat (lipid) makes up 37% of the calories in the American diet. Fat is energy rich and provides
9 kcal/gm. Dietary lipids are primarily (90%) triacylglycerols (Fig. 1) but also include cholesterol
esters, phospholipids, essential unsaturated fatty acids and fat-soluble vitamins (A, D, E, K). In
normal individuals, essentially all (98%) of the fat consumed in the diet is absorbed, and most is
transported to adipose for storage. Triacylglycerols in the diet are hydrolyzed in the intestine by lipases
released from the pancreas, but are then reassembled in the intestinal cells. The poor water solubility of
lipids presents problems for digestion because the substrates are not easily accessible to the digestive
enzymes in the aqueous phase, and the lipolytic products tend to aggregate to larger complexes that make
poor contact with the cell surface and therefore are not easily absorbed. This latter problem is overcome
by "solubilization" of the lipid products with bile acids. Aside from the solubility aspects, the general
principle of dietary lipid assimilation is that of hydrolyzing large non-absorbable molecules into smaller
units. Dietary fats are transported to tissues as triacylglycerol or cholesterol via chylomicrons. On arrival
at peripheral tissues (e.g., adipose or muscle), the fatty acids are removed from the triacylglycerol by a
lipase (lipoprotein lipase) located in the walls of capillaries, and the released fatty acids diffuse into the
cell.

Steps of lipid digestion/absorption (Fig. 2/Table 1)

Step 1: Minor digestion of triacylglycerols begins in the mouth by the action of the enzyme lingual
lipase followed by gastric lipase that is acid-stable and hence is functional even in the acidic
environment of the stomach. These lipases remove one of the three fatty acids leaving diacylglycerol and
free fatty acid as a product.
triacylglycerol → fatty acid + diacylglycerol (lingual or gastric lipase)

Step 2: The major digestion of all lipids occurs in the lumen of the small intestines (specifically in the
duodenum and jejunem). The pancreas releases into the intestine lipases to carry out this process. The
major enzyme in this process is pancreatic lipase. Pancreatic lipase removes the fatty acids from carbons
one and three of the glycerol backbone. Pancreatic lipase requires for its activity colipase, a small protein
that binds to both the water-lipid interface and to lipase, to anchor and activate the enzyme. The
pancreatic secretion also includes cholesterol esterase, which removes the ester portion of dietary
cholesterol ester to facilitate its uptake across the intestinal wall. Another minor component of the dietary
lipids are phospholipids derived from cell membranes. These are processed by the pancreatic enzyme
phospholipase A2. The enzyme removes a fatty acid from carbon 2 of the glycerol backbone leaving a
fatty acid on carbon 1 and the polar headgroup on carbon 3 (lysophospholipid).
triacylglycerol → 2 fatty acids + monoacylglycerol (pancreatic lipase)
cholesterol ester → cholesterol + ester (cholesterol esterase)
phospholipids → fatty acid + lysophospholipid (phospholipase A2)

Step 3: Because of the poor solubility of lipids, they must be solubilized by a “biological detergent”.
This solubilizing agent is bile acid that is produced by the liver from cholesterol and converted to bile
salts by intestinal bacteria. Bile salts act in the absorption of lipids by reversibly forming micelles. These
micelles are structures with well-defined sizes and are much smaller than emulsion droplets. The
arrangements of the bile salts in micelles is such that the hydrophobic (water-hating) portions are
removed from contact with water by interacting with the lipids they are solubilizing, while hydrophilic
(water-loving) groups remain exposed to the water. Bile salts form mixed micelles with lipids, such as 2-
monoacylglycerol, phospholipids, fatty acids, cholesterol and fat soluble vitamins. These mixed micelles
have disk-like shapes, wherein the phospholipids and fatty acids form a bilayer and the bile salts occupy
the edge positions. During triacylglycerol digestion by pancreatic lipase, free fatty acids and
monoacylglycerols are released at the surface of fat emulsion droplets. Thus the products of
triacylglycerol hydrolysis are continuously transferred from emulsion droplets to the mixed micelles.

Lipid Transport -2
 Lipids:
Triacylglycerol Lingual Lipase
Cholesterol esters
Phospholipids
STEP 1
Figure 1 Five
Gastric Lipase steps of lipid
digestion and
absorption. Step
stomach
1 is relatively
STEP 2 minor. Most
Pancreas releases: hydrolysis of
lipase small intestine triacylglycerols,
cholesterol esterase pancreas as well as
phospholipase A2 phospholipids
STEP 4
Lipids absorbed and cholesterol
from micelles into esters occurs in
STEP 3 epithelial cells Step 2.
Liver releases bile
acids to solubilize liver
STEP 5
lipid products in
Chylomicrons form and
mixed micelles
travel through lymphatics

Step 4: Uptake of lipids by the epithelial cells lining the intestines occurs by passive diffusion from the
mixed micelles through the membrane. Absorption is virtually complete for fatty acids and
monoacylglycerols, which are slightly water-soluble due to carboxyl and hydroxyl groups. This process is
less efficient for water-insoluble lipids such as cholesterol ester only 30% of which is absorbed. After
absorption of lipid digestion products, the micelles remain behind to solubilize other lipid products, thus
acting as a type of "shuttle."

Step 5: Cholesterol is converted back to its cholesterol ester form and fatty acids are reattached to
glycerol backbones to form new molecules of triacylglycerol. These lipid products are then incorporated
into a lipoprotein known as a chylomicron (see description of lipoproteins below). The chylomicrons are
then exported into the lymphatic system. You may wonder why chylomicrons do not enter the blood. This
occurs because the fatty acids contained in the triacylglycerol of the chylomicron must be first delivered
to tissues such as adipose tissue and muscle rather than to the liver. If the chylomicrons entered the blood
they would go straight to the liver where large amounts of fatty acids would be dropped off. This would
be harmful to the liver since it could become clogged with fat.

Lipid Transport -3
Table 1. Summary of the steps of lipid digestion and absorption

Step Location Important Enzymes

1. Minor digestion (triacylglycerols) mouth and stomach lingual/ gastric lipase
pancreatic lipase
lumen of the small intestines
2. Major digestion (all lipids) cholesterol esterase
phospholipase A2
3. Formation of mixed micelles lumen of the small intestines N/A
4. Passive absorption of lipolytic
into intestinal epithelial cell N/A
products
5. Assembly and export of from intestinal cells to the
N/A
chylomicrons lymphatics

LIPOPROTEINS:

Lipoprotein general structure

Lipoproteins are characterized according to their densities. The lipoprotein densities vary depending on
their lipid composition. The structure of a lipoprotein (specifically LDL) includes a lipid core that varies
with each type of lipoprotein (see Table 2) surrounded by phospholipids with their hydrocarbon tails
facing the interior and their polar head groups exposed to the surface for interaction with water (Fig 2;
Stryer 26.16). The free hydroxyl group of cholesterol is exposed on the surface of the lipoprotein to
interact with water. Likewise, proteins (see Table 3) sit on the outer surface of the lipoprotein. These
proteins are referred to as apoproteins.

Figure 2. Model of low

density lipoprotein. Other
lipoproteins have a similar
structure differing in the core
content of lipid and the type
of apoproteins on the surface
of the molecule.

Lipid Transport -4
Lipoprotein composition (Figure 3)

Chylomicrons are the least dense of the lipoproteins because they contain the greatest amount of lipid
(Figure 3). In keeping with the composition of the diet, the vast amount of lipid in the chylomicron is
triacylglycerol. The lipid:protein ratio is 50:1. Note that the chylomicrons are the only lipoprotein derived
exclusively from dietary lipids and are therefore considered the exogenous system.

Very low density lipoprotein (VLDL) is made in the liver from triacylglycerol and from cholesterol that
is mostly made in the body (90%) with a small portion (10%) derived from the diet. Because these lipids
are made in the body, this constitutes part of the endogenous system. Triacylglycerol still predominates
over cholesterol but the proportion is now less than 4:1. Lipids relative to protein is just 9:1 in VLDL
accounting for the increase in density.

Low density lipoprotein (LDL) are actually derived from the very low density (VLDL) form after the
latter has dumped most of its triacylglycerols in tissues such as adipose and muscle. Cholesterol is now
the predominant lipid being almost 3-fold greater than triacylglycerol. This is considered the so-called
“bad cholesterol” because it is carried out to the periphery of the body. Overall, the ratio of lipids to
protein is just 3:1. Like VLDL, LDL is part of the endogenous system.

High density lipoprotein (HDL), as the name suggests, carry the most protein which is present at a 1:1
ratio with lipids. Cholesterol predominates over triacylglycerol at just over 2:1. This is the so-called
“good cholesterol” because it is carried to the liver for disposal. HDL interfaces with both the exogenous
and endogenous lipoprotein systems because it donates apoproteins in the formation of both chylomicrons
and VLDL

P
C
100%
P
P
80% C Figure 3. The major classes of
lipoproteins and their relative
Composition

P contents of triacylglycerol (T),

60% cholesterol (C) and protein (P).
C
40% T
T C

20%
T
T
0%
Chylo- VLDL LDL HDL
microns Lipoprotein Type

Lipid Transport -5
Transport and processing of lipids in the exogenous system

The functions of the lipoproteins are summarized in Table 2 and the functions of the apoproteins are
summarized in Table 3 at the end of the notes. These table summaries correlate with events shown in
figures 4, 5 and 6. The exogenous system includes chylomicrons, chylomicron remnants and to a small
extent HDL (Fig. 4). Chylomicrons are distributed into the body through the lymphatic system to various
non-hepatic tissues, primarily adipose tissue and muscle. The chylomicrons must deliver fatty acids (from
the triacylglycerols) to these tissues. Apoprotein B-48 forms a shell around the lipids to facilitate
formation of the chylomicron. The surface protein is hydrophilic to aid in solubilization of the
chylomicrons. From the lymphatics, chylomicrons enter the capillaries of target tissues (Figure 4).
Lipoprotein lipase, attached to the inner wall of the capillaries, catalyzes the hydrolysis of the
triacylglycerols to release fatty acids that enter the tissues. Apoprotein CII, associated with the
chylomicrons, activates the lipoprotein lipase molecules. After their release from the triacylglycerols, the
fatty acids traverse the cell membrane to be taken up by the tissue. The remainder of the molecule,
chylomicron remnants, contains largely dietary cholesterol and is taken in by the liver by binding of
apoprotein E to receptors on the surface of the liver (Figure 4). This formation and processing of
chylomicrons is termed the exogenous system for lipoprotein metabolism.

Lymph system: In primarily adipose or muscle chylomicrons interact

nascent chylomicrons with lipoprotein lipase (LPL) activated by apo CII;
travel to capillaries LPL hydrolyzes TAGs; fatty acids enter tissues;
remnants produced with the return of apo CII to HDL
Nascent chylomicrons
mature by acquiring apo
INTESTINE

CII (C) and E (E) from non-hepatic tissues

HDL in the blood
CE CE CE
CE CE E
CE CE E
CE CE C
E C

E
ApoB48 helps with chylomicron remnants travel C
chylomicron assembly through blood to liver E E E

= chylomicron Liver:
apo E on chylomicron remnants
= HDL
bind to receptor
= lipoprotein lipase
TAG = triacylglycerols cholesterol from remnants
= fatty acid from TAG deposited in liver
LIVER
= chylomicron remnant
= liver apo E receptor
= cholesterol
Figure 4. Exogenous pathway of lipid transport. Chylomicrons carry dietary fatty acids to tissues and the
remnants take cholesterol to the liver. HDL in the exogenous system provides in the blood apo CII and
apo E to create a mature chylomicrons molecule.

Lipid Transport -6
Medical Scenario:

T.C., a 59 year old male, entered the hospital suffering from chronic gnawing, epigastric pain. He
reported that he had endured mild symptoms of this type since age 18, but had become worse in the last
year, with bloating, flatulence and several loose bowel movements daily, but without blood, pus or
mucus. In the last few years he had reduced his food intake in an attempt to control pain and diarrhea.
His weight fell from 62 to 44 kg. The past history showed that he had been healthy as a boy while living
in the Caribbean on a diet relatively low in fat. At 18 he became a merchant sailor and began to
experience abdominal pain. He described the diet on board ship as higher in fat. At 24, he had a
perforated duodenal ulcer, and four years later, a partial small-bowel obstruction due to adhesions, both of
which were treated surgically. At the time of the second surgery, his blood serum was described as
“lipemic,” with a serum triglyceride of 1100 mg/100 ml (nl<165), but serum cholesterol was only 214
mg/100 ml (nl=150-220). He continued to have recurrent epigastric pain, but did not seek medical advice
until the present admission.

Physical examination showed a pale and wasted man. His stool contained 22.6 g of fat per day on a diet
containing 100 g of fat. Nutrition therapy had been initiated earlier, and there was no evidence from blood
tests of a residual nutritional deficiency or abnormality of folic acid, ascorbic acid, carotene, vitamins A,
B12 and E, essential fatty acids or amino acids. Because of a sudden fall in the patient’s hemoglobin, he
received a transfusion of packed red blood cells. The hemoglobin rose to normal and also had a
remarkable effect on the plasma triglycerides. The levels that had previously been 1750 mg/100 ml, fell to
just 196 mg/100 ml two days after the transfusion. However over the next 2 weeks, the triglycerides rose
once again to 1750mg/100 ml. This patient has a defect of his apo CII. The transfusons temporarily
provided him with normal apo CII that influenced his plasma lipoprotein levels causing the patient’s
triglyceride-rich lipoproteins to decrease. After two weeks the normal apo CII was replaced with his
defective form and the hypertriglyceridemia returned.

Transport and processing of lipids in the endogenous system

The endogenous system is displayed in Figure 5. The endogenous system involves three lipoproteins –
VLDL, LDL and HDL. Very low density lipoprotein (VLDL) is formed in the liver from triacylglycerol
and cholesterol provided by the tissue and with the assistance of apoprotein B-100 (Figure 5). The VLDL
is exported and picks up apoprotein CII and E in the blood. Note that since LDL is derived from VLDL,
the VLDL molecule contains the apoproteins needed for both VLDL and LDL functions. The role of
VLDL is to transport and deliver fatty acids to peripheral tissues. Triacylglycerols contained in VLDL, as
in chylomicrons, are substrates for lipoprotein lipase in capillaries. Therefore VLDL contains apo CII for
the same reason, and in this way fatty acids, synthesized from excess sugar in liver, are delivered to other
tissues.

After triacylglycerol hydrolysis by lipoprotein lipase, VLDL converts to the low-density lipoprotein
(LDL) (Figure 5). The density increases because of the loss of the triacylglycerols. The role of LDL is to
carry cholesterol, derived from the liver, to other tissues (Table 2). LDL travels through the blood to a
variety of tissues. Apo B100, on the LDL particle, binds to protein receptors (LDL receptors) on the
tissues (Table 3), and LDL deposits its cholesterol in the cell. The processing of LDL will be discussed
further below, as well as the reason the cholesterol contained in LDL is considered to be “bad
cholesterol”.

Finally, high-density lipoprotein (HDL) contains the smallest percentage of lipids and benefits the body
by being a scavenger of cholesterol from peripheral tissues (Table 2). Cholesterol from peripheral tissues
leaves the cell (Figure 5). HDL contains apo A1 (Table 3) that facilitates the formation of cholesterol
ester, via an acyltyransferase (LCAT). It is cholesterol ester that is stored in the core of the HDL particle.

Lipid Transport -7
 mature VLDL created At primarily adipose or muscle VLDL
by acquiring apo CII interact with lipoprotein lipase (LPL)
B100 (B) helps assemble (C) and E (E) from activated by apo CII; LPL hydrolyzes TAGs;
and export TAG, and HDL in blood fatty acids enter tissues; LDL are produced
cholesterol in nascent with the return of apo CII and apo E to HDL
VLDL
non-hepatic tissues
LIVER
B B
CE CE CE CE CE CE
CE CE CE
CE
CE
B

bile acids B

B
LDL travels through blood to various
tissues (70% liver; 30% other)
B B B

Various tissues:
apo B100 on LDL bind to receptor

LDL taken into the cell to deliver

cholesterol (see Fig 6 for details)

apo E on HDL binds to liver receptor

receptor to deliver cholesterol for
processing and excretion as bile acids

HDL scavenges cholesterol from non-hepatic

= fatty acid from TAG
tissues via CERP facilitating efflux and apo
= cholesterol A1 activating LCAT promoting the formation
of cholesterol ester
TAG = triacylglycerols
= VLDL = lipoprotein lipase
= LDL
= apo B100 receptor
= HDL = liver apo E

Figure 5. Endogenous pathway of lipid transport. VLDL carries fats to other tissues from the liver. LDL
takes cholesterol to tissues. HDL collects cholesterol from the body back to the liver and also transfers
apo E and CII to form mature VLDL molecules. Excess cholesterol in liver is excreted as bile acids for
lipid digestion.

Lipid Transport -8
HDL attaches to liver cell receptors via its apo E protein, analogous to the chylomicron remnants.
Because HDL is a scavenger, it is considered to be “good cholesterol”. Consequently higher levels of
HDL and a greater ratio of HDL:LDL can be protective against the deleterious effects of excess
cholesterol. Individuals with a lower than normal ratio of HDL:LDL should receive appropriate treatment
through diet modification and/or inhibition of cholesterol biosynthesis in the body. This is important
because of the dangers of atherosclerosis, discussed further below.

Role of LDL receptor in uptake of cholesterol

Within the endogenous system, LDL is cleared from the blood by uptake into cells (Figure 6). This uptake
increases the supply of cholesterol in peripheral tissues but at the same time decreases cholesterol
synthesis in these tissues. The apo B-100 protein of LDL attaches (binds) to a specific LDL receptor
protein on the surface of the cell (Fig. 6; step 1). The receptors aggregate to form coated pits containing
clathrin (step 2), which facilitates LDL internalization into the cell via endocytosis (step 3). A sorting
endosome forms by combination of the LDL vesicle and an acidic (5.0) (step 4). Acidity causes
dissociation of LDL from its receptor, which is then recycled to the cell membrane (step 5). The LDL and
cholesterol ester accumulate in transport vesicles that then fuse with the lysosome in which apo B100
protein is degraded to its component amino acids (step 6). Also in the lysosome, the cholesterol ester is
hydrolyzed to the free cholesterol form (step 6). Free cholesterol then moves via Niemann-Pick C (NPC)
protein to the Golgi apparatus for storage (step 7) followed by esterification catalyzed by ACAT (acyl
CoA:cholesterol acyltransferase) and storage in droplets (step 8).

ligand and receptor

recycling of dissociate at pH 5 in transport vesicle and
LDL sorting endosome lysosome fuse to form
receptor receptor and
clathrin step 4 late endosome

Clathrin- step 5 lysosome

coated pit step 6
ooo

step 2 late endosome NPC-1 Golgi

mediated free pool of step 8
ACEH transfer
ooo

CE Æ cholesterol cholesterol
ACAT
step 1 endocytosis B100 Æ amino
step 7
step 3 CE stored in
step 6
vesicle droplets

LDL
CE CE
CE

Figure 6. Steps in the cellular uptake of cholesterol via the LDL receptor. NPC-1 (Niemann-Pick C)
protein is used to move cholesterol to the Golgi. ACAT (acyl CoA:cholesterol acyltransferase) esterifies
cholesterol to the ester (CE) form

Lipid Transport -9
Relationship of LDL receptor to hypercholesterolemia and atherosclerosis

Cholesterol content in the cell is kept low, in part, by decreasing the number of LDL receptors. When
cholesterol levels become excessive, the number of LDL receptors may be markedly become decreased to
limit extraction of LDL from the blood. This results in an accumulation of LDL and hence of cholesterol
in the blood. Buildup of cholesterol in blood vessels can cause blockage, which if occurring in arteries
leads to atherosclerosis. Individuals who have a significant genetic defect for the LDL receptor develop
atherosclerosis early in development and often die at a young age from heart disease. Such disorders fall
into the class of familial hypercholesterolemia and are caused by an inability of the defective receptors
to take LDL into the cells.

Table 2. Summary of lipoprotein functions

Lipoprotein Function
Chylomicron deliver fatty acids as part of triacylglycerol, from dietary fat to muscle,
adipose
Chylomicron deliver dietary cholesterol to the liver
remnants
VLDL deliver fatty acids, attached to triacylglycerol, derived from liver
synthesis to non-hepatic tissues (e.g., muscle, adipose)
LDL from VLDL; delivers cholesterol, derived from liver synthesis to
various tissues
HDL collects (scavenges) cholesterol from non-hepatic tissues and delivers to
the liver

Table 3. Summary of apoproteins and their characteristics

Lipoprotein Apoprotein Function

Chylomicron B-48 chylomicron formation
CII activates lipoprotein lipase
E transferred to remnants
Chylomicron E binds to liver receptor for chylomicron remnants to enter the cell
remnants
VLDL B-100 assembly of VLDL
CII activates lipoprotein lipase
E binds to liver receptor for VLDL to enter the cell
LDL B-100 binds to LDL receptor for LDL to enter the cell
HDL A1 facilitates cholesterol efflux from cells
CII stored on HDL for transfer to chylomicrons and VLDL
E binds to liver receptor for HDL to enter the cell

Lipid Transport -10