BIOM 209/CHEM 210/PHARM 209
Lipoprotein, Fat Soluble Vitamin, and Prenol
Metabolism, Signaling and Lipidomics
Phospholipid
monolayer
ApoB-100
Figure: Nelson DL, Cox MM (2005),
Lehninger Principles of Biochemistry, 4th
ed. W.H. Freeman & Co.
Triacylglycerols
Free (unesterified)
cholesterol
Cholesteryl esters
Professor Edward A. Dennis
Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego
Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or
individual slides (without alteration) for academic, non-profit and non-commercial purposes.
Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org
E.A. DENNIS 2016 ©
Some Definitions
Lipoprotein = a macromolecular
complex of proteins with XOL,
XOL esters, triglycerides and
phospholipids.
Apoprotein = a specific protein that
is integrated into a lipoprotein
complex. Often, it has enzymatic
or receptor-ligand functions.
Chylomicron = a large lipoprotein
made chiefly of triglycerides that
is made in the GI track to ship
dietary lipids to the liver. Marked
by ApoB-48.
Blood plasma
after fast
Blood plasma
after meal
After eating, chylomicrons flood the
bloodstream visibly changing the
color of plasma.
Figure: Nelson DL, Cox MM (2005), Lehninger Principles
of Biochemistry, 4th ed. W.H. Freeman & Co.
E.A. DENNIS 2016 ©
Some More Definitions
VLDL = a lipoprotein made in the
liver to ship lipids to peripheral
tissue. Marked by large size,
triglycerides and Apo B-100.
Phospholipid
monolayer
ApoB-100
LDL = a VLDL from which most
of the triglycerides have been
removed. Small and high in
cholesterol esters.
HDL = smallest lipoprotein
consisting mostly of protein
and some cholesterol esters.
“Good cholesterol” marked by
Apo A and D.
Triacylglycerols
Free
(unesterified)
cholesterol
Cholesteryl esters
Model of a VLDL lipoprotein
Figure: Nelson DL, Cox MM (2005),
Lehninger Principles of Biochemistry, 4th
ed. W.H. Freeman & Co.
E.A. DENNIS 2016 ©
Lipoprotein Composition
Kritchevsky, Nutr Int, 1986, 2, 290-7
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Size Matters (For Lipoproteins)
Chylomicrons (50-200 nm diameter) VLDL (28-70 nm diameter)
LDL (20-25 nm diameter)
HDL (8-11 nm diameter)
Figures: Nelson DL, Cox MM (2005), Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman & Co.
E.A. DENNIS 2016 ©
The Cast of Apoproteins
Apolipoprotein
Molecular
weight
Lipoprotein
association
ApoA-I
28,331 HDL
ApoA-II
17,380 HDL
ApoA-IV
44,000 Chylomicrons,
HDL
ApoB-48
240,000 Chylomicrons
ApoB-100
513,000 VLDL, HDL
Function (if known)
Activates LCAT; interacts with ABC
transporter
Binds to LDL receptor
ApoC-I
7,000 VLDL, HDL
ApoC-II
8,837 Chylomicrons,
VLDL, HDL
Activates lipoprotein lipase
ApoC-II
8,751 Chylomicrons,
VLDL, HDL
Inhibits lipoprotein lipase
ApoD
32,500 HDL
ApoE
34,145 Chylomicrons,
VLDL, HDL
Triggers clearance of VLDL and
chlyomicron remnants
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Lipoproteins in Circulation
• Chylomicrons
– Formed in gut
– Go to liver
• VLDL’s
– Formed in liver
– Go to periphery
• LDL’s & IDL’s
– Formed in periphery
• “VLDL remnants”
– Return to Liver
• HDL’s
– Precursors are made in
liver
– Formed in periphery
– Go to liver
Figures: Nelson DL, Cox MM (2005), Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman & Co.
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Processing of LP’s - LCAT
Cholesterol
Phosphatidylcholine
(lecithin)
Lyso-phosphatidylcholine
(lyso-lecithin)
•
•
•
•
•
lecithincholesterol
acyltransferase
(LCAT)
Cholesteryl ester
Lecithin is also called phosphatidylcholine (PC)
LCAT always takes the 2-position acyl group
Activated by Apo A-I
Net effect: free cholesterol turns into cholesterol ester
Found associated with HDL particles
– The reason HDL has lots of cholesteryl esters.
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Processing of LP’s - Lipoprotein Lipase
Triacylglycerol (part of LP)
lipoprotein
lipase
• Located on surface of
capillary endothelium
• Degrades TG’s in the
chylomicrons and
VLDLs that go by
– Activated by Apo C-II
– Inhibited by Apo C-III
glycerol
• Works to export lipids
from VLDL to tissues
Free fatty acids (FFA)
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Hormone Sensitive Lipase
Triacylglycerol
(adipocyte cytosol)
hormonesensitive lipase
glycerol (reused)
• Made in adipose
tissue
• Triggered by PKA
• Works much like LP
lipase
• Export lipids from the
adipocyte to albumin
Free fatty acids (released onto albumin)
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Processing LP’s - The Basic Scheme
Chylo
more TG
B48, CII, CIII, E
VLDL
LP Lipase
LP Lipase
more TG, CE
B100, CII, CIII, E
IDL
Chylo
Liver
TG
B48, E
repackaging
“remnants”
HDL
LDL
CE, less TG
B100
Peripheral
Tissues
cholesterol
consumption &
atherosclerosis
CE, TG
B100, E
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Hormonal Controls: LIVER
Two hormonal
mechanisms
– Insulin (“store fat”)
• surface receptor
• tyrosine kinase
• Promotes protein
dephosphorylation
– cAMP (“release fat”)
• chiefly triggered by
epinephrine, glucagon
• Acts mainly via PKA
• Promotes AMPdependent protein
phosphorylation
Figure: Fundamentals of Biochemistry: Life at the molecular level, 2 nd ed, by Voet, D Voet JG, Pratt
CW. 2006. Reprinted with permission of John Wiley & Sons, Inc.
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Hormonal Controls: ADIPOCYTE
Two hormonal
mechanisms
– Insulin (“store fat”)
• Induces lipoprotein lipase
• Promotes
dephosphorylation of
hormone sensitive lipase
– cAMP (“release fat”)
• Promotes phosphorylation
of hormone sensitive
lipase leading to activation
Figure: Fundamentals of Biochemistry: Life at the molecular level, 2 nd ed, by Voet, D Voet JG, Pratt
CW. 2006. Reprinted with permission of John Wiley & Sons, Inc.
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Review of Lipoproteins and Cholesterol
Principle 1
– Cholesterol is both absorbed from
the diet and made in the liver.
Principle 2
– Where LDL goes, there goes Cholesterol!
• Cholesterol and its esters are a small part of
VLDL and chylomicrons, but a large part of LDL
Principle 3
A model of an LDL particle.
– Elevated levels of serum LDL are associated with heart disease and
atherosclerosis
How does the cholesterol inside LDL’s know where to go (liver cells vs.
serum vs. peripheral cells) and what happens to it when it gets there?
Figure reprinted with permission of John Wiley & Sons, Inc. from Fundamentals of Biochemistry: Life at
the Molecular Level, 4th ed, by Voet, D Voet JG, Pratt CW. 2006.
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Developing the Brown & Goldstein Model
Drs. Brown and Goldstein:
1. Postulated there was an LDL
receptor on cells
2. Looked for patients who might
lack it
• That is, people with high serum
LDL cholesterol
• “FH” patients
Dr. Michael
Brown
Dr. Joseph
Goldstein
3. Compared biochemical activities
in normal people versus people
without the LDL-R
• they found differences proving a
receptor must exist
1985 Nobel Prize
in Medicine
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LDL and Cholesterol Uptake by Cells
Figure: Brown, PNAS, 76, 3330-7 (1979).
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Supporting Evidence: Biochemical Activity
Figures to Left:
A. Surface Binding of LDL
B. Cellular Uptake of LDL
C. Hydrolysis of Apoprotein B
D. Hydrolysis of Cholesterol Esters
E. HMG-CoA Reductase activity
Circles = normal
Triangles =FH patients
– note it is the opposite of all the others!
F. XOL Esterficiation
Figures adapted from Brown, PNAS, 76, 3330-7 (1979).
[LDL]
[LDL]
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LDL and LDL Receptors in the Liver
• Receptor-mediated
endocytosis
• Serum LDL depends
on:
– VLDL produced
– VLDL turned into
LDL
– # of LDL receptors
• more receptors
means less
serum LDL
Figure reprinted with permission of John Wiley & Sons, Inc. from Fundamentals of Biochemistry, 1 st ed,
by Voet, D Voet JG, Pratt CW. 2002.
E.A. DENNIS 2016 ©
Regulation of Cholesterol & LDL Receptors
Increased free cholesterol
concentration in liver cell cytosol has
3 effects:
1. HMG-CoA reductase is inhibited
• less new cholesterol is synthesized
2. ACAT activity increases
• more free cholesterol esterfied into a
storable form
3. LDL Receptor protein translation is
inhibited
• Fewer receptors are placed on the cell’s
surface
• Fewer LDL particles are endocytosed
• Serum LDL and cholesterol levels
increase
Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of
Biochemistry, 2nd ed. Worth Publishers, Inc.
E.A. DENNIS 2016 ©
Normal LDL Receptor Levels
• LDL receptors on liver
• Most LDL is
reabsorbed by liver
– low serum LDL levels
– little atherosclerosis
• Liver produces small
amounts of cholesterol
Figure reprinted with permission of John Wiley & Sons, Inc. from Fundamentals of
Biochemistry, 1st ed, by Voet, D Voet JG, Pratt CW. 2002.
E.A. DENNIS 2016 ©
LDL in Familial Hypercholesterolemia
• No working LDL
receptors on liver
– genetic defect
• All LDL created stays
in circulation
– high serum LDL levels
– lots of atherosclerosis
• Liver produces
cholesterol
Figure reprinted with permission of John Wiley & Sons, Inc. from Fundamentals of
Biochemistry, 1st ed, by Voet, D Voet JG, Pratt CW. 2002.
E.A. DENNIS 2016 ©
LDL and High Dietary XOL
• High dietary cholesterol
• High cytosolic
cholesterol in liver cells
– HMG-CoA inhibited
– LDL-R’s not produced
• LDL not picked up
• All the LDL stays in
circulation
– elevated serum LDL
– lots of atherosclerosis
Figure reprinted with permission of John Wiley & Sons, Inc. from Fundamentals of
Biochemistry, 1st ed, by Voet, D Voet JG, Pratt CW. 2002.
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Atherosclerosis Biochemistry
Elevated Serum LDL
LDL acetylation &
oxidation
Macrophage chemotaxis &
LDL uptake
Foam cells
& Fatty Streaks
Advanced Lesions
Rupture, Thrombosis
& Infarction
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Biochemistry in Early Atherosclerosis
LDL
Acetylation
Oxidation
• Mf’s have many receptors
• Oxidized LDL (OxLDL) is a
chemotaxin
– 40-70% less atherosclerosis with
antioxidant therapy
OxLDL
OxLDL
AcetylLDL
• Only proven in mice!
• Human studies inconclusive
• Glycosylation also effects
• Scavenger Receptor A (SR-A)
is not regulated by cholesterol
Classic
LDL
receptor
gets down
regulated
Chemotactic
receptor
reacts to
OxLDL
“SR-A”
unregulated
by increasing
cholesterol in
cytosol
Macrophage (Mf)
– Mf’s keep eating more & more
– They become “foam cells”
• Full of cholesterol and cholesterol
esters
– SR-A knockout mice have 50%
less atherosclerosis
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Subsequent Atherosclerosis
Progression
1. Macrophage chemotaxis &
LDL uptake
2. Formation of
“Fatty Streaks”
3. Advanced
Lesions
4. Rupture, Thrombosis
& Infarction
Figure: Ross, NEJM, 1999, 340:2.
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Vitamins: The Family Tree
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Preview: Vitamin A - Retinol
Various “retinoids”
– retinol, retinal & retinoic acid
– retinyl esters
Retinol
• transport in blood
– beta carotene
All-trans retinal
Numerous Functions
– Vision
•
•
•
•
Retinoic acid
11
12
11-cis retinal
(formed by photoisomerization
of all-trans retinal)
all-trans retinal
11-cis-retinal
Growth & Wound healing
especially epithelium
– Reproduction
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Vision- How it works…
• Cis-Retinal acts as a cofactor of the protein opsin to form
rhodopsin.
• It functions in a similar way in rods and cones. But we’ll use rods
as a model.
• Rhodopsin and transducin are embedded in the cell membrane
of the outer rod segment.
• Rhodopsin is the photoreceptor. It is an integral membrane
protein with 7 membrane spanning segments. When a photon
of light hits rhodopsin, it causes the isomerization of cisretinal to trans-retinal. This activates the receptor, causing
it to bind to the heterotrimeric G protein, transducin.
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Retinol from b-Carotene
• Retinal and retinol are readily
interconverted
• Most dietary vitamin A comes
in the form of b-carotene.
– 1 carotene = 2 retinal
– But: inefficient conversion
– Less toxic than retinol
Cleavage
Site
2x
• Safer form of the vitamin
– Found in yellow and dark green
vegetables
• carrots, doc.
b-carotene
Retinal
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Vitamin A Deficiency
• Incidence: Rare in the US
– Seen in people who don’t eat well
• Worldwide, it is the third most common
nutritional deficiency, accounting for
500,000 cases of blindness annually.
• Symptoms
– Loss of acuity & night blindness,
blindness
– Lesions on corneal surface
– Eventually, dermatology problems
• Mechanism: Lack of retinoids
Trivia: Ancient Egyptians
recognized that night blindness
could be treated by eating liver.
– Poor epithelial growth
– Rhodopsin synthesis is impaired
• Treatments: A supplementation
– Better diet
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Vitamin A Toxicity
There are three syndromes of vitamin A toxicity:
• Acute Toxicity (very rare)
– occurs in adults when >200 mg are ingested
– symptoms include nausea, vomiting, vertigo, and blurry
vision.
• Chronic Toxicity (rare)
– occurs with long-term ingestion of doses higher than 10
times the RDA.
– symptoms include problems talking, hair loss,
hyperlipidemia, hepatotoxicity, bone and muscle pain,
and vision problems.
– In postmenopausal women, it has been associated with
increased fracture risk.
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Vitamin A Toxicity
• Teratogenic Effects:
– Synthetic retinoids can be used to treat severe
dermatological conditions including severe psoriasis
and acne vulgaris.
– Synthetic retinoids, like acitretin, cause spontaneous
abortions and severe life-threatening congenital
malformations.
• Women treated with retinoids must not get pregnant at
the time of treatment or become pregnant for up to 3
years after treatment.
• Patients receiving treatment with retinoids must not give
blood for up to three years after treatment.
– The presence of these drugs in plasma can be
demonstrated for up to several years after a person
stops taking them. It could be disastrous if an
unsuspecting pregnant woman received one in a
transfusion, hence the ban.
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Vitamin D - Cholecalciferol
Ergocalciferol
Diet
• Vitamin D is a cholesterol-like
molecule
• Important to bone and calcium
regulation
– Acts more like a steroid
hormone rather than a enzyme
cofactor
• Cholecalciferol (D3) has two sources:
Cholecalciferol
synthesis in skin
– Diet
• plants have ergocalciferol (D2), which
easily becomes D3
• animal flesh has ready-made D3
– Sunlight (UV)
• Converts 7-dehydrocholesterol into
D3 in the skin
7-dehydrocholesterol
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Metabolism of Cholecalciferol (D)
Calcitriol is the active form of the vitamin
– aka: 1,25 (OH)2-cholecalciferol or 1,25-D
Its precursor (provitamin) is cholecalciferol,
native vitamin D
– aka: D or D3
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Vitamin D Toxicity
• Vitamin D can build to toxic levels!
– Most dangerous vitamin!
– Messes up serum Ca++ leading to
hypercalcemia, hypercalciuria and
bone demineralization.
– Intoxication may occur in fad
dieters who consume
"megadoses" of supplements, in
Trivia: In the 1940s and 1950s, a number
patients on vitamin D replacement
of children developed hypercalcemia and
some even had hypercalcemic-induced
therapy for malabsorption, or in
brain injury. This was felt to be a result of
children who overdose on
the high concentrations of vitamin D in
fortified milk products
supplements.
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Vitamin K - Phylloquinone
Vitamin K1
• Made by normal intestinal flora
– Antibiotics can cause a loss of K!
• Two forms exist
– phylloquinone (K1) - plant sources
• spinach, cabbage, cauliflower
– menaquinone (K2) - bacterial sources
• Important in blood clotting
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Vitamin K Function
• Important in Hematology
• Catalyzes g -carboxylation
• Required by liver to make
clotting factors
– Factors II, VII, IX & X
– K = Klotting
– No K = No Klotting
• Inhibited by anticoagulants
like warfarin
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Vitamin K Deficiency
• Incidence: Common in certain groups
– Malnourished people on antibiotics
– Babies whose intestines are still sterile
– Patients on anticoagulants like warfarin
• Symptoms: Spontaneous bleeding
– Hematemesis (bloody vomit)
– Hemarthrosis (blood in joint capsules)
– Spontaneous bruising & bleeding gums
• Mechanism: Lack of vitamin K
– Factors II, VII, IX & X depend on K
• Treatments: K supplements
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Warfarin Overdose
Warfarin
Trivia: Warfarin was
originally developed
as a rodenticide.
• Warfarin has been the standard oral
anticoagulant used in a variety of clinical
settings.
• Patients treated with warfarin frequently
become overly anticoagulated. The most
common causes include drug interaction,
dietary changes and superimposed illnesses.
• If the clotting time is significantly impaired or if
the patient is at a high risk for bleeding Vitamin
K is given to reverse the effects.
• Mechanism of Action: Warfarin is similar in
structure to vitamin K and interrupts the vitamin
K dependent carboxylation cycle by blocking
reduction of the inactive K 2,3 epoxide to the
active form of the vitamin.
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Vitamin E - Tocopherols
Vitamin E
• Several variations exist
– D-a-tocopherol is most potent
• Lipid-media antioxidants
– contrast with vitamin C: a water-media antioxidant
• Found in nuts and meats
• No clear deficiency disease
• Long term effects and safety of
supplementation are unclear.
E.A. DENNIS 2016 ©
Summary of Fat Soluble Vitamins
Vitamin
Active Forms
Function
Sources
Disease
Toxic?
Retinol
ll-cis retinal
many others
Vision, growth
veggies
A deficiency
Somewhat
Cholecalciferol
Calcitriol
Bone and Ca++
regulation
UV light
Dairy
Rickets
Very!
Tocopherols
same
Antioxidant
Nuts, meat
Atherosclerosis?
Not really
Phylloquinones
same
Blood clotting
Colonic flora
K deficiency
Somewhat
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Isoprene Units - A Common Thread
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Example: Recognizing Isoprene Units
2
1
3
Isoprene
(2-methyl-1,3-butadiene)
• Retinol=4 x
Isoprene
• Look for 5-carbon
units
• Double bonds may
be removed or
rearranged
• C2 connects
always to at least 3
other carbons
Retinol
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What About Vitamin Supplements?
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Review of Lipid Metabolism
LIPIDS
CARBOHYDRATES
PROTEINS
fatty acids
glucose
amino acids
Acetyl CoA
malonyl CoA
citrate
fatty acid
neutral lipids
phospholipids
sphingolipids
cholesterol esters
acetoacetyl CoA
HMG-CoA
CO2 + ATP
(energy)
ketone bodies
cholesterol
Oversimplified picture
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Organ Localization
BLOOD TRANSPORT
Brain
•
•
•
•
TG on lipoproteins
FA on albumin
Glucose dissolved
Ketone Bodies dissolved
TG
Glucose
(Ketones)
Adipose
FA
Liver
Glucose, FA
(Ketones)
Bile
Muscles
Intestine
Oversimplified picture
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Intracellular Localization
FA
Acetyl CoA
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