CHOLESTEROL

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LIPID METABOLISM
FATS ARE WELL-SUITED TO SERVE AS STORAGE FUELS,
BUT THEIR MOBILIZATION IS CHALLENGING
TRIACYLGLYCEROL
CHOLESTEROL
PHOSPHOLIPID
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PROCESSING OF DIETARY LIPIDS IN VERTEBRATES
BILE SALT cholesterol deriv, amphipathic,
solubilize fat particle to micelles
Insolubility of fats leads to need to emulsify
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Target sites for lipase action
Micelle formation allows lipases access for
hydrolysis
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• Lipase action also releases glycerol
• The glycerol portion of triacylglycerol
• also is metabolized to yield energy
• (5 of total in the triacylglycerol)
• 2 steps to glycolysis
• Glycerol 3P dehydrogenase is
• the cytosolic enzyme

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PROCESSING OF DIETARY LIPIDS IN VERTEBRATES
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Apolipoprotein Lipids ? Chylomicron, LDL,
VLDL, HDL, etc
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PROCESSING OF DIETARY LIPIDS IN VERTEBRATES
Muscle oxidation Adipocyte storage
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Adipose tissue (fat cells)

• Epinephrine and glucagon bind
• fat cell receptor to trigger cAMP-
• mediated activation of PKA.
• PKA activates lipase by phosphorylation
• Lipase interacts with stored lipids in
• intracellular droplet, to generate
• fatty acids and glycerol
• Perilipin phosphorylation also induces
• mobilization of stored lipids.
• Mobilized fatty acids in the blood
• are bound to serum albumin for
• transport to muscle cells

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NOTE IMPORTANCE OF FLEXIBLE BIOTIN ARM
ROLE OF BIOTIN IS SIMILAR TO THAT IN PYRUVATE
CARBOXYLASE
NOTE MALONYL-COA IS UNIQUE TO BIOSYNTHESIS AND
IS NOT PART OF FATTY ACID CATABOLISM
Fatty acid biosynthesis Begins with the
irreversible reaction catalyzed by acetyl-CoA
carboxylase. 3 functional subunits BCP,
carboxylase, and transcarboxylase
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There is a repeating four-step sequence for fatty
acid biosynthesis Each step extends by two
carbons the product leaves after C16 is made
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STRUCTURES OF FATTY ACID SYNTHASES
Fungal FAS I
Mammalian FAS I
Mammalian enzyme dimer, 480 kD, 7 active sites
per subunit (FAS I) E. coli enzyme 7 activities
are on separate diffusible proteins
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• Both acetate and malonate groups are linked to
  the
• enzyme by thioesters
• The reduction sequence amounts to adding 4
  electrons per
• step, and decarboxylating the 3C malonate to
  2C,
• also at every step.
• The chain grows from the carboxyl terminal end
• Palmitate (160) is released

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• First step the CO2 released
• is the same carbon added
• from HCO3- by acetyl-CoA
• carboxylase
• Condensation coupled to
• decarboxylation becomes
• thermo. favorable
• The extra energy needed to
• make fatty acid synthesis
• favorable is provided by ATP
• (acetyl-CoA ? malonyl-CoA)

• 4-step sequence
• Condensation of activated acyl groups, with
• decarboxylation, gives a b-keto
  product
• Reduction to alcohol
• Elimination of water to create double bond
• Reduction of double bond to saturation

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• 7 protein chains are needed in the
• overall synthetic scheme
• ACP acyl carrier protein, thioester linkage
• KR, HD, ER do steps 2, 3, 4 of the synthetic
• pathway within each individual cycle

• In the overall scheme, the first step is the
  activity of the
• transacetylase to transfer a 2C unit from
  acetyl-CoA
• Uses acetyl-CoA pool in the cytoplasm

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First step of fatty acid synthesis AT
activity transfers acetate from acetyl-CoA
onto the KS subunit Second step MT
activity transfers malonyl group from
malonyl-CoA to ACP
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PRIMED COMPLEX WITH BOTH 2C ACETATE AND 3C
MALONATE ESTERIFIED
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• After priming, the KS
• subunit performs the
• first step of the
• 4-step synthesis by
• condensing the acetyl
• and malonyl groups
• Loss of CO2
• 4-carbon unit remains
• attached to the ACP

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• The next three steps
• (steps 2, 3, 4 in the
• synthesis) are
• performed in turn by
• the KR, HD, and
• ER subunits of the
• large complex
• Substrate channeling
• End-product is butyryl-
• group attached on
• ACP (butyryl-ACP)
• Last step is the AT
• activity transfers the
• 4C butyryl group from
• ACP to KS to prepare
• for the next round

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• Beginning of the second round
• another malonate is transferred
• onto ACP by MT activity
• KS does condensation of malonate
• with butyryl group to form
• 6-carbon fatty acid.
• Note growth is at the carboxyl end
• And so on
• A hydrolytic activity releases
• palmitate (16C stage)

OVERALL REACTION 8Ac-CoA 7 ATP 14 NADPH 14
H ? palmitate 8 CoA 7 ADP 7 Pi 14
NADP 6 H2O

• Note that CO2 is taken up in production of
  malonyl-CoA
• but is released in the condensation step no
  net use
• Malonyl-CoA is synthesized and then used
  immediately
• Only 6 H2O produced because one is needed in
  final hydrolysis

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• NADH/NAD low (glycolysis)

Compartmentalization Fatty acid synthesis,
other biosynthesis is in the cytosol
Note fatty acid elongation is in mitochondria and
ER Some other lipid syntheses go on in
the ER Plant cells compartmentalize
lipid synthesis/oxidation differently
Fatty acid synthesis occurs where NADPH is
high
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NOTE IMPORTANCE OF FLEXIBLE BIOTIN ARM
ROLE OF BIOTIN IS SIMILAR TO THAT IN PYRUVATE
CARBOXYLASE
NOTE MALONYL-COA IS UNIQUE TO BIOSYNTHESIS AND
IS NOT PART OF FATTY ACID CATABOLISM
Fatty acid biosynthesis Begins with the
irreversible reaction catalyzed by acetyl-CoA
carboxylase. 3 functional subunits BCP,
carboxylase, and transcarboxylase
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cytosolic

• NADPH synthesis for fatty acid synthesis
• pentose P pathway and malic enzyme

• Citrate is transported into the cytosol as a
• means of moving acetyl-CoA 2C units
• Citrate lyase (cytoplasm) generates acetyl-CoA
• Oxaloacetate is converted to malate and
• pyruvate to shuttle back into mitochondria
• TCA and gluconeogenesis enzymes regenerate
• oxaloacetate in the mitochondria
• Energy cost 2 ATP per acetyl-CoA

Making acetyl-CoA available to fatty acid
synthesis
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Regulation of fatty acid synthesis acetyl-CoA
carboxylase End-product fatty acid is a
feedback inhibitor (palmitoyl-CoA) Insulin
activates response to high blood sugar ?
synthesize fats rather than oxidize them
Citrate activates transported out of
mitochondria when acetyl CoA up Glucagon,
epinephrine signals of low glucose
concentration ? cellular response is to shut
down energy-demanding fatty acid synthesis
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• Control mechanism at the level of fatty acid
  transport
• malonyl-CoA inhibits carnitine
  acyl-transferase I
• Stops fatty acid oxidation and synthesis from
  occurring simultaneously

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Fatty acid elongation system used to make
stearate (180) or longer fatty acids Found in
both mitochondria and in smooth endoplasmic
reticulum Elongation steps are interspersed
with desaturation to introduce double bonds
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DESATURATION OF FATTY ACIDS
Reduction of O2 is accomplished with electrons
derived from both saturated fatty-acyl CoA and
from NADPH

• Introduction of the double bond into a fatty acid
  chain is oxidative
• Electrons are transferred through redox cofactors
  from NADPH, which
• is also oxidized
• Oxygen is reduced to water
• This enzyme is a mixed-function oxidase
• Oxidase enzyme family catalyze oxidations where
  molecular oxygen
• is the electron acceptor, but no oxygens
  appear in the oxidized product
• (occurs in b-oxidation in plant peroxisomes
• Mono/dioxygenases oxygens are directly
  incorporate into substrate

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• Introduction of double bonds in
• fatty acids is species-dependent
• Plants can introduce double-bonds
• beyond the C9 position, but
• mammals cannot
• Linoleic and linolenic acids must be
• obtained from the diet these are
• essential fatty acids for mammals
• Arachidonic acid, with 4 double-bonds,
• is essential as a precursor for
• regulatory lipids (eicosanoids) and
• must be made from linoleic acid

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• Eicosanoids short-range
• signaling molecules in tissues
• Arachidonate is released by lipase
• in response to hormonal signals
• Prostaglandins such as PGG2
• and PGH2, and thromboxanes are
• among the eicosanoids
• Eicosanoids induce narrowing of
• blood vessels suppressing their
• synthesis alleviates blood
• clotting and heart attack/stroke
• Low doses of aspirin may reduce
• heart attacks and strokes
• Cyclooxygenase (COX) is the
• target of aspirin, ibuprofen
• This is the cyclic pathway another
• linear pathway to other classes
• of eicosanoids also exists

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• Mechanisms of COX inhibition
• Aspirin acetylates critical SER in COX
• Naproxen (Aleve) and Ibuprofen
• (Tylenol) mimic substrate/intermediate
• structure
• These are nonsteroidal antiinflammatory
• drugs (NSAIDs)
• Unfortunately, aspirin etc also regulate
• secretion of mucins that protect
• stomach lining from acid/proteolysis
• Stomach irritation is a side effect ?
• seek new drugs that alleviate this

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COX1

• Mammals have 2 COX isozymes
• COX1 synthesizes prostaglandins
• that regulate mucin secretion
• COX2 synthesizes prostaglandins
• that mediate inflammation
• Hydrophobic channels that bind
• substrate differ subtly in the two
• isozymes and are the targets for
• binding by new NSAIDs

COX1 COX 2
COX1 and COX2 are very similar, but drugs
targeting only COX2 have been developed
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Cholesterol synthesis all carbons come from
acetate Isoprene unit is the essential
intermediate
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• Essential elements of cholesterol synthesis
• 3 acetates condense to mevalonate (6C)
• Mevalonate is converted to an isoprene unit
• 6 5C isoprenes polymerize to squalene
• Squalene cyclizes to cholesterol

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• Pathway to mevalonate from acetyl CoA
• HMG-CoA reductase is the major
• regulatory point in cholesterol synthesis
• Uses NADPH reducing equivalents to
• generate the alcohol function on
• mevalonate

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Second stage 3 ATP are required to generate
activated intermediates, from which activated
isoprenes are made by decarboxylation and
phosphorolysis
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HEAD
TAIL
HEAD
TAIL
Third stage isoprene condensations first,
two head to tail condensations get to 15C
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Third stage (continued) a head-to-head
condensation of 2x 15C farnesyl groups
creates the 30C squalene
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NOTE SIMILARITY TO CHOLESTEROL
20 STEPS
STEROID 4 FUSED RINGS
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• The activated isoprene is the
• precursor of many other
• important metabolites
• All these products are isoprenoids
• There are gt20,000 isoprenoids (!)

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For transport from liver (site of most
synthesis) to other tissues ? Convert to
cholesteryl ester through OH group, makes
even more hydrophobic Transport is then in
secreted lipoprotein particles
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Lipoprotein particles are macromolecular
complexes carrier proteins (apoliporoteins)
together with lipids phospholipids,
cholesterol/esters, triacylglycerols
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Functions of lipoprotein particles Chylomicrons
largest/least dense/high lipid carry dietary
fatty acids to tissue VLDL very low-density
excess liver fatty acids are packaged this way,
transport to store LDL formed from VLDL by
removal of triacylglycerol low density rich in
cholesterol, carry cholesterol to extrahepatic
tissue HDL small, protein-rich, dense, little
cholesterol
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• LCAT enzyme is associated with HDL particles
  forms cholesteryl esters
• Cholesteryl esters convert the nascent HDL
  particle into a mature particle
• The cholesterol-rich HDL returns to the liver,
  where the cholesterol is unloaded
• Some cholesterol is converted to bile salts
  (cholesterol-like w/steroid nucleus)
• Depleted HDL recirculates to pick up more
  cholesterol (reverse cholesterol transport)

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• The apoB-100 protein on LDL
• is recognized by the LDL receptor
• This mechanism removes cholesterol
• from the bloodstream
• LDL-LDL receptor binding is
• followed by endocytosis,
• lysosome fusion, hydrolysis of
• the cholesteryl esters.
• Cholesterol may be reesterified for
• storage or incorporated into
• membranes
• The apoB-100 is degraded but the
• LDL receptor recirculates to
• the cell surface
• Familial hypercholesterolemia is
• a genetic disease in which there
• is a deficiency of LDL receptors

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• Drugs developed to treat familial
• hypercholesterolemia rely on
• inhibition of HMG-CoA reductase
• Drug structure resembles mevalonate
• Competitive inhibitors, reduce cholesterol
• synthesis
• Reduce blood serum cholesterol by 30
• In familial hypercholesterolemia, the
• cholesterol is not taken into the cell
• so there is no down-regulation of its
• synthesis

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If sufficient cholesterol comes in from the
blood in the form of LDL, then the accumulation
in the cell inhibits HMG-CoA reductase Hormone
control insulin (high blood sugar) stimulates
synthesis, glucagon has opposite effect High
cholesterol in the cell also stimulates ester
formation and inhibits further LDL uptake
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Cholesterol is the precursor for synthesis of
steroid hormones of 3 classes Synthesized at low
levels by adrenal cortex
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• Pathways for steroid hormone
• synthesis many oxidases
• and oxygenases are involved
• Energy-requiring syntheses