BIOM 209/CHEM 210/PHARM 209
Lipid Cell Signaling Genomics, Proteomics, and
Metabolomics
Fatty Acid Metabolism, Signaling, and Lipidomics
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 ©
The Need for “Metabolomics”
“Premiums in the shape of sensational discoveries may
be hoped for, but cannot be assured even to the greatest
genius. But what has to penetrate, relative to this
question, more completely into the consciousness of
pathologists, is this, that to understand zymoses, to be
able to counteract them by rational, as distinguished from
empirical or accidentally discovered means, is only
possible by the aid of a complete knowledge of the
chemical constitution of all the tissues, organs and juices
of the body, and of all their possible products.”
Johann Ludwig Wilhelm Thudichum (1829-1901)
A Treatise on the Chemical Constitution of the Brain (1884)
Quoted from [Joseph Needham (1971) The Chemistry of Life,
Cambridge Univ Press p. 199]
LIPID MAPS
T
M
E.A. DENNIS 2016 ©
WHY LIPIDS AND LIPIDOMICS?
•
Kinds of Biological Molecules
1.
2.
3.
4.
Nucleic Acids
Amino Acids
Carbohydrates
Lipids (Fats)
•
Lipid Categories
1.
2.
3.
4.
5.
6.
7.
8.
Fatty Acyls
Glycerolipids
Glycerophospholipids
Sphingolipids
Sterol Lipids
Prenol Lipids
Saccharolipids
Polyketides
E.A. DENNIS 2016 ©
www.lipidmaps.org
Screenshot 1-5-16
LIPID MAPS Quick Search
3) Select the structure you wish to view
1) Enter item in Quick Search
(upper right hand corner of home page)
2) Select the structure database list
4) View the structure & details
Lipidomics Components
Lipid
Data
Genes
Pathways
Informatics
Infrastructure
Annotations
Proteins
www.lipidmaps.org
Mass Spec
Lipid
Standards
E.A. DENNIS 2016 ©
LIPID MAPS
www.lipidmaps.org
TM
Metabolism and Energy Overview
•
Proteins
Carbohydrates
Lipids
•
•
Amino
Acids
Simple
Sugars
Fatty
Acids
•
Many biomolecules are
degraded to Acetyl CoA
Acetyl CoA provides
biologic energy
Excess acetyl CoA is
stored as Fatty Acids
(FA’s)
FA’s are assembled into
more complex lipids like
triglycerides (TG’s)
Pyruvate
Acetyl CoA
Energy
(CO2, H2O)
E.A. DENNIS 2016 ©
What is a “Fatty Acid”?
Palmitic acid
Fatty acid: a carboxylic acid with a long hydrocarbon chain. Usually,
they have an even number of carbons. Reactive and toxic.
Ester group
Fatty acid ester: a fatty acid in which the carboxylic acid
group has reacted with the alcohol group of another molecule
(often glycerol) to form a stable, less reactive ester bond.
E.A. DENNIS 2016 ©
What is a “Triglyceride”?
Glycerol: common name for
1,2,3-trihydroxy-propane.
Glycerol
Triglyceride: a glycerol
molecule with three esterfied
fatty acid side chains. Also
known more correctly as a
“triacylglycerol”. Stable, nonpolar, hydrophobic.
Triacylglycerol
E.A. DENNIS 2016 ©
Common Saturated Fatty Acids
Saturated FA’s have no double bonds
16 Carbons = Palmitic Acid (Palmitate)
18 Carbons = Stearic Acid (Stearate)
E.A. DENNIS 2016 ©
Common Unsaturated Fatty Acids
Unsaturated FA’s have at least one double bond,
usually in the Z (cis) conformation
18 Carbons, 1 double bond at c9 = Oleic Acid (Oleate)
18
9
1
18 Carbons, 2 double bonds at c9 and c12 = Linoleic Acid (Linoleate)
18
12
9
1
E.A. DENNIS 2016 ©
More Unsaturated Fatty Acids
18 Carbons, 3 cis double bonds at 9, 12 & 15 =
a-Linolenic Acid (a-Linolenate)
18
15
12
9
1
20 Carbons, 4 cis double bonds at 5,8,11 & 14
Arachidonic Acid (Arachidonate)
(5Z,8Z,11Z,14Z-Eicosatetraenoic Acid)
E.A. DENNIS 2016 ©
What are Essential Fatty Acids?
• Two “Essential” FA’s
cannot be synthesized
by humans
Diet
Linoleic
acid
Linolenic
acid
Arachidonic
acid
EPA
– Linoleic acid
– Linolenic acid
• Used in the
biosynthesis of
polyunsaturated fatty
acid
• Must come from diet
E.A. DENNIS 2016 ©
Key Enzyme: Acetyl-CoA Carboxylase
• Acetyl-CoA Carboxylase is
a key enzyme
• Converts acetyl-CoA into
malonyl-CoA
– The CO2 is released
later
– Biotin is a cofactor
• It is the “committed step”
in FA synthesis
• It is the regulated, ratelimiting enzyme in FA
synthesis
“E” above is the enzyme acetyl-CoA
carboxylase, which is conjugated to biotin.
E.A. DENNIS 2016 ©
Key Enzyme Complex: FA Synthase
8 Acetyl CoA
Palmitic Acid + 8 CoASH
HS
Cytosolic complex
of enzymes.
Steps 1 - 6 happen
iteratively in a colocated complex of
enzymes.
ACP** is the carrier
protein that holds the
carbon backbone.
Contains 4’phosphopantetheine.
ACP stands for Acyl Carrier Protein. (10,000 kDa, Ser 36)
Figure adapted from Nelson DL, Cox MM (2005), Lehninger Principles of Biochemistry, 4th ed. W.H. Freeman & Co.
E.A. DENNIS 2016 ©
FA Synthesis: Step 1
Step 1: Set up acetyl-ACP
Acetyl-CoA-ACP
Transacylase
ACP is “acyl carrier protein” and is
a part of a large enzyme complex.
It holds the reactants in place
while other enzymes catalyze the
subsequent reaction steps.
E.A. DENNIS 2016 ©
FA Synthesis: Step 2
Step 2: Set up malonyl-ACP
Acetyl-CoA-ACP
Transacylase
HCO3-
Acetyl-CoA
Carboxylase
Malonyl-CoA-ACP
Transacylase
This step will iterate many times, adding carbons to the growing FA backbone.
E.A. DENNIS 2016 ©
FA Synthesis: Step 3
Step 3: Condense them, giving Acetoacetyl-ACP and CO2
condensing enzyme
CO2
ACP
The condensing enzyme is also known as b-ketoacyl-ACP
synthase. It is part of the FA synthase complex
E.A. DENNIS 2016 ©
FA Synthesis: Step 4
Step 4: Use NADPH to reduce the b-carbonyl to a hydroxyl group
H+ + NADPH
b-ketoacyl-ACP
reductase
NADP+
E.A. DENNIS 2016 ©
FA Synthesis: Step 5
Step 5: Remove the hydroxyl group as H2O leaving a double bond
b-hydroxylacyl-ACP
dehydratase
E.A. DENNIS 2016 ©
FA Synthesis: Step 6
Step 6: Use another NADPH to reduce the double bond
H+ + NADPH
Enoyl-ACP
reductase
NADP +
E.A. DENNIS 2016 ©
FA Synthesis: Repeat Cycle
Repeat from step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP
recycle reactions 2-6
six more times
thioesterase
• 6 iterations
makes
Palmitoyl-ACP.
• Finally, the
enzyme
thioesterase
cleaves the ACP
from palmitoyl
• Palmitate is
released.
E.A. DENNIS 2016 ©
Fatty Acid Synthesis Summary
One iteration:
Step 1: Set up acetyl-ACP
Step 2: Set up malonyl-ACP
Step 3: Condense them, giving Acetoacetyl ACP
Step 4: Use NADPH to reduce distal carbonyl to a hydroxyl group
Step 5: Remove the hydroxyl group as H2O leaving a double bond
Step 6: Use another NADPH to reduce the double bond
REPEAT: From step 2 using the new 4-carbon butyryl-ACP in
place of acetyl-ACP in step 3
E.A. DENNIS 2016 ©
[2a]
Acetyl-CoA
Carboxylase
Acetyl-CoA enters cycle
Acetyl-CoA
[1]
Malonyl-CoA
Acetyl-CoA-ACP transacylase
Malonyl-CoA-ACP transacylase
[2b]
Initiation
Acetyl-ACP
Malonyl-ACP
Fatty acid
synthase cycle
[3]
b-ketoacylACP synthase
Release from
FA synthase complex
Acetoacetyl-ACP
[4]
b-ketoacyl-ACP reductase
Elongation
Palmitoyl-ACP
b-hydroxybutyrylACP
Butyryl-ACP
thioesterase
[6]
Palmitate
enoyl-ACP reductase
b-hydroxyacyl-ACP dehydratase
2-trans-butenoyl-ACP
[5]
E.A. DENNIS 2016 ©
The FA Synthase Enzyme
• FA synthase is an enzyme
complex
• It includes all the FA synthesis
enzymes except for acetyl-CoA
carboxylase
• Actually exists as a dimer of two
complete, anti-parallel complexes
-- like Ying and Yang
• Cytosolic
Figure: Voet, D, Voet JG, Pratt CW (2002),
Fundamentals of Biochemistry: Life at the
Molecular Level, 2nd ed. Reprinted with
permission of John Wiley & Sons, Inc.
Figures: Nelson DL, Cox MM (2005), Lehninger Principles of
Biochemistry, 4th ed. W.H. Freeman & Co.
E.A. DENNIS 2016 ©
Tuberculosis cases (thousands)
Tuberculosis
Reported Tuberculosis in the US 1982-2008
AIDS increase
50% decrease
• Incidence: one of the leading
causes of death due to infectious
diseases
– Often follows HIV infection
• Symptoms: pulmonary infection
– cough, sputum, pleural effusions
• see picture below left
– urogenital & brain affects also seen
Year
• Mechanism: Mycobacterium
tuberculosis infection
• Treatments:
– First-line combination:
• Pyrazinamide and Isoniazid
– stop mycobacterial FA synthase!
• Rifampin (RNA transcription inhibitor)
– Various second-line agents
– If needed, HIV treatment
Normal lung
Source: CDC
Tuberculosis infection
E.A. DENNIS 2016 ©
Treating Tuberculosis
• Mycobacteria
– make their outer membrane with
mycolic acids using FA synthases
• FAS-1 is a single, eukaryote-like
enzyme with multiple actions
• FAS-2 is a multi-unit, prokaryote-like
enzyme with multiple actions
• Pyrazinamide
Figure: Draper, Nat. Med. 6, 977-8 (2000).
Mycolic acid;
R1 and R2 are
long-chain
aliphatic
hydrocarbons
(“peer-ah-ZIN-a-mide”)
– Inhibits FAS-I
– Relatively specific for M. tuberculosis
– Arrests synthesis of both fatty acids
and mycolic acids
• Isoniazid (“eye-so-NYE-a-zid”)
– Inhibits FAS-II
– Stops synthesis of mycolic acids
E.A. DENNIS 2016 ©
Bacteria vs. Mammals
FA Synthesis in Bacteria
FA Synthesis in Mammals
• Acyl carrier protein (ACP)
is a separate molecule, as
are each of the six
enzymes.
• ACP is part of the FA
synthase complex, which
is one large protein
present as a dimer.
• “Acetyl CoA carboxylase”
is two separate enzymes,
plus a biotin cofactor
joined to a third enzyme.
• Acetyl-CoA carboxylase is
one enzyme conjugated to
a biotin cofactor.
E.A. DENNIS 2016 ©
Regulation of FA Synthesis
Regulation occurs primarily at acetylCoA carboxylase, the rate limiting step
Feedback Mechanisms
Hormonal Mechanisms
• Citrate, which builds up
when acetyl-CoA is
plentiful, accelerates FA
synthesis.
• Insulin, which signals a resting,
energy rich state, dephosphorylates and accelerates the enzyme.
• Palmitoyl-CoA weakly
inhibits FA synthesis.
• Glucagon, epinephrine and
norepinephrine, which signal
immediate energy needs, phosphorylate and slow the enzyme
[via AMP -dependent protein
kinase and also via CMPdependent PKA].
E.A. DENNIS 2016 ©
Metabolism and Energy Overview
Proteins
Amino
Acids
Carbohydrates
Lipids
Simple
Sugars
Fatty
Acids
Pyruvate
Acetyl CoA
• Triglycerides (TG’s) are
decomposed into Fatty
Acids (FA’s).
• FA’s are b-oxidized to
release energy and create
acetyl CoA fragments.
• Acetyl CoA enters the
power-producing Krebs
cycle and electron
transport chain.
Energy
(CO2, H2O)
E.A. DENNIS 2016 ©
Naming Conventions: Palmitic
Acid
16
6
e

d
5
4
g
b
3
2
1
a
Carbonyl carbon

e
d
g
b
a
omega, always the last alkyl carbon
epsilon, fifth carbon after the carbonyl
delta, fourth carbon after the carbonyl
gamma, third carbon after the carbonyl
beta, second carbon after the carbonyl
alpha, first carbon after the carbonyl
E.A. DENNIS 2016 ©
Beta Oxidation In a Nutshell...
Fatty Acid
One iteration of b-Oxidation:
Make fatty acyl CoA.
Acyl CoA
(1)
Step 1: Oxidize the b-carbon (C3)
Enoyl CoA
(2)
L-hydroxyacyl CoA
(3)
Ketoacyl CoA
(4)
Step 2: Hydrate the b-carbon
Step 3: Oxidize the b-carbon, again!
Step 4: Thiolyze a-b bond, releasing acetyl CoA
REPEAT from step 1, w/ 2 fewer carbons
Acyl CoA
(shorter)
Acetyl CoA
TCA Cycle
E.A. DENNIS 2016 ©
Make activated acyl CoA
Triglycerides
Hormone sensitive
lipase
Free FA’s
adipocyte
Albumin
• Hormone-sensitive lipase
– decomposes triglycerides
– removes FA groups
– free FA’s diffuse through
membrane
• Albumin
Free FA’s
– carries FA’s to target tissue
bloodstream
Fatty acyl CoA
synthase
Free FA’s
– FA’s diffuse into tissue cells
Acyl CoA
• Fatty acyl CoA synthase
– adds CoA to the free FA
mitochondria
Acyl CoA
• Carnitine shuttle
Carnitine
shuttle
– moves fatty acyl CoA into
the mitochondria
target tissue cell
E.A. DENNIS 2016 ©
Regulation
• Hormone Sensitive Lipase is activated by cAMP
• Cyclic AMP also turns off acetyl CoA
carboxylase, stopping FA synthesis
• Hormones like glucagon and epinephrine
increase cAMP
– FA synthesis slows
– Triglycerides are broken down
– FA’s enter b-oxidation faster
E.A. DENNIS 2016 ©
Location, Location, Location
• Fatty Acyl CoA is oxidized inside the
mitochondrial matrix
• The carnitine shuttle moves it into
the matrix
– Free carnitine is exchanged for acyl
carnitine
• Carnitine acyl transferases catalyze
reactions on both sides of membrane
– driven by concentration gradient
• CAT-1 is inhibited by high levels of
malonyl CoA generated by acetyl CoA
carboxylase in the pathway to Fatty
Acid Synthesis.
Figure: Voet, D, Voet JG, Pratt CW (2006), Fundamentals of Biochemistry: Life at the Molecular Level, 2 nd ed. Reprinted
with permission of John Wiley & Sons, Inc.
E.A. DENNIS 2016 ©
Step 1: Oxidize the b-carbon
• Acyl CoA dehydrogenase
oxidizes the b-carbon
Fatty acyl CoA
FAD
Acyl CoA
dehydrogenase
FADH2
– 3 versions of the enzyme
exist
– Specific to short, medium
and long chain substrates
• One FADH2 is generated
– makes 2 ATP’s
• A trans double bond is
created at the 2-carbon
• Product is an Enoyl CoA
D2-trans-enoyl CoA
E.A. DENNIS 2016 ©
Medium-chain dehydrogenase deficiency
• Incidence: 1 in 10,000 live births
Fatty Acid
Acyl CoA
(1)
Enoyl CoA
(2)
L-hydroxyacyl CoA
(3)
Ketoacyl CoA
(4)
Acyl CoA
(shorter)
– More common than phenylketonuria!
• Symptoms:
– severe hypoglycemia >> lethargy, coma
• little energy from FA’s
• glucose reserves are immediately burned
– contributes to sudden infant death syndrome (10%
of cases)
• Mechanism:
– Normally, there are 3 separate fatty acyl CoA
dehydrogenase enzymes for STEP 1 of b-oxidation
• Specific for short, medium and long acyl chains, respectively
– Autosomal recessive lack of medium chain enzyme
• Treatment: Special diet and supportive care
E.A. DENNIS 2016 ©
Step 2: Hydrate the b-carbon
• Enoyl CoA hydratase
adds a water molecule
across the double bond
• Product is b-hydroxyacyl
CoA
– S- stereoisomer
E.A. DENNIS 2016 ©
Step 3: Oxidize the b-carbon,
Again!
• Beta-hydroxyacyl CoA
dehydrogenase
oxidizes the b-carbon
again
• One NADH is created
– makes 3 ATP’s
• A ketoacyl CoA is
produced
E.A. DENNIS 2016 ©
Step 4: Thiolyze off acetyl CoA
CoA-SH
Acyl CoA:
Acyltransferase
(“thiolase”)
• Thiolase (aka: Acyl
CoA acyltranferase)
splits the ketoacyl
• A new CoASH is
consumed
• Acetyl CoA is released
• A new, shorter Acyl
CoA remains and reenters the cycle
E.A. DENNIS 2016 ©
REPEAT with a Shorter Acyl CoA
• Palmitoyl CoA (16 carbons) becomes
myristoyl CoA (14 carbons).
• Each iteration releases another acetyl CoA.
• 7 iterations will release 8 acetyl CoA
fragments from the original palmitoyl CoA.
• Net equation:
Palmitoyl CoA + 7CoASH + 7FAD+ + 7NAD+ + 7H20
yields: 8 Acetyl CoA + 7 FADH2 + 7NADH + 7H+
E.A. DENNIS 2016 ©
b-Oxidation Cycle
Removal of 2 carbons
from acyl chain
Fatty acyl-CoA
Acyl CoA
dehydrogenase
[4]
Acyl CoA:
acyltransferase
[1]
D2-trans-enoyl CoA
b-ketoacyl CoA
Acetyl-CoA
[3]
b-hydroxyacyl CoA
dehydrogenase
enoyl CoA
hydratase
[2]
TCA cycle
b-hydroxyacyl CoA
E.A. DENNIS 2016 ©
How Much Energy?
Each palmitoyl CoA group released from a triglyceride:
– directly produces 7 NADH & 7 FADH2
• which generate 21 and 14 ATP’s, respectively
– releases 8 acetyl CoA molecules for TCA Cycle
• which each generate 12 ATP
Grand total is 131 ATP per fully oxidized palmitoyl CoA
Efficiency of Energy Recovery = 40%
** Recently, some have calculated energetically less ATP
equivalents per NADH/FADH2 [2.5 and 1.5 instead of 3 and 2]
which lowers the numbers somewhat (Berg).
E.A. DENNIS 2016 ©
Efficiency of Fat Storage
Fat has 9 kcal/gram = 9 kcal/cc
Both Carbohydrate and Protein have 4
kcal/gram = 4 kcal/3cc or 1.33 kcal/cc
Fat/Carbohydrate or Protein = 6x/cc!!!
E.A. DENNIS 2016 ©
Problem: Oxidizing Unsaturated FA’s
Linoleic Acid
C12 (Even #Unsat)
C9 (Odd # Unsat)
3 NAD+ + 3FAD + CoA-SH
3 rounds of b- oxidation
3 NADH + 3FADH2 + 3Acetyl-CoA
Problem: b-g cis
double bond
E.A. DENNIS 2016 ©
Special Case 1: Odd Unsaturations
The GOOD News:
• Enzyme enoyl-CoA
isomerase moves the double
bond over 1 position.
• Oxidation process resumes
The BAD News:
• The first oxidation step is
skipped
• One less FADH2 is made
– 2 fewer ATPs
E.A. DENNIS 2016 ©
Special Case 2: Even Unsaturations
4
2
The GOOD News:
• Enzymes 2,4 dienoyl-CoA
reductase and 3,2 dienoyl-CoA
isomerase convert the g-d and
a-b double bonds into a single
a-b unsaturation.
• Oxidation process resumes
3
H+ + NADPH
NADP+
3
The BAD News:
4
2
3
4
• The conversion costs one
NADPH directly.
• Net effect is the loss of one
NADH
– 3 fewer ATPs
2
E.A. DENNIS 2016 ©
FA Synthesis vs. FA Oxidation
Synthesis
Oxidation
Cytosol
Mitochondria
ACP
CoA
Malonyl CoA
Acetyl CoA
b-hydroxyl acyl step
R-config
S-config
Electron carriers
NADPH
NADH, FADH
Liver
Muscle, liver
Cell Location
Acyl carrier
2-Carbon Piece
Primary tissue site
E.A. DENNIS 2016 ©
FA Synthesis vs. FA Oxidation
(cont)
1. They are not the reverse of one another.
– Different subcellular locations
– L and D isomers of b-hydroxyacyl intermediates
cannot easily jump to the other pathway
– Electrons donated from oxidation (NADH, FADH2)
cannot directly enter synthesis, which uses NADPH.
2. Separate, semi-independent pathways allow
more sophisticated regulation.
– Accelerating one pathway does not mean slowing
the other.
E.A. DENNIS 2016 ©
Dennis et al (2010)
J. Biol. Chem, 51, 39976-85