Chapter 5.
Metabolism of Lipids

Lipids
Insoluble or immiscible

Triacylgerols
store and supply energy for metabolism.

Lipoids: phospholids, glycolipids, cholesterol and
cholesterol ester
membrane components
Metabolism of lipid

Fatty acids
esterified to some
backbone
molecules
glycerol
sphingosine
cholesterol
Metabolism of Lipids

Fats
store in adipose tissue


Essential fatty acids: formation of
membrane, regulation of chollesterol
metabolism, precursors of eicosanoids
(protaglandins, thromboxanes and
leukotrienes.
Necessary unsaturated fatty acids
Fat Facts
Dietary lipids are 90% triacylglycerols; also include
cholesterol esters, phospholipids, essential unsaturated fatty
acids; fat soluble vitamins (A,D,E,K)
Fat is energy rich and provides 9 kcal/gm
Normally essentially all (98%) of the fat consumed is
absorbed, and most is transported to adipose for storage.
SIX STEPS OF LIPID DIGESTION AND ABSORPTION
Minor digestion of triacylglycerols in mouth and stomach by lingual (acidstable) lipase
Major digestion of all lipids in the lumen of the duodenum/jejunum by
pancreatic lipolytic enzymes
Bile acid facilitated formation of mixed micelles that present the lipolytic
products to the mucosal surface, followed later by enterohepatic bile
acid recycling
Passive absorption of the lipolytic products from the mixed micelle into the
intestinal epithelial cell
Reesterification of 2-monoacylglycerol, lysolecithin, and cholesterol with
free fatty acids inside the intestinal enterocyte
Assembly and export from intestinal cells to the lymphatics of
chylomicrons coated with Apo B48 and containing triacylglycerols,
cholesterol esters and phospholipids
Summary of the physiologically important lipases
Lipase
Site of
Action
Regulation
Preferred
Substrate
C
cleaved
Product(s)
----
TAGs with med.
chain FAs
3
FFA+DAG
lingual/acidstable lipase
mouth,
stomach
pancreatic
lipase
small
intestine
colipase (+)
milk lipase
small
intestine
bile acids (+) TAGs with med.
chain FAs
phospholipase small
A2 (PLA2)
intestine
bile acids (+) PLs with unsat.
Ca2+ (+)
FA on position 2
lipoprotein
lipase
apo CII (+)
insulin (+)
TAGs in chylomicron or VLDL
insulin (-)
glucagon (+)
Epineph. (+)
TAG stored in
adipose cells
capillary
walls
Hormone-sens. adipose cell
Lipase
TAGs with longchain FAs
1 and 3 FFA+2MG
1 and 2 FFA+
and 3
glycerol
2
1 and 2
and 3
3
Unsat FFA
lysolecithin
FFA+
glycerol
FFA+DAG
Absorption of Lipids

Metabolism of Triacylglyerols

LIPOLYSIS
Mobilization of fats from triacylglycerols
Hormone sensitive lipase
Rate-determining step
Specific for removing first fatty acid
Phosphorylated form is active
cell
membrane
Fatty acid +
Diacylglycerol
Triacylglycerol
HORMONES
Epinephrine
Glucagon
RECEPTORS
ATP
ADP
Adenylyl
cyclase
cyclic
AMP
HSL-a
OP
Insulin
+
active
+
protein
kinase A
protein
phosphatase
inactive
ATP
+ = activation
- = inhibition
HSL-b
phosphodiesterase
OH
- caffeine
(inactive form)
theophylline
AMP
HSL = hormone-sensitive lipase
Pi
Figure 1. Hormonal activation of triacylglycerol (hormone-sensitive) lipase.
Phosphorylation brings about activation to HSL-a.
lipolysis

Glycerols and fatty acids
diffuse out of adipose cells and enter into circulation

Free fatty acids (FFA)
form fatty acid-albumin complexes

Glycerols
to form dihydroxyacetone phosphate (DHAP)
Figure. Page 176
Beta-Oxidation of Fatty Acids

Beta Oxidation Part I
The break down of a fatty acid to acetyl-CoA
units…the ‘glycolysis’ of fatty acids
STRICTLY AEROBIC
Occurs in the mitochondria
Acetyl-CoA is fed directly into the Krebs cycle
Overproduction causes KETOSIS
Exemplifies Aerobic Metabolism
at its most powerful phase
CH3CH2CH2COOH
ATP
PPi
[CH3CH2CH2CO-AMP]
HS-CoA
AMP
Acyl-CoA
synthetase
CH3CH2CH2CO~SCoA
Fatty acyl CoA
Prepares a Fatty Acid for transport and metabolism
Knoop’s Experiment
Diet
(even chain)
(odd chain)
CH2CH2CH2COO
CH2CH2COO
Urine
CH2COO
Phenylpyruvate
Phenylacetate
COO
Benzoate
Benzoate
Beta-Oxidation of Fatty Acids

THE ENERGY STORY
Glucose
C6H12O6 + 6O2  6CO2 + 6H2O Ho = -2,813 kJ/mol
= - 672 Cal/mol
= 3.74 Cal/gram
Stearic Acid
C18H36O2 + 26O2  18CO2 + 18 H2O Ho = -11,441 kJ/mol
= - 2,737 Cal/mol
= 9.64 Cal/gram
On a per mole basis a typical fatty acid is
4 times more energy rich that a typical hexose
Sample calculation of energy produced
for the cell via b-oxidation of palmitate
(a C16 fatty acid):
Palmitoyl-CoA
Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O
8 Acetyl-CoA
80 ATP
7 FADH2
10.5 ATP
7 NADH + 7H+
17.5 ATP
108 ATP
-2 ATP
Total 106 ATP
Beta Oxidation Part II
3 Obstacles
Unsaturated fatty acid
Obstacle of cis double bonds
Polyunsaturated fatty acid
Obstacle of position of double bond
Odd number chain fatty acid
Obstacle of 3 carbons at the end
Oleic Acid
C18:cis9
4
3
2
1
H H
C=C
CH3CH2CH2CH2CH2C
CH2CH2CH2CH2CH2CH2CH2CO~SCoA
Whoops!
A cis D.B. will interfere
Linoleic
5
4
3
2
1
H H H H
C=C C=C
CH3CH2CH2CH2 CH2 CH2CH2CH2CH2CH2CH2CH2CO~SCoA
Unsaturated and Polyunsaturated Require
Additional Enzymes
New b carbon
Cleavage here
CH3CH2CH2
Enoyl CoA
Isomerase
H H 9
C=C 8 7
CH2CH2CH2-CO~SCoA
New COO group
9
H 8 7
CH2C
CH3CH2CH2
C-CO~SCoA
H
Trans double bond
4
H H
C=C
CH2-CH2
3
2
1
H H 9
C=C
CH2
CH2C~SCoA
-CH2-CH
CH22-CH
C~SCoA
CH22C~SCoA
-CH2CH2C~SCoA
2-CH
O
O
O
O
Linoleic Acid C18 cis 9,12
6
5
CH3C~SCoA
O
4
3
CH3C~SCoA
O
2
1
CH3C~SCoA
O
Poly Unsaturated (Continued)
9
H H
H H
C=C C=C
-CH2
CH2
CH2CO~SCoA
H H
H H
C=C C-C
-CH2
CH2
C-CO~SCoA
Round 4
starter
Enoyl-CoA
isomerase
H
H H
C=C CH2CO~SCoA
-CH2
CH2
Beta carbon to be
Round 5
starter
H H
C=C CH2CO~SCoA
-CH2
CH2
FAD
beta 6
FADH2
H
C-CO~SCoA
H H
C=C
C
H
Round 5
starter
Acyl-CoA
dehydrogenase
H H
C=C
CO~SCoA
CH2
Dead end
New Strategy
Reduce near (bond), Shift far (bond)
H
C-CO~SCoA
H H
C=C
C
H
NADPH + H+
NADP+
beta 6
2,4 dienoyl-CoA
reductase
H
CH2 C
beta 6
C
H
CH2CO~SCoA
-CH2 CH2
C
H
beta 6
H
C-CO~SCoA
Continue Beta Oxidation
3,2 enoyl-CoA
isomerase
Ketone bodies formation and
utilization
What is Ketosis?
An excessive production of ketones in the blood
3 derivatives of acetyl-CoA
Acetoacetate
b-hydroxybutyrate
Acetone
CH3CCH2COOO
b
H
CH3CCH2COOOH
O
CH3-C-CH3
What is the Significance of
ketosis
Acidosis
Excessive acid in the blood
Overflow
Excessive oxidation of fatty acids
Metabolic Problem
Faulty Carbohydrate Metabolism
Metabolic fate of Acetyl CoA
Pyruvate
minor
Fatty Acids
Acetyl-CoA
major
Citrate
Ketone Bodies
CH3C~SCoA
O
CH3C~SCoA
O
HS-CoA
CH2C~SCoA
CH3C + O
rearrangement
OH
b-Ketothiolase
CH3CCH2C~SCoA
O
O
Acetoacetyl-CoA
CH3CCH2C~SCoA
O
O
HS-CoA
CH3C~SCoA
O
CH2C-OO
CH3CCH2C~SCoA
HO
O
OH
OOC-CH2-C-CH2-C~SCoA
CH3
O
HMG-CoA
Synthase
b-hydroxy-b-methyl
glutaryl-CoA
(HMG-CoA)
OH
OOC-CH2-C-CH2-C~SCoA
HMG-CoA
Acetoacetate
CH3
O
CH3-C~SCoA
O
OH
+ 2-C~SCoA HMG-CoA
OOC-CH2-C-CH
Lyase
CH3 O
OOC-CH2-C-CH3
O
CO2
NADH + H+
NAD+
CH3-C-CH3
OOC-CH2-CH-CH3
O
Acetone
OH
b-hydroxybutyrate
Utilization of ketone bodies
1.
2.
3.
Acetoacetate/succinyl-CoA CoA
transferase
Acetoacetyl-CoA thiokinase
Acetoacetyl-CoA thiolase
Page 180
Pysiological Significance of
ketogenesis


Ketone bodies produced by the liver are
excellent fuels for a variety of extrahepatic
tissues, especially during times of
prolonged starvation.
Reconversion of ketone bodies to acetylCoA inside the mitochondria provides
metabolic energy.
Regulation of Ketogenesis

Feeding status
In the hungry state, higher glucagon and other
lipolytic hormones trigger the lipolytic process
in adipose tissue with the result that free fatty
acids pass into the plasma for uptake by liver
and other tissues. This promotes fatty acid
oxidation and ketogenesis in the liver.
Regulation of Ketogenesis

Metabolism of glycogen in the hepatic cells
once fats enter the liver, they have two distinct fates:
activated to acyl-Co-A and oxidized, or esterified to
glycerol in the production of triacylglycerols in
cytoplasm. If the liver has sufficient supplies of glycerol3 phosphate by glucose metabolism, most of the fats will
be turned to the production of triacylglycerols. In
contrast, glucose deficiency will cause a lower
triacylglycerols and ATP generation, with the majority of
the FAs entering beta-oxidation leading to a increased
production of ketone bodies.
Regulation of Ketogenesis

The fall in malonyl-CoA concentration can terminate
the inhibition on carnitine acyltransferase I, such that
long-chain fatty acids can be transported through the
inner mitochondrial membrane to the enzymes of fatty
acid oxidation and ketogenesis. This may happen during
a hungry state. In contrast, administration of food after
a fast, or of insulin to the diabetic subject, reduces
plasma free fatty acid concentrations and increases liver
concentration of malonyl-CoA, this will inhibit carnitine
acyltransferase I and thus reverses the ketogenic process.
Fatty Acid Biosynthesis

Not exactly the reverse of degradation
by a different set of enzymes , in a different part of the cell

Primarily in the cytoplasm of the following
tissues: liver, kidney, adipose, central nervous
system and lactating mammary gland

Liver is the major organ for fatty
acid synthesis
LIPID BIOSYNTHESIS






Fatty acid biosynthesis-basic fundamentals
Fatty acid biosynthesis-elongation and
desaturation
Triacylglycerols
Phospholipids
Cholesterol
Cholesterol metabolism
Fatty Acid Biosynthesis
Synthesis






Cytosol
Requires NADPH
Acyl carrier protein
D-isomer
CO2 activation
Keto  saturated
Beta Oxidation






Mitochondria
NADH, FADH2
CoA
L-isomer
No CO2
Saturated  keto
Rule:
Fatty acid biosynthesis is a stepwise assembly
of acetyl-CoA units (mostly as malonyl-CoA)
ending with palmitate (C16 saturated)
3 Phases
Activation
Elongation
Termination
Cofactor
ACTIVATION
Biotin
O
CH3C~SCoA
HN
NH
O
CH2CH2CH2CH2CO
S
NHCH2CH2CH2CH2 ENZYME
HCO3-
ATP
Biocytin
ADP + Pi
-OOC-CH
2C~SCoA
O
O
LYS
CO2
O
NH
C N
O
active carbon
S
Acetyl-CoA carboxylase
CH2CH2CH2CH2CO
Carboxybiocytin
Acetyl-CoA Carboxylase
The rate-controlling enzyme of FA synthesis



In Bacteria -3 proteins
(1) Carrier protein with Biotin
(2) Biotin carboxylase
(3) Transcarboxylase
In Eukaryotes - 1 protein
(1) Single protein, 2 identical polypeptide
chains
(2) Each chain Mwt = 230,000 (230 kDa)
(3) Dimer inactive
(4) Activated by citrate which forms
filamentous form of protein that can be seen in
the electron microscope
Yeast Fatty Acid Synthase Complex
2,500 kDa Multienzyme Complex
6 molecules of 2 peptide chains called A and B
(6b6)
A: (185,000)
Acyl Carrier protein
b-ketoacyl-ACP synthase (condensing enzyme)
b-ketoacyl-ACP reductase
B: (175,000)
b-hydroxy-ACP dehydrase
enoyl-ACP reductase
palmitoyl thioesterase
Fatty Acid
Synthase
Complex
Acyl Carrier Protein
Phosphopantetheine
H
H HO CH3
O
HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-CH2-SerO
OH H
O
Cysteamine
ACP
Acyl carrier protein
10 kDa
H
H HO CH3
O O
HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-P-O-CH2
O
O
OH H
O O
Coenzyme A
O
O-P-O
OH
Adenine
H
OH
Initiation
Overall Reaction
Malonyl-CoA + ACP
-OOC-CH
CH3C~SCoA
2C~S-
O
O
CO2
HS-CoA
CH3C-CH2C~SO
ACP
+ HS-CoA
Acyl Carrier
Protein
ACP
O
NOTE:
Malonyl-CoA carbons become new COOH end
Nascent chain remains tethered to ACP
CO2, HS-CoA are released at each condensation
b-Carbon
Elongation
CH3C-CH2C~S-
NADPH
D isomer
O
Reduction
O
b-Ketoacyl-ACP reductase
H
CH3C-CH2C~SHO
O
-H2O
NADPH
ACP
ACP
Dehydration
b -Hydroxyacyl-ACP dehydrase
H
CH3C-= C- C~S- ACP
H
O
Enoyl-ACP reductase
CH3CH2CH2C~S- ACP
O
Reduction
TERMINATION
-KS
Transfer to Malonyl-CoA
Ketoacyl ACP
Synthase
Transfer to KS
-S-ACP
-CH2CH2CH2C~S- ACP
Free to bind
Malonyl-CoA
O
Split out CO2
CO2
When C16 stage is reached, instead of transferring to KS,
the transfer is to H2O and the fatty acid is released
Fatty Acid Synthase
O
S-C-CH2-CH2-CH3
b-Ketoacyl
-ACP synthase
KS
O
CH3-CH2-CH2-C-S
Acetyl-CoA
HS
CoA-SH
NADP+
Enoyl-ACP
reductase NADPH
H+
O
CH3-CH=CH-C-S
b-Hydroxyacyl-ACP H2O
KS
ACP
Initiation or
priming
O
S-C-CH3
SH
Malonyl-CoA
Malonyl-CoACoA-SH ACP transacylase
O
O
S -C-CH2-COO-
CH3-CH -CH2-C-S
b-Ketoacyl
-ACP reductase
O
S -C-CH3
KS -SH
dehydrase
OH
Acetyl-CoAACP transacylase
KS
NADP+
NADPH
H+
S
C=O
CH2
C=O
CH3
b-Keto-ACP
synthase (condensing enzyme)
CO2
KS -SH
Elongation
Overall Reactions
Acetyl-CoA + 7 malonyl-CoA + 14NADPH + 14H
7H++
Palmitate + 7CO2 + 14NADP+ + 8 HSCoA + 6H2O
7 Acetyl-CoA + 7CO2 + 7ATP
7 malonyl-CoA +7ADP + 7Pi + 7H+
8 Acetyl-CoA + 14NADPH + 7H+ + 7ATP
Palmitate + 14NADP+ + 8 HSCoA + 6H2O + 7ADP + 7Pi
PROBLEM:
Fatty acid biosynthesis takes place in the
cytosol. Acetyl-CoA is mainly in the
Mitochondria
acetyl-CoA
How is acetyl-CoA made available to the cytosolic
fatty acyl synthase?
SOLUTION:
Acetyl-CoA is delivered to cytosol from the
mitochondria as CITRATE
CH2COO
HO-C-COO
mitochondria
CH2COO
CH2COO
HO-C-COO
OAA
CO2
Pyr
Acetyl-CoA
Citrate lyase
COO
C=O
OAA
Malate
CH2
dehydrogenase
COO
NADH
CH2COO
Acetyl-CoA
HS-CoA
L-malate
CO2
COO
HO-C-H L-malate
CH2
COO Malic enzyme
NADP+
NADPH + H+
COO
C=O Pyruvate
CH3
Cytosol
Post-Synthesis Modifications

C16 satd fatty acid (Palmitate) is the product

Elongation
Unsaturation
Incorporation into triacylglycerols
Incorporation into acylglycerol phosphates



Elongation of Chain (two systems)
R-CH2CH2CH2C~SCoA Malonyl-CoA*
O
(cytosol)
HS-CoA
OOC-CH2C~SCoA CH3C~SCoA
CO2
O
O
Acetyl-CoA
(mitochondria)
R-CH2CH2CH2CCH2C~SCoA
O
O
1 NADPH
Elongation systems are
NADH
found in smooth ER and
2 - H2O
3 NADPH
mitochondria
R-CH2CH2CH2CH2CH2C~SCoA
O
Desaturation
Rules:
The fatty acid desaturation system is
in the smooth membranes of the endoplasmic
reticulum
There are 4 fatty acyl desaturase enzymes in
mammals designated 9 , 6, 5, and 4 fatty
acyl-CoA desaturase
Mammals cannot incorporate a double bond
beyond 9; plants can.
Mammals can synthesize long chain unsaturated
fatty acids using desaturation and elongation
Triacylglycerol
Synthesis






O
O-C-R
O
R-C-O
O
O-C-R
Fatty acyl-CoA
DHAP reduction to glycerol-PO4
or
Glycerol kinase to glycerol-PO4
Two esterifications
Diacylglycerol-PO4 intermediate
Triacylglycerol
Triacylglycerol Biosynthesis
glycolysis
CH2OH
C=O
CH2OP
DHAP
NADH + NAD+
CH2OH ADP ATP
CH2OH
HO-C-H
HO-C-H
glycerol kinase
CH
OP
CH2OH
2
glycerol-PO4
dehydrogenase
Glycerol-PO4
O
Not in adipose
tissue
O
CH2O-C-R
2 R-C~CoA
Phosphatidic
acid
O
R-C-O-C-H
CH2OP
H2O
PO4
O
R-C~CoA
O
CH2O-C-R
O
Phospholipid
R-C-O-C-H
biosynthesis
1,2 Diacylglycerol CH2OH
(DAG)
O
O CH2O-C-R
R-C-O-C-H O
CH2O-C-R
Question

Can a triacylglycerol (triglyceride) storage
fat be synthesized entirely from glucose,
i.e., every carbon in the fat comes from a
sugar?

Answer:
YES
Metabolism of Phospholipids

Phospholipid
phosphorous-containing lipids
fatty acids, a phosphate group, and a simple organic
molecule

Glycerolphospholipids (phosphoglycerides)
glycerol

Sphingolipid
sphingosine
Classification of structural
features of glycerolphospholipids
Table 8-2

Phospholipids
hydrophilic head , hydrophobic tail

Membrane
phospholipid bilayer
Glycerolphospholipids
Phospholipid
Biosynthesis
(smooth ER)
O
O CH2O-C-R
R-C-O-C-H
O
CH2OPO
O
Ester linkage
- or +
Phosphatidic
Acid
- or +
Polar component
= choline, serine, ethanolamine, etc
Glycerophospholipids
O
O CH2O-C-R
R-C-O-C-H O
+
CH2OPO-CH2CH2-N(CH3)3
O
Phosphatidylcholine or lecithin
Strategy of Glycerophospholipid
Biosynthesis


Activate diacylglycerol
NH2
Activate appending moiety (salvage)
N
O
N
PPP- Ribose
CTP
Eukaryotes
DHAP
1
Glycerol-3-PO4
2
ATP
Glycerol
FA-CoA
1-Acyl-DHAP
NADPH
1-Acyl-glycerol-3-PO4
O
O CH2O-C-R Phosphatidic acid
3
R-C-O-C-H
DAG
CH2OP
ATP
CTP
O
O CH2O-C-R
R-C-O-C-H
CH2OH
1,2 DAG
Pi
CDP-diacylglycerol
ethanolamine (CDP-ethanolamine)
choline (CDP-choline)
Serine (phosphatidylethanolamine)
Glycerol (CDP-diacylglycerol)
Inositol (CDP-diacylglycerol)
Cardiolipin (phosphatidylglycerol)
NH2
N
O
O
O
N
+
(CH3) N CH2CH2 O P O P O CH2
3
O
O
O
Cytidine diphosphate (CDP) choline HO
OH
NH2
N
O
+
O
H N CH2CH2 O P O P
3
O
O
Cytidine diphosphate (CDP)ethanolamine
O
N
O CH2
O
HO
OH
Regulation of Triacylglecerol
Metabolism

Pancreas
primary organ involved in sensing the organism’s
dietary and energetic states.
monitoring glucose concentrations in the blood.


Low blood glucose stimulates the secretion
of glucagon
Elevated blood glucose calls for the
secretion of insulin
Acetaly-CoA carboxylase (ACC)

Committed enzyme in fatty acid synthesis
activated by citrate
inhibited by palmitoyl-CoA, long-chain fatty acyl-CoAs

Affected by phosphorylation
glucagon or epinephrine
decreased activity of ACC by phosphorylation
insulin
increases the synthesis of triacylglycerols
Important Derivatives of Unsaturated
Fatty Acids- Arachidonic Acid
EICOSANOID FACTS
20-carbon compounds
Include prostaglandins, prostacyclins, thromboxanes, leukotrienes
Physiological effects at very low concentrations
Many of their effects mediated by cyclic AMP or calcium second
messengers
Unlike hormones, not transported in the blood
Local mediators that act where synthesized or in adjacent cells
The Actions of Prostaglandins and Leukotrienes
a.
the inflammatory response involving primarily the joints (rheumatoid
arthritis) and skin (psoriasis);
b.
the production of pain and fever;
c.
the regulation of blood pressure (vaso-constrictors/dilators) and blood
clotting (platelet function);
d.
decreased gastric acid secretion (prostacyclins may be an ideal way to
control the symptoms of peptic ulcer, but prostanoid synthesis inhibitors, like
aspirin, increase acid secretion causing peptic ulcer);
e.
the control of several reproductive functions such as the induction of labor
and delivery - this has led to the use of PGF2 as a mid-trimester
abortifacient drug or as a labor-inducing agent;
f.
the regulation of the sleep/wake cycle;
g.
hypersensitivity allergic reactions (a primary action of leukotrienes).
Dietary linoleic acid
metabolism
Arachidonic acid
esterification
Membrane phospholipids

Cell Activation Events:
mechanical trauma,
cytokines
growth factors

Phospholipase A2
(PLA2)
Arachidonic acid
Anti-inflammatory glucocorticoids
GC induce lipocortin that
inhibits
PLA2
Aspirin,
Indomethacin,
Ibuprofen
NSAIDs

Cyclooxygenase
(COX)
Prostaglandins
and thromboxanes
(Cyclic/ring product)
Aspirin inhibits irreversibly
Indomethacin forms a salt bridge in the binding site
Ibuprofen competes for substrate binding
Zyflo

Lipooxygenase
(LOX)
Leukotrienes
(Linear product)
Zyflo competes with
AA for binding
Figure 1. Liberation of arachidonic acid and its metabolism to prostaglandins/
thromboxanes or to leukotrienes
LEUKOTRIENE FACTS
leukotriene synthesis inhibited by Zyflo, a lipooxygenase inhibitor
leukotriene action blocked by accolate, a receptor antagonist
peptidoleukotrienes:
leukotrienes with short peptides added
components of slow reacting substances of anaphylaxis (SRS-A)
anaphylaxis violent (potentially fatal) allergic reaction
10,000 times more potent than histamine
SRS-A released from lung following immunological stress
SRS-A contract smooth muscle causing constriction of bronchi
implicated in hypersensitivity reaction – such as insect sting
Arachidonic Acid (6)
derived from membrane phospholipids
aspirin
indomethacin
ibuprofen
X
O2
Cyclooxygenase
Prostaglandin
endoperoxide
synthase
PGG2
2GSH
Hydroperoxidase
GSSG
PGH2
central intermediate
(Head of pathway)
Figure 3. Conversion of arachidonic acid to PGH2
Figure 4. Structure and
mechanism of action of aspirin
CH2 OH
Ser
COOH O
O C CH3 
O
C CH3
Cyclooxygenase
O
(active)
C OCH3
O
C CH
O O
3
C CH3
CH3CH3
CH2 C
O C
COOH
OH

Ser
Acetylated
Cyclooxygenase
(inactive)
COX-1 VS COX-2 DRUG ACTION
Aspirin:
works on both isoforms
COX-1 effect reduces platelet aggregation (TXA2)
COX-2 effect reduces inflammation
Side effects due to COX-1 inhibition – stomach irritation
Specific COX-2 inhibitors
Celebrex/Vioxx
Target inflammatory response
No COX-1 inhibition to produce aspirin-induced side effects
Metabolism of Cholesterols
Biosynthesis of Cholesterol
Introduction


Functions of cholesterol.
 Important cell membrane component.
 Precursor for 3 biologically active compounds.
 Bile.
 Steroid hormones.
 Vitamin D.
Disease implications.
 Cardiovascular disease.
 Diet control and synthesis manipulation = < heart
disorders.
Biosynthesis of Cholesterol
Introduction

Disease implications.



Gall stones.
Steroidogenic enzyme deficiency.
Source of cholesterol.




Meat.
Eggs.
Dairy products.
De novo liver synthesis.
Cholesterol Synthesis
O

O
C
OH
CH2
C
O
CH2
C
SCoA
CH3
hydroxymethylglutaryl-CoA
Hydroxymethylglutaryl-coenzyme A (HMG-CoA)
is the precursor for cholesterol synthesis.
HMG-CoA is also an intermediate on the pathway for
synthesis of ketone bodies from acetyl-CoA.
The enzymes for ketone body production are located
in the mitochondrial matrix.
HMG-CoA destined for cholesterol synthesis is made
by equivalent, but different, enzymes in the cytosol.
O
H3C
H2O  O
H3C
C
CH2
C
SCoA
SCoA
HMG-CoA
Synthase
HSCoA
OH
O
O
C
acetoacetyl-CoA
acetyl-CoA

O
C
CH2
C
O
CH2
C
SCoA
CH3
hydroxymethylglutaryl-CoA


HMG-CoA is formed by condensation of acetyl-CoA
& acetoacetyl-CoA, catalyzed by HMG-CoA Synthase.
HMG-CoA Reductase catalyzes production of
mevalonate from HMG-CoA.
The carboxyl of HMG
that is in ester linkage to
the CoA thiol is reduced
to an aldehyde, and then
to an alcohol.
NADPH serves as
reductant in the 2-step
reaction.
Mevaldehyde is thought
to be an active site
intermediate, following
the first reduction and
release of CoA.
HO
C
H2C
CH3
CH2
C

O
C
H2C
O
HMG-CoA
HMG-CoA
Reductase
2NADP+
+ HSCoA
HO

SCoA
O
O
2NADPH
C
CH3
CH2
H2
C
OH
C
O
mevalonate
HMG-CoA Reductase is an integral protein of
endoplasmic reticulum membranes.
The catalytic domain of this enzyme remains active
following cleavage from the transmembrane portion
of the enzyme.
The HMG-CoA Reductase reaction, in which
mevalonate is formed from HMG-CoA, is ratelimiting for cholesterol synthesis.
This enzyme is highly regulated and the target of
pharmaceutical intervention.
Mevalonate is
phosphorylated by 2
sequential Pi transfers
from ATP, yielding
the pyrophosphate
derivative.
ATP-dependent
decarboxylation, with
dehydration, yields
isopentenyl
pyrophosphate.
HO
CH3
C
H2C
CH2
mevalonate
C

O
O
HO
H2C
2 ATP
(2 steps)
2 ADP
CH3
C

CH2 OH
CH2
O
CH2
O
C
O
O
O
O
P
O
O
5-pyrophosphomevalonate
ATP
ADP + Pi
CO2
CH3
O
C
H2 C
P
O
CH2
CH2
O
P
O
O
O
P
O
O
isopentenyl pyrophosphate
CH3
Isopentenyl
pyrophosphate is
the first of several
compounds in the
pathway that are
referred to as
isoprenoids, by
reference to the
compound isoprene.
C
H2 C
H2
C
C
H2
O
O
P
O
O
O
O
isopentenyl pyrophosphate
CH3
C
H2C
C
H
isoprene
P
CH2
O
CH3
O
C
H2C
CH2
CH2
O
isopentenyl
pyrophosphate
P
O
O
O
P
O
O
CH3
O
C
H3C
CH
CH2
dimethylallyl
pyrophosphate
O
P
O
O
O
P
O
O
Isopentenyl Pyrophosphate Isomerase inter-converts
isopentenyl pyrophosphate & dimethylallyl pyrophosphate.
Mechanism: protonation followed by deprotonation.
Condensation Reactions
Prenyl Transferase catalyzes head-to-tail condensations:

Dimethylallyl pyrophosphate & isopentenyl
pyrophosphate react to form geranyl pyrophosphate.

Condensation with another isopentenyl pyrophosphate
yields farnesyl pyrophosphate.

Each condensation reaction is thought to involve a
reactive carbocation formed as PPi is eliminated.
CH3
H3C
C
O
CH
CH2
O
O
P
O
P
O
O
O
CH3
dimethylallyl pyrophosphate
H2C
C
O
CH2
CH2
O
P
O
O
O
P Pi
CH3
CH3
H3C
C
CH
CH2
CH2
C
CH2
O
P
O
O
P
O
CH3
H2C
O
O
O
geranyl pyrophosphate
C
O
CH2
CH2
O
P
O
O
O
CH3
H3C
C
CH
CH2
CH2
farnesyl pyrophosphate
C
CH3
CH
CH2
CH2
P
O
O
isopentenyl pyrophosphate
P Pi
CH3
O
isopentenyl pyrophosphate
O
CH
P
C
O
CH
CH2
O
P
O
O
O
P
O
O
Each condensation involves a carbocation formed as PPi is eliminated.
CH3
CH3
2 H3C
C
CH
NADPH
CH2
CH2
C
CH3
CH
CH2
CH2 C
O
CH
CH2
2 farnesyl pyrophosphate
O
P
O
O
O
P
O
O
NADP+ + 2 PPi
NADP+
NADPH
O2
H2O
O
squalene
H+
2,3-oxidosqualene
HO
lanosterol
Squalene Synthase: Head-to-head condensation of 2 farnesyl
pyrophosphate, with reduction by NADPH, yields squalene.
NADP+
NADPH
O2
H2O
O
squalene
H+
2,3-oxidosqualene
HO
lanosterol
Squaline epoxidase catalyzes conversion of squalene to
2,3-oxidosqualene.
This mixed function oxidation requires NADPH as
reductant & O2 as oxidant. One O atom is incorporated
into substrate (as the epoxide) & the other O is reduced to
water.
Squalene
Oxidocyclase
catalyzes a series
of electron shifts,
initiated by
protonation of the
epoxide, resulting
in cyclization.
H+
O
2,3-oxidosqualene
HO
lanosterol
Structural studies of a related bacterial enzyme have
confirmed that the substrate binds at the active site in a
conformation that permits cyclization with only modest
changes in position as the reaction proceeds.
The product is the sterol lanosterol.
19 steps
HO
HO
lanosterol
cholesterol
Conversion of lanosterol to cholesterol involves 19
reactions, catalyzed by enzymes in ER membranes.
Additional modifications yield the various steroid
hormones or vitamin D.
Many of the reactions involved in converting lanosterol
to cholesterol and other steroids are catalyzed by
members of the cytochrome P450 enzyme superfamily.
Regulation of cholesterol synthesis
HMG-CoA Reductase, the rate-limiting step on the
pathway for synthesis of cholesterol, is a major control
point.
Short-term regulation:
HMG-CoA Reductase is inhibited by phosphorylation,
catalyzed by AMP-Dependent Protein Kinase (which
also regulates fatty acid synthesis and catabolism).
This kinase is active when cellular AMP is high,
corresponding to when ATP is low.
Thus, when cellular ATP is low, energy is not expended
in synthesizing cholesterol.
Long-term regulation is by varied formation and
degradation of HMG-CoA Reductase and other enzymes
of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:
•
Degradation of HMG-CoA Reductase is
stimulated by cholesterol, oxidized derivatives of
cholesterol, mevalonate, & farnesol
(dephosphorylated farnesyl pyrophosphate).
•
HMG-CoA Reductase includes a transmembrane
sterol-sensing domain that has a role in activating
degradation of the enzyme via the proteasome
(proteasome to be discussed later).
Long-term regulation is by varied formation and
degradation of HMG-CoA Reductase and other enzymes
of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:
•
Degradation of HMG-CoA Reductase is
stimulated by cholesterol, oxidized derivatives of
cholesterol, mevalonate, & farnesol
(dephosphorylated farnesyl pyrophosphate).
•
HMG-CoA Reductase includes a transmembrane
sterol-sensing domain that has a role in activating
degradation of the enzyme via the proteasome
(proteasome to be discussed later).
Lipid
transport
• triacylglycerides, cholesterol, phospholipids
• dietary lipid transport –chylomicron
• endogenous lipid transport (VLDL, IDL,
LDL, HDL)
Dietary uptake and distribution of fatty acids
intestinal lumen
triacylglycerols
epithelial cells
triacylglycerols
pancreatic
lipases
FFA + monoacylglycedrols
bile acids
absorbed by intestinal
cholesterol
epithelial cells and
micelles
reconverted to
triacylglycerols
•Packaged into chylomicron
•Released into lymphatic system and then via
capillaries to blood stream
chylomicron
•acted upon by lipases on cell walls of
capillaries in tissues
energy production
FFA
•taken up by
tissues
reconversion to TAGs
in adipocytes for storage
hormone sensitive lipases
FFA
released to circulatory system
and combine with albumin for
delivery to tissues
Why do we need lipoproteins?



Triacylglycerides (TAGs) + cholesterol (Chol)
are nonpolar molecules → insoluble in H2O
TAG + Chol must be packaged within a polar
shell in order to be transported through the
blood to the various tissues
This is accomplished by combining nonpolar
lipids w/ amphipathic lipids → (a polar
water-soluble terminal group attached to an
H2O -insoluble hydrocarbon chain)
Lipoproteins & Apolipoproteins
Lipoproteins (LP)
 function: transport of cholesterol + esterified lipids in
blood
 structure:
1) polar shell ---single phospholipid (PL) layer:
head groups directed outward
-Chol
-apolipoproteins
2) nonpolar lipid core
-hydrophobic TAG(triacylglycerol)
-cholesteryl ester (CE)
apolipoproteins
• Provide structural stability to Lp
• Act as cofactors for enzymes involved in
plasma lipid and Lp metabolism
• Serve as ligands for interaction w/Lp receptors
that help determine disposition of individual
particles
There are many types of apolipoproteinsa
Apoprotein
Lipoproteins
Function(s)
Secretion of VLDL from liver
2) Structural protein of VLDL, IDL, and HDL
3) Ligand for LDL receptor (LDLR)
Apo B-100 VLDL, IDL, LDL
1)
Apo B-48
Chylomicrons,
remnants
Secretion of chylomicrons from intestine;
lacks LDLR binding domain of Apo B-100
Apo E
Chylomicrons, VLDL,
IDL, HDL
Ligand for binding of IDL & remnants to
LDLR and LRP
Apo A-I
HDL, chylomicrons
1)
Apo A-II
HDL, chylomicrons
Unknown
Apo C-I
Chylomicrons, VLDL,
IDL, HDL
Modulator of hepatic uptake of VLDL and
IDL (also involved in activation of LCAT)
Apo C-II
Chylomicrons, VLDL,
IDL, HDL
Activator of LPL
Apo C-III
Chylomicrons, VLDL,
IDL, HDL
Inhibitor of LPL activity
Major structural protein of HDL
2) Activator of LCAT
Lipoprotein Structure
Lipoproteins
•
•
hydrophobic core (TAGS, cholesterol esters)
hydrophilic surface (P-lipids, cholesterol, and
apolipoproteins)
• Function
transport of lipids in blood
• Types of lipoproteins
(classified according to density)
• very low density (VLDL)
• intermediate density (IDL)
• low density (LDL)
• high density (HDL)
Protein content increase, lipid decreases as density increases.
85%
Chylomicron
2%
% TAGS
VLDL
IDL
% Protein
8%
LDL
HDL
33%
nm
Lipoproteins
• Chylomicron:
• 85% TAG, 4% chol., 8% protein
•formed in intestinal epithelial cells
• deliver exogenous TAGS to tissue
• 80 -500nm
• ApoCII activates lipases in capillary cell
walls releasing FFA to tissue
• chylomicron remnants return to liver where
they bind to ApoE receptor and are taken up
• 1/2 life in blood - 4-5 minutes
• VLDL:
• 50% TAGs, 22% choles., 10% protein
• 30 -100 nm
• formed in liver
• deliver endogenous lipids to other tissues
(mainly muscle and fat cells)
• ApoCII activates lipases in capillary cell
walls releasing FFA to tissue
• converted to IDLs and LDL as lipids are
released
Lipoproteins
• IDL: (31% TAGs, 29% choles., 18% protein)
• formed from VLDLs as lipids removed
• some IDLs return to liver
• rest converted to LDLs by further removal
of lipids
• LDL: “bad” cholesterol
•
•10% TAGs, 45% choles., 25% protein
• 25 - 30 nm
• formed as lipids removed from VLDLs
and IDLs.
• all apolipoproteins lost except ApoB100
• bind to LDL receptor via ApoB100 and
taken up by endocytosis by hepatic and other
tissues (50-75% taken up by liver).
• Primary mode of cholesterol delivery to tissues.
• Synthesis of LDL receptor is inhibited by
high levels of intracellular cholesterol and
stimulated by low levels of cholesterol.
Therefore, cholesterol uptake is closely
matched to intracellular cholesterol levels.
Lipoproteins
• HDL: “good” cholesterol
•
•
•
•
8% TAGs, 30% choles., 33% protein
7.5 - 10 nm
formed in liver
scavenge cholesterol from cell surfaces
and other lipoproteins and deliver it to liver.
• Convert cholesterol to cholesterol ester
• bind to “scavenger receptor” on liver cell
surface - cholesterol esters taken up and
HDLs released and reenter circulation.
Liver
Intestine
Dietary lipids
Triacylglycerols
cholesterol
Cholesterol esters
HDL
chylomicron
VLDLs
LDLs
Triacylglycerols
FFA
monoacylglycerols
Cholesterol
Cholesterol esters
Peripheral tissues
HDLs
Distribution of endogenous lipids The Exogenous Pathway
Liver
Intestine
Dietary lipids
ApoE/LDLR
mediated
uptake
chylomicron
acquire
ApoE, CII
and others
Chylomicron
remnants
Peripheral tissues
LPLs activated by ApoCII
Triacylglycerols
FFA
monoacylglycerols
Cholesterol
Cholesterol esters
Distribution of endogenous lipids The Endogenous Pathway
Liver
Triacylglycerols
cholesterol
Cholesterol esters
acquire
ApoE, CII
and others
VLDLs
IDLs
LDLR/ApoE
LDLR/ApoB100
LDLs
LPLs activated
by ApoCII
Peripheral
tissues
Triacylglycerols
FFA
monoacylglycerols
Cholesterol Ester
Cholesterol
Distribution of endogenous lipids The
HDL Pathways
Transport of excess cholesterol from peripheral
tissues back
to liver for excretion in bile
HDLs act as acceptors for excess chol, Apo, PL
derived from
CM, VLDL and LDL
HDLs synthesized by both liver and intestine
Distribution of endogenous lipids The HDL Pathways
Liver
scavenger receptor
uptake of cholesterol
Triacylglycerols
cholesterol
Cholesterol esters
HDL
VLDLs
IDLs
CEs
LDLs
Peripheral
tissues
Triacylglycerols
FFA
monoacylglycerols
TAGs
Cholesterol Ester
Cholesterol
HDLs
VLDL
Choles.
Abnormal Metabolism of
Lipoprotein


Hyperlipoproteinemia
Genetic diseases