BIOM 209/CHEM 210/PHARM 209
Lipid Cell Signaling Genomics, Proteomics, and Metabolomics
Essential Fatty Acids, Fish Oils, Eicosanoid Signaling, Inflammation, and Lipidomics
Figure: Lawrence et
al. , Nat Rev Immun,
2002, 2, 787-795.
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 ©
Fatty Acid Elongation & Desaturation
• FA Synthase makes
palmitate (C16)
• Several enzymes then
add double bonds
– fatty acyl CoA
desaturases
• 4 versions: D9, D6, D5,
D4
• Other enzymes
elongate the chain
– elongases
• add 2 carbons at a time
• occurs in the
mitochondria and ER
E.A. DENNIS 2016 ©
Desaturase Electron Transport
Figure: Lehninger AL, Nelson DL, Cox MM (1993), Principles of Biochemistry, 2nd ed. Worth Publishers, Inc.
E.A. DENNIS 2016 ©
Key Point
Observation 1
– Palmitic acid is the shortest FA available in mammals (C16)
Observation 2
– The D-9 bond is the farthest “desaturatable” site in mammals
Therefore:
– Mammals cannot make double bonds in the last 6
bond positions (-1 to -6) of a fatty acid chain
Cannot desaturate
-carbon
Can desaturate
a-carbon
E.A. DENNIS 2016 ©
Making EFA’s from Palmitate
• Only happens in plants
• Humans need linoleate
and linolenate from diet
(-3)
(-6)
E.A. DENNIS 2016 ©
Important pathways of unsaturated fatty acid biosynthesis in plants and animals. Note the conversion of dietary 18:2 from
plant sources to g-18:3 and 20:4 in animals, and the further desaturation of plant derived a-18:3 by animals.
E.A. DENNIS 2016 ©
What are Essential Fatty Acids?
• Two “Essential” FA’s
cannot be synthesized
by humans
Linoleic
acid
Linolenic
acid
– Linoleic acid (-6)
– Linolenic acid (-3)
• Obtained from diet
– Fish oils
– Plant sources
Arachidonic
acid
EPA
• Religious tradition of
fish on Fridays?!
E.A. DENNIS 2016 ©
Fish Oils and Epidemiology
Incidence of select diseases by population group
• Different Diets
– Danes eat “western”
diet
– Eskimos eat a lot of
fish and fish oils
• Population Studies
– Suggest fish oils
help
Figure: Lands, Fish and Human Health, Academic Press, 1986
E.A. DENNIS 2016 ©
-3 vs. -6 Fatty Acids
Fatty acid composition by source.
SOURCE
• Fish oils
– Hi -3/-6 ratio
• Few other good
sources of -3
– all have lots of -6
– Low -3/-6 ratio
CONTENT (%)
Linseed Oil
-3
13-35
26-58
-6
1-4
5-23
Soybean
Sunflower
Olive
Coconut
Butter
Margarine
2-10
-
49-52
44-68
4-15
1-3
3
11-48
Fish
E.A. DENNIS 2016 ©
Cardiac Arrythmias & Fish Oils
• Incidence: Common
• Symptoms:
– Chest pain, fainting, anxiety
– If cardiac output drops: shock
and death
• Mechanism
– Various cellular and conduction
system anomalies
– Sometimes, acquired damage
Figure: Dubin, The Rapid Interpretation of EKG’s, 6th Ed., Cover Publishing, 2000.
• Treatments:
– Diet: omega-3 fatty acids
– Anti-arrhythmia medications
– Implantable pacemakers &
defibrillators
– Surgical ablation of focal tissue
Figure: Rosenberg, NEJM, 346, 1102-3 (2002).
E.A. DENNIS 2016 ©
Fatty Acids in the US Diet
50%
Carbohydrate
Protein 15%
7%
52
Polyunsaturated
38
10
5%
Other
11%
12%
Saturated
Monounsaturated
33% Fat
Current US Diet (USDA 2008)
E.A. DENNIS 2016 ©
Back to the Romans and Greeks….
12
Inflammation in Clinical Medicine
Classic Inflammatory
Response
• Symptoms
–
–
–
–
Redness (rubor)
Swelling (turgor)
Heat/Fever (calor)
Pain (dolor)
Inflammatory Diseases
• Inappropriate
Inflammatory response
• Many Triggers:
– Musculo-skeletal Injuries
– Arthritis
• Rheumatoid
• Gouty (Uric Acid crystals)
• Rapid Innate Defense
– immobilizes injuries
• pain & swelling
– accelerates immune responses
• blood flow (redness & warmth)
• fever
–
–
–
–
–
Headaches & Colds
Asthma
Toxic Shock Antigens
Debated: Pre-eclampsia?
Other, rarer conditions
E.A. DENNIS 2016 ©
Inflammatory Biomolecules
Signal Molecules
– Histamine
– Eicosanoids
• Prostaglandins
• Thromboxanes
• Leukotrienes
– Bradykinins
– Cytokines
• Made in almost all tissues
• Very short half-life
• Act locally on neighbors
• Not usually stored up
• 20-carbon backbones
• Made from arachidonic acid
• Interferons
• Interleukins
• Chemotaxins
– Other minor molecules...
E.A. DENNIS 2016 ©
COX-1 and COX-2 Pathways
Figure: Fitzgerald and Patrano , NEJM , 2001, 345, 433-42.
E.A. DENNIS 2016 ©
Key Definitions
Arachidonic Acid (AA) = the precursor
fatty acid that is used to make most
prostaglandins, thromboxanes and
leukotrienes.
Prostaglandins (PG) = eicosanoids
having a bridge making a 5-carbon ring,
and either 1, 2 or 3 double bonds. Roughly
a dozen different PG’s have widely
different effects (not all covered here).
Arachidonic Acid
PGE2
PGI 2
PGF2a
E.A. DENNIS 2016 ©
More Definitions
Thromboxanes (TX) = eicosanoids that
have a 6-member oxygen-containing
ring. TXA2, a platelet activator, is the
main one.
COX = cyclooxygenase, the enzyme that
makes AA into PG’s and TX’s.
TXA2
Leukotriene (LT) = eicosanoids that
have an open backbone. LTA4 is the
intermediate. LTB4 is a major
chemotaxin and LTC4, D4 and E4 are
important in asthma.
5-LIPOX= 5-lipoxygenase, the enzyme
that makes AA into LT’s.
LTA4
E.A. DENNIS 2016 ©
Synthesis of PG’s and TX’s
Phospholipid containing
arachidonate
Phospholipase A2
cyclooxygenase
activity of COX
Lysophospholipid
Arachidonate
20:4(D5,8,11,14)
2O2
X
Aspirin, ibuprofen
• Key enzyme is
cyclooxygenase (COX)
• PGG2 is a transition state
• PGH2 is a stable
intermediate
– Rapidly made into specific
PG and TX end products
PGG2
peroxidase
activity
of COX
• Various conversion
enzymes make PGG2
and PGH2 into all the
other PG’s and TX’s
PGH2
Other prostaglandins
Thromboxanes
E.A. DENNIS 2016 ©
Structure & Naming of the Core PG’s
PGF1a
PGF2a
PGE2
PGF3a
E.A. DENNIS 2016 ©
Aspirin & Cyclooxygenase
• Aspirin donates an acetyl group
– Covalent binding and inhibition
• Enzyme is permanently wrecked
– Cell must translate more enzyme copies from
scratch
E.A. DENNIS 2016 ©
Aspirin vs. Heart Attacks & Strokes
• Incidence: Very common, a daily
event
• Symptoms: Tissue ischemia
– Strokes: loss of neurological function
– Heart attacks: chest pain & shock
• Mechanism: Platelet thrombosis
– TXA2 is a potent platelet activator
• Treatments: Block platelet
aggregation
– Aspirin prevents platelet TXA2
production
• Small daily dose safely cuts risks
• Note, PGI2 made in endothelial cells
causes vasodilation-opposite of TXA2
– Anticoagulants (e.g. thrombin inhibitors)
• Heparin, Coumadin, tPA
– Other anti-platelet drugs
– Fish oil (n-3) makes TXA3 (inactive)
Platelet thrombosis (clotting) biochemistry.
Figure: Arch of Internal Med, Weitz et al, 2000 ,160, 749-58.
E.A. DENNIS 2016 ©
Introduction to COX-1 and COX-2
Somewhat oversimplified
Figure: Wolfe et al NEJM, 1999, 340:1888-9.
E.A. DENNIS 2016 ©
NSAIDs
•
•
•
•
NSAID = “non-steroidal anti-inflammatory drug”
All inhibit PG production
Non-selective NSAIDs
COX-2 selective NSAIDS
– Only slightly inhibit COX-1
– Fewer GI side effects
The selective COX-2 inhibitors introduced a novel
strategy for the prevention of NSAID-related
gastroduodenal toxicity in high-risk patients.
However, cardiovascular toxicity has limited the
use of these drugs, and make it likely that they will
have a diminishing clinical role. Currently, there is
only one available.
• celecoxib (Celebrex) is approved by Food and Drug
Administration (FDA) but carries a new boxed
warning about GI and cardiovascular risk.
• valdecoxib (Bextra) was removed because of concerns
of cardiovascular risk and reports of Stevens-Johnson
Syndrome.
• refocoxib (Vioxx) was removed by Merck due to an
increased risk of stroke and myocardial infarctions with
long-term use.
E.A. DENNIS 2016 ©
Peptic Ulcers and NSAIDs
• Incidence: Common
• Symptoms: Stomach problems
– Stomach pain
– Vomiting, often with blood
– Sometimes: anemia and death
• Mechanism: No mucosal layer
– NSAIDs block production of protective
layer of mucous
• Treatments: In patients requiring long
term a/o high dose NSAID therapy:
– Give acid-lowering drugs like the
proton pump inhibitors (PPI).
– Give Misoprostol, a synthetic
prostaglandin E1 analog. It replaces
the protective prostaglandins
consumed with NSAID therapies
– Consider H. pylori testing before
beginning long term a/o high dose
NSAID therapy. (More during Micro.)
Direct and, in some cases, indirect
– In certain patients, a selective COX-2
(metabolite) effects of NSAIDs reduce
inhibitors might still be considered.
mucosal protection and increase ulcer risk.
Figure: Wolfe et al NEJM, 1999, 340:1888-9.
E.A. DENNIS 2016 ©
“Relative Selective” Among COX-2 Inhibitors
• 50% inhibition
concentration of
several NSAIDs.
• COX-2 inhibitors:
– Celecoxib
– Rofecoxib
• Relative, not
absolute
selectivity
Figure: Fitzgerald and Patrano , NEJM , 2001, 345, 433-42.
E.A. DENNIS 2016 ©
Synthesis of LT’s
5-lipoxygenase
FLAP
Arachidonate
20:4(D5,8,11,14)
O2
O2
5-lipoxygenase
FLAP
12S-Hydroperoxyeicosatetraenoate
(12S-HpETE)
Other Leukotrienes
• 5-Lipoxygenase (5-LIPOX) is the key
enzyme
– competes with COX for substrate
• 5-HPETE vs. 12-HPETE pathways
5S-Hydroperoxyeicosatetraenoate
(5S-HpETE)
Leukotriene A4
(LTA4)
LTC4
LTD4
E.A. DENNIS 2016 ©
Structure & Naming of Core LT’s
LTA4
Gly-Cys-Glu
LTE4
SH
LTC4
LTD4
E.A. DENNIS 2016 ©
Leukotrienes & Asthma
Figure: NEJM:“Drug Therapy: Treatment of Asthma with Drugs Modifying the Leuktriene Pathway” Drazen et al. Vol 340:3; 1999.
E.A. DENNIS 2016 ©
Aspirin-Triggered Asthma
• Incidence: ~1 in 7 children have
asthma
– Triggers include allergens, exercise & aspirin
• Symptoms: Bronchoconstriction
• Mechanism: Surge in LT production
– Aspirin blocks COX, leaving 5-LIPOX open
– Arachidonic acid substrate becomes LT’s
Arachidonic Acid
Arachidonic
LT’s
Asthma
PG’s
• Treatments: Reduce LT effects
–
–
–
–
Discontinue Aspirin and other NSAIDs
Give 5-LIPOX and LT receptor inhibitors
Use direct bronchodialators (e.g. albuterol)
Corticosteroids and anti-IgE antibodies
E.A. DENNIS 2016 ©
Eicosanoid Biosynthesis and Receptor Signaling
Dennis & Norris (2015) Eicosanoid Storm in Infection and Inflammation 15: 511-523
Therapeutics Targeting Eicosanoid Pathways
Dennis & Norris (2015) Eicosanoid Storm in Infection and Inflammation 15: 511-523
Arachidonic Acid Serves as a
Precursor for Many Eicosanoids
?
?
?
?
EPA/DHA
?
Are eicosanoids less pro-inflammatory when derived from EPA/DHA?
Proposed Steps in 2-carbon Elongation of AA and the
Subsequent Production of Dihomoprostaglandins
Harkewicz & Dennis
JBC 282, 2899 (2007)
Eicosanoid Signaling Pathways
in RAW264.7 Macrophage
ATP
ATP
LPS
(KLA)
Numerous
eicosanoid
metabolites
Buczynski et. al. (2007) JBC, 282, 22834
Effects of PUFA Supplementation on Fatty Acid
Membrane Distribution and Release by PLA2
Effects of PUFA Supplementation on Fatty Acid Release
KLA
ATP
Effects of PUFA Supplementation on TLR-4
Stimulated Eicosanoid Signaling
Fold Increase
Fold
Decrease
Not
detected
Effects of PUFA Supplementation on
TLR-4 Stimulated COX-2 Signaling
PNAS (2012) 109, 8517
Effects of PUFA Supplementation on ATP
Stimulated Eicosanoid Signaling
Fold Increase
Fold
Decrease
Not
detected
Effects of PUFA Supplementation on ATP
Stimulated COX-1 and 5-LOX Signaling
PNAS (2012) 109, 8517
Inhibition of COX Activity Correlates with
Degree of 22-carbon Saturation
COX Inhibition
15 min ATP
AdA
8 hr KLA
DPA
DHA
PNAS (2012) 109, 8517
Majority of Supplemented EPA is Incorporated in Membranes
as DPA in Primary Resident Macrophages
Global Summary of Fish Oil Effects
Supplement
Control
Membrane
AA
EPA
DHA
AA
AA AdA
AA EPA DPA
AA DHA
AA
AA AdA
AA EPA DPA
AA DHA
PLA2
TLR-4
COX-2
P2X7
COX-1 5-LOX
PG2
5-HETE
PG3
LT4
DihomoPG2
5-HEPE
TLR-4
COX-2
P2X7
COX-1 5-LOX
PG2
PG3
DihomoPG2
Decrease
5-HETE
LT4
5-HEPE
TLR-4
COX-2
P2X7
P2X7
TLR-4
COX-1 5-LOX COX-2
COX-1 5-LOX
PG2
PG3
5-HETE
LT4
DihomoPG2
5-HEPE
Dihomo- 5-HEPE
PG2 4/7-HDoHE
Increase
PNAS (2012) 109, 8517
No Change
PG2
5-HETE
PG3
LT4
Conclusions on Omega-3 Study
• The global effects of EPA and DHA on normal lipid
metabolism can be quantitatively studied.
• EPA and DHA affect the overall eicosadome
decreasing production of some, but not all, AAderived eicosanoids.
• There is a concomitant increase in specific EPAand DHA-derived metabolites.
• cPLA2 releases EPA/DHA from membrane
phospholipids.
• Deciphering the role of fish oil-derived ω-3 EPA
and DHA in inflammatory eicosanoid signaling
provides insight as to their role as therapeutic
agents in human disease.