Molecular Biochemistry II

Lipid Digestion & Transport

Copyright © 1999-2008 by Joyce J. Diwan.

All rights reserved.
 Lipid Digestion & Transport

Digestion & transport of lipids poses unique problems

relating to the insolubility of lipids in water.
Enzymes that act on lipids are soluble proteins or
membrane proteins at the aqueous interface.
Lipids, & products of their digestion, must be
transported through aqueous compartments within the
cell as well as in the blood & tissue spaces.
 Bile Acids O

C O R2
R1
CH3

CH3

HO OH
H R1 = OH or H
R2 = H or NH−CH2−COOH or NH−CH2−CH2−SO3−

Bile acids (bile salts) are polar derivatives of cholesterol,

formed in liver and secreted into the gall bladder.
They pass via the bile duct into the intestine, where they
aid digestion of fats & fat-soluble vitamins.
Bile acids are amphipathic, with detergent properties.
 Bile Acids O

C O R2
R1
CH3

CH3

HO OH
H R1 = OH or H
R2 = H or NH−CH2−COOH or NH−CH2−CH2−SO3−

Bile acids emulsify fat globules into smaller micelles,

increasing the surface area accessible to lipid-hydrolyzing
enzymes.
They also help to solubilize lipid breakdown products (e.g.,
mono- & diacylglycerols from triacylglycerol hydrolysis).
Secretion of bile salts & cholesterol into the bile by liver
is the only mechanism by which cholesterol is excreted.
Most cholesterol & bile acids are reabsorbed in the
small intestine, returned to the liver via the portal vein, &
may be re-secreted. This is the enterohepatic cycle.
Agents that interrupt the enterohepatic cycle are used to
treat high blood cholesterol. Examples:
 Synthetic resins, as well as soluble fiber (e.g., oat bran
fiber and fruit pectin), that bind bile acids &/or
cholesterol, prevent absorption/reabsorption.
 A recently introduced drug ezetimibe acts on cells
lining the lumen of the small intestine to inhibit
absorption of cholesterol.
 O O

H2C O C R1 H2C O C R1
O O O

HC O C R2
H2O
HC O C R2 + −
O C R3
O

H2C O C R3 H2C OH

triacylglycerol 1,2-diacylglycerol fatty acid

Pancreatic lipase (secreted into the intestine), catalyzes

hydrolysis of triacylglycerols at positions 1 & 3, forming
1,2-diacylglycerols, & then 2-monoacylglycerols.
A protein colipase is required to aid binding of the enzyme
at the lipid-water interface.
Monoacylglycerols & fatty acids are absorbed by intestinal
epithelial cells. Within intestinal epithelial cells
triacylglycerols are resynthesized.
 O
Phospholipase A2 is
H2C O C R1
secreted by the O
pancreas into the R2 C O C H O
intestine.
CH2O P O X
It hydrolyzes the ester
linkage between the phospholipid O−
fatty acid & the H2O
hydroxyl on C2 of O
phospholipids.
H2C O C R1 O
Lysophospholipids,
HO C H O + R2 C O−
the products of
Phospholipase A2 CH2O P O X
reactions, are O−
powerful detergents. lysophospholipid fatty acid
Lysophospholipids, produced from phospholipids
via Phospholipase A2, aid digestion of other lipids
by breaking up fat globules into small micelles.
Some phospholipid (lecithin) is secreted by the liver
in the bile, presumably to provide substrate for
Phospholipase A2 within the intestine and thus aid in
fat digestion.
Cobra & bee venoms contain Phospholipase A2.
These venoms, injected into the blood, produce
lysophospholipids that disrupt cell membranes and
lyse blood cells.
 Cholesteryl Ester

R C O

Within intestinal cells (and other body cells) some of the

absorbed cholesterol is esterified to fatty acids, forming
cholesteryl esters.
(R = fatty acid hydrocarbon in diagram above)
The enzyme that catalyzes cholesterol esterification is
ACAT (Acyl CoA: Cholesterol Acyl Transferase).
 PDB 1ICM
Within intestinal cells, fatty
acids (which are poorly
soluble & have detergent
properties) are sequestered
from the cytosol by being
bound with intestinal fatty
acid binding protein
(I-FABP). I-FABP

Fatty acid-binding proteins, which are in several cell

types, have a ''β -clam" structure.
A fatty acid is carried in a cavity between 2 approx.
orthogonal β -sheets, each consisting of 5 antiparallel
β -strands.
 Apoprotein B-100

monolayer of
Free fatty acids phospholipid &
cholesterol
are transported in
the blood bound core: cholesteryl
to albumin, a esters & some
serum protein triacylglycerols
secreted by liver. LDL

Most other lipids are transported in the blood as part of

lipoproteins, complex particles whose structure includes:
 a core consisting of a droplet of triacylglycerols and/or
cholesteryl esters
 a surface monolayer of phospholipid, cholesterol, &
specific proteins (apolipoproteins), e.g., B-100.
Lipoproteins differ in the ratio of protein to lipids, & in
the particular apoproteins & lipids that they contain.
They are classified based on their density:
 Chylomicron (largest; lowest in density due to high
lipid/protein ratio; highest % weight triacylglycerols)
 VLDL (very low density lipoprotein; 2nd highest in
triacylglycerols as % of weight)
 IDL (intermediate density lipoprotein)
 LDL (low density lipoprotein, highest in cholesteryl
esters as % of weight)
 HDL (high density lipoprotein; highest in density due
to high protein/lipid ratio)
Apolipoprotein Structure:
Amphipathic α -helices (polar along one surface of a
helix and hydrophobic along the other side) are common
structural motifs.
One view is that these α -helices may float on the
phospholipid surface of the lipoprotein.
Some domains of apolipoproteins have roles in
interaction of lipoproteins with cell surface receptors.
 N
Apolipoprotein A-I (apoA-I) of human HDL, in the
absence of lipid, is found consist of an N-terminal
antiparallel 4-helix bundle & a C-terminal domain that
is also α -helical.
A truncated apoA-I, engineered
to lack the first 43 amino acids,
was found to have a more open
structure, with a horseshoe shape
(dimer shown at right).
Lack of the first α -helix at the
N-terminus may prevent
stabilization of the 4-helix bundle. Truncated ApoA-I PDB 1AV1

On interacting with lipid, the compact structure of intact

apoA-I is assumed to open up into a structure resembling the
horseshoe shape observed for the truncated protein.
In the open form, proline residues interrupt α -helical
segments, providing curvature that would be appropriate for
wrapping around a spherical or elliptical lipid micelle.
 PDB 1AV1

A strip of
hydrophobic
residues runs
along one edge of
the amphipathic
α -helix. Lipid-binding domain of HDL Apolipoprotein A-I

In the crystal, antiparallel dimers were found to be formed

by association of these hydrophobic residues.
At right the apoA-I dimer is displayed as spacefill with
hydrophobic residues magenta; polar residues cyan.
For more diagrams, see the article by Ajees et al.
Apolipoprotein E (apoE), a constituent of several
classes of lipoproteins, also has an N-terminal domain
that folds as a 4-helix bundle in the absence of lipid.
Based in part on a low resolution structure determined
in the presence of phospholipids, it has been proposed
that interaction with lipids converts apoE to an
α -helical hairpin that wraps around the lipid
particles.
Diagrams (Weisgraber website, Gladstone Institute)
There is special interest in the structure and stability of
apolipoprotein E.
In addition to being a constituent of various lipoproteins,
e.g. VLDL & HDL, a variant of apolipoprotein E,
designated apoE4, is implicated in Alzheimer's disease
and other neurological conditions.
Having the apoE4 isoform is a major risk factor for
Alzheimer's disease.
Fragments of apoE4 are found to generate intracellular
deposits resembling the neurofibrillary tangles seen in
Alzheimer's disease.
Formation of lipoproteins:
Intestinal epithelial cells synthesize triacylglycerols,
cholesteryl esters, phospholipids, free cholesterol, and
apoproteins, and package them into chylomicrons.
Chylomicrons are secreted by intestinal epithelial cells,
and transported via the lymphatic system to the blood.
Apoprotein CII on the chylomicron surface activates
Lipoprotein Lipase, an enzyme attached to the lumenal
surface of small blood vessels.
Lipoprotein Lipase catalyzes hydrolytic cleavage of fatty
acids from triacylglycerols of chylomicrons.
Released fatty acids & monoacylglycerols are picked up
by body cells for use as energy sources.
As triacylglycerols are removed by hydrolysis,
chylomicrons shrink in size, becoming chylomicron
remnants with lipid cores having a relatively high
concentration of cholesteryl esters.
Chylomicron remnants are taken up by liver cells, via
receptor-mediated endocytosis (to be discussed later).
The process involves recognition of apoprotein E of the
chylomicron remnant by receptors on the liver cell surface.
Liver cells produce, and secrete into the blood, very low
density lipoprotein (VLDL).
• The VLDL core has a relatively high triacylglycerol
content.
• VLDL has several apoproteins, including apoB-100.
MTP (microsomal triglyceride transfer protein), in the
lumen of the endoplasmic reticulum in liver, has an
essential role in VLDL assembly.
MTP facilitates transfer of lipids to apoprotein B-100 while
B-100 is being translocated into the ER lumen during
translation.
Control of VLDL production:
 VLDL assembly is dependent on availability of lipids.
Transcription of genes for enzymes that catalyze lipid
synthesis is controlled by SREBP.
 Availability of apoprotein B-100 for VLDL assembly
depends at least in part on regulated transfer of B-100
out of the ER for degradation via the proteasome.
As VLDL particles are transported in the bloodstream,
Lipoprotein Lipase catalyzes triacylglycerol removal
by hydrolysis.
With removal of triacylglycerols and some proteins, the
% weight that is cholesteryl esters increases.
VLDL are converted to IDL, and eventually to LDL.
VLDL  IDL  LDL
The lipid core of LDL is predominantly cholesteryl esters.
Whereas VLDL contains 5 apoprotein types (B-100, C-I,
C-II, C-III, & E), only one protein, apoprotein B-100, is
associated with the surface monolayer of LDL.
 LDL
Cells take up LDL by extracellular space
receptor-mediated
cytosol LDL
endocytosis, involving receptor
formation of a clathrin-
coated pit & pinching off
of a vesicle incorporating
receptor-mediated
the receptor & LDL cargo. endocytosis
After the clathrin coat disassembles, the vesicle fuses with
an endosome.
LDL is released from the receptor within the acidic
environment of the endosome, and the receptor is returned
to the plasma membrane.
After LDL is transferred to a lysosome, cholesterol is
released & may be used, e.g., for membranes synthesis.
 LDL
extracellular space
cytosol LDL
receptor

receptor-mediated
endocytosis

The LDL receptor was identified by M. Brown & J.

Goldstein, who were awarded the Nobel prize.
The LDL receptor is a single-pass transmembrane
glycoprotein with a modular design.
 LDL
extracellular space
The cytosolic domain cytosol LDL
at the C-terminus of the receptor
LDL receptor binds
adapter proteins that
mediate formation of receptor-mediated
the clathrin coat. endocytosis

The LDL-binding domain on the exterior side of the

plasma membrane recognizes & binds apoprotein B-100.
Once the receptor with bound LDL is taken into a cell by
endocytosis, the LDL-binding domain faces the lumen
of the vesicle and later the lumen of the endosomal
compartment.
 The N-terminal LDL-binding (apoprotein B-100-
binding) domain of the receptor consists of a series of

N- R1 R2 R
cysteine-rich repeats (R1-R7), each of which is
stabilized by 3 disulfide linkages and has a bound Ca++ .
 Between the cysteine-rich repeats & the transmembrane
(TM) segment are 3 epidermal growth factor-like
domains (EGF-A, B, C) & a β -propeller.
 A domain subject to O-linked glysosylation (GD),
between the innermost EGF domain & the trans-
membrane α -helix, may act as a spacer to extend the
LDL-binding region out from the cell surface.
 PDB 1N7D

The long, flexible, β-propeller

modular structure
allows association of
N-terminal domains of
the receptor with ligand
on the surface of a
lipoprotein that may
vary in size. LDL receptor: LDL-binding domain
Under acidic conditions of the endosome the β -propeller
forms a complex with two of the cysteine-rich repeats.
This is what causes the receptor to release LDL, which is then
carried via a vesicle to a lysosome to be degraded.
Control of LDL Receptor activity:
 Synthesis of LDL Receptor is suppressed by high
intracellular cholesterol.
This process involves decreased release of SREBP.
Members of the SREBP family of transcription factors
activate transcription of genes for the LDL receptor,
as well as for enzymes essential to cholesterol
synthesis such as HMG-CoA Reductase.
The decreased synthesis of LDL receptor prevents
excessive cholesterol uptake by cells.
It has the deleterious consequence that excess dietary
cholesterol remains in the blood as LDL.
 A secreted protease PCSK9 degrades the LDL
receptor in liver.
Naturally occurring mutations that increase PCSK9
activity lead to increased plasma LDL because LDL
is not taken up by liver cells.
Other mutations that lead to decreased PCSK9
activity are associated with low plasma LDL.
Drug companies are evaluating feasibility and
consequences of inhibiting PCSK9.
The lowered intracellular cholesterol that results from
treatment with statin drugs, leads to SREBP activation,
increasing transcription of the gene for LDL receptor.
Thus statins lower plasma cholesterol both by inhibiting
HMG-CoA Reductase (decreasing cholesterol synthesis)
and by promoting removal of LDL from the blood.
However, the statin-induced SREBP activation also
leads to increased expression of PCSK9 in liver, limiting
the effect of statins on LDL uptake from the blood.
Mutations affecting the LDL receptor are associated
with the most common form of the disease familial
hypercholesterolemia (high blood cholesterol).
Cells lacking functional LDL receptors cannot take up
LDL.
As a result, the amount of circulating LDL increases,
leading to enhanced risk of developing atherosclerosis.
Other hereditary hypercholesterolemias relate to genetic
defects in structure of apolipoproteins.
E.g., familial defective apoprotein B100 leads to
impaired binding of LDL to cell surface receptors, with
elevated levels of circulating LDL.
HDL (high density lipoprotein) is secreted as a small
protein-rich particle by liver (and intestine).
One HDL apoprotein, A-1, activates LCAT (Lecithin-
Cholesterol Acyl Transferase), which catalyzes
synthesis of cholesteryl esters using fatty acids cleaved
from the membrane lipid lecithin.
The cholesterol is scavenged from cell surfaces & from
other lipoproteins.
HDL may transfer cholesteryl esters to other lipoproteins.
Some remain associated with HDL, which may be taken up
by liver & degraded.
HDL thus transports cholesterol from tissues & other
lipoproteins to the liver, which can excrete excess
cholesterol as bile acids.
High blood levels of HDL ("good" cholesterol) correlate
with low incidence of atherosclerosis.
Bacterial & viral infections, & some inflammatory disease
states decrease HDL & increase VLDL production by the
liver.
These & other changes associated with inflammation can
lead to increased risk of atherosclerosis if prolonged.
 blood vessel lumen endothelial cells

elastic
lamina smooth muscle cells
Cell layers adjacent to the lumen of arterial blood vessel.
Development of an atherosclerotic plaque:
Various conditions can initiate formation of a lesion in the
endothelium lining the arterial lumen.
 Inflammatory response, including cytokine production
that may be activated by oxidized lipids present in LDL.
 Risk factors include elevated circulating LDL, high
blood pressure, exposure to nicotine, etc.
 blood vessel lumen endothelial cells

elastic
lamina smooth muscle cells

Monocytes in the blood adhere to endothelial cells at sites

of injury/inflammation, & then pass into the subendothelial
space where they differentiate into macrophages.
Lipoproteins (e.g., LDL) leak across the endothelium &
accumulate in the subendothelial space, in part through
binding to proteoglycans.
Over time, exposure to oxygen radicals results in oxidation
of polyunsaturated fatty acids within LDL & modification
of the apolipoprotein.
 blood ves
Macrophages have on their surface scavenger receptors
that cause them to take up oxidized lipoproteins becoming
"foam cells" that have many cytoplasmic lipid droplets.
Although in humans foam cells mainly develop from
macrophages, smooth muscle cells may also migrate into
the subendothelial space & become foam cells.
 blood ves
Foam cells aggregate within the developing arterial
plaque.
Within the plaque core foam cells eventually undergo
necrotic death, releasing harmful cellular contents
that can promote plaque rupture & development of
blood clots.