Lipid Metabolism 1: Overview of lipid transport in animals, fatty acid oxidation, ketogenesis in liver mitochondria

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Lipid Metabolism 1Overview of lipid transport
in animals, fatty acid oxidation, ketogenesis in
liver mitochondria
Bioc 460 Spring 2008 - Lecture 35 (Miesfeld)
Adipose tissue is the primary triacylglycerol
storage depot in animals, fats are an excellent
form of redox energy
Stored fat comes from the conversion of
carbohydrates into fatty acids in the liver
Prime rib contains large amounts of saturated
fats in the form of triacylglycerols
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Key Concepts in Lipid Metabolism

• Stored lipids is the primary source of energy in
  most organisms. Lipids, such as
  triacylglycerols, are much more reduced than
  carbohydrates and are hydrophobic, which makes
  them ideal storage forms of high energy
  compounds.
• The three sources of triacylglycerols in animals
  are dietary lipids, stored triacylglycerols in
  adipose tissue, and the conversion of carbon from
  either carbohydrate or protein into fatty acids
  in the liver.
• ?-oxidation is the mitochondrial process by which
  fatty acids are oxidized to yield NADH, FADH2,
  and acetyl-CoA. These metabolites are oxidized
  by the citrate cycle and electron transport
  system to yield large amounts of ATP.
• Ketogenesis takes place in liver mitochondria
  when acetyl-CoA levels are high and oxaloacetate
  levels are low. Acetoacetate and
  D-?-hydroxybutyrate are exported are exported and
  converted back into acetyl-CoA by peripheral
  tissues.

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Overview of Lipid Transport in Animals

• There are three basic sources of fatty acids in
  animals that can be used for energy conversion
  processes
• 1) fatty acids present in triacylglycerols
  obtained from the diet,
• 2) fatty acids stored as triacylglycerols in
  adipose tissue that are released by hydrolysis
  following hormone stimulation (glucagon or
  epinephrine signaling)
• 3) fatty acids synthesized in the liver from
  excess carbohydrates and exported as
  triacylglycerols.

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• Fat is stored in fat cells (adipocytes). Obesity,
  especially childhood obesity, can be due to both
  more fat storage per cell, and to a larger number
  of adipocytes.
• In contrast, in normal healthy adults, the onset
  of old age and reduced metabolic rates leads to
  weight gain resulting primarily from storing more
  fat per cell (although adults can also add more
  fat cells if they become obese).

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• Review of lipid structures
• Fatty acids are stored as triacylglycerols

Glycerol esterification of fatty acids protects
cell membranes from the amphipathic nature of
fatty acids. Soap is made out of fatty acids and
works well to remove oils from hands and clothes
by forming micelles that trap the lipids in a
water soluble particle.
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Lipid metabolism is critical to animals who
depend on lipids as a major energy source. Plants
only use seeds as a major lipid storage depot.
Conversion of carbohydrates to fatty acids is
thought to be a major contributing factor to
obesity and diabetes in developed countries over
the last 30 years. The primary source of these
carbohydrates are soft drinks, and processed
foods (snack foods) that have been prepared with
refined sugar and flour. Another dietary demon
contributing to obesity has been transesterified
fats.
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Pathway Questions

• 1. What purpose does fatty acid metabolism serve
  in animals?
• Fatty acid oxidation in mitochondria is
  responsible for providing energy to cells when
  glucose levels are low. Triacylglycerols stored
  in adipose tissue of most humans can supply
  energy to the body for 3 months during
  starvation.
• Fatty acid synthesis reactions in the cytosol of
  liver and adipose cells convert excess acetyl CoA
  that builds up in the mitochondrial matrix when
  glucose levels are high into fatty acids that can
  be stored or exported as triacylglycerols.

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Pathway Questions

• 2. What are the net reactions of fatty acid
  degradation and synthesis for the C16 fatty acid
  palmitate?
• Fatty acid oxidation
• Palmitate 7 NAD 7 FAD 8 CoA 7 H2O ATP
  ? 8 acetyl CoA 7 NADH 7 FADH2 AMP 2
  Pi 7 H
• Fatty acid synthesis8 Acetyl CoA 7 ATP 14
  NADPH 14 H ? Palmitate 8 CoA 7 ADP
  7 Pi 14 NADP 6 H2O

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Pathway Questions

• 3. What are the key enzymes in fatty acid
  metabolism?
• Fatty acyl CoA synthetase enzyme catalyzing the
  "priming" reaction in fatty acid metabolism which
  converts free fatty acids in the cytosol into
  fatty acyl-CoA using the energy available from
  ATP and PPi hydrolysis. Carnitine
  acyltransferase I - catalyzes the commitment step
  in fatty acid oxidation which links fatty
  acyl-CoA molecules to the hydroxyl group of
  carnitine. The activity of carnitine
  acyltransferase I is inhibited by malonyl-CoA,
  the product of the acetyl-CoA carboxylase
  reaction, which signals that glucose levels are
  high and fatty acid synthesis is favored.

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Pathway Questions

• 3. What are the key enzymes in fatty acid
  metabolism?Acetyl CoA carboxylase - catalyzes
  the commitment step in fatty acid synthesis using
  a biotin-mediated reaction mechanism that
  carboxylates acetyl-CoA to form the C3 compound
  malonyl-CoA. The activity of acetyl CoA
  carboxylase is regulated by both reversible
  phosphorylation (the active conformation is
  dephosphorylated) and allosteric mechanisms
  (citrate binding stimulates activity,
  palmitoyl-CoA inhibits activity). Fatty acid
  synthase - this large multi-functional enzyme is
  responsible for catalyzing a series of reactions
  that sequentially adds C2 units to a growing
  fatty acid chain covalently attached to the
  enzyme complex. The mechanism involves the
  linking malonyl-CoA to an acyl carrier protein,
  followed by a decarboxylation and condensation
  reaction that extends the hydrocarbon chain.

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Pathway Questions

• 4. What are examples of fatty acid metabolism in
  real life?
• A variety of foods are prominently advertised as
  "non-fat," even though they can contain a high
  calorie count coming from carbohydrates. Eating
  too much of these high calorie non-fat foods
  (e.g., non-fat bagels) activates the fatty acid
  synthesis pathway resulting in the conversion of
  acetyl-CoA to fatty acids, which are stored as
  triacylglycerols.

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• Transport and storage of fatty acids and
  triacylglycerols

Much of the triacylglycerol stored in adipose
tissue originates from dietary lipids. Fats that
enter the small intestine from the stomach are
insoluble and must be emulsified by bile acids
such as glycocholate which are secreted by the
bile duct and function as detergents to promote
the formation of micelles. Lipases are water
soluble enzymes in the small intestine that
hydrolyze the acyl ester bonds in
triacylglycerols to liberate free fatty acids
which then pass through the membrane on the
lumenal side of intestinal epithelial cells.
Pancreatic lipase cleaves the ester bond at the
C-1 and C-3 carbons to release two free fatty
acids and monoacylglyclerol.
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• Transport and storage of fatty acids and
  triacylglycerols

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• Transport and storage of fatty acids and
  triacylglycerols

Chylomicrons transport the triacylglycerols to
adipose tissue for storage, and to muscle cells
for energy conversion processes.
Apolipoprotein C-II on the surface of
chylomicrons binds to and activates lipoprotein
lipase on endothelial cells which leads to the
release of fatty acids and glycerol. Fatty
acids diffuse into the endothelial cells and then
enter nearby adipose and muscle cells where they
are stored or used for energy conversion
pathways. The glycerol produced by lipoprotein
lipase returns to the liver where it is converted
to dihydroxyacetone phosphate.
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• Transport and storage of fatty acids and
  triacylglycerols

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• Fatty acids are synthesized in the liver from
  carbohydrates

Dietary lipids are not the only source of
triacylglycerols stored in adipocytes. The liver
synthesizes triacylglycerols from fatty acids
when glucose levels are high and the amount of
acetyl CoA produced exceeds the energy
requirements of the cell. Glucose provides the
necessary substrates for triacylglycerol
synthesis (acetyl CoA for fatty acid synthesis
and glycerol) using reactions in the glycolytic
pathway and the citrate cycle.
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• The fatty acid ? oxidation pathway in mitochondria

Fatty acids must first be activated by a two step
reaction catalyzed by medium chain fatty acyl CoA
synthetase. In the first step, the carboxylate
ion of the fatty acid attacks a phosphate in ATP
to form an acyl-adenylate intermediate and
release pyrophosphate (PPi) which is quickly
hydrolyzed by the enzyme inorganic
pyrophosphatase to form 2 Pi.
In the second step of the fatty acyl CoA
synthetase reaction, the palmitoyl-adenylate
intermediate is attacked by the thiol group of
CoA to form the thioester palmitoyl-CoA product
and release AMP.
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• Fatty acid are transported into mitochondria by
  carnitine

The fatty acyl-CoA products of the fatty acyl CoA
synthetase reaction have two fates. If the
energy charge of the cell is low, then they will
be imported into the mitochondrial matrix by the
carnitine transport cycle and degraded by the
fatty acid oxidation reactions to yield acetyl
CoA, FADH2 and NADH. However, if the energy
charge is high, and fatty acid synthesis is
favored, then mitochondrial import of fatty
acyl-CoA is inhibited and the fatty acyl-CoA
molecule is used instead for triacylglycerol or
membrane lipid synthesis in the
cytosol. Carnitine acyltransferase I is located
in the outer mitochondrial membrane and replaces
CoA with carnitine to form fatty acyl carnitine
which is translocated across the inner
mitochondrial membrane. The carnitine
translocating protein is an antiporter that
exchanges a fatty acyl carnitine molecule for a
carnitine. Once inside the mitochondrial matrix,
fatty acyl carnitine is converted back to fatty
acyl CoA in a reaction catalyzed by carnitine
acyltransferase II releasing the carnitine so
that it can be shuttled back across the membrane.
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• Fatty acid are transported into mitochondria by
  carnitine

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• ?-oxidation yields large amounts of ATP

Once the electron-rich carbons of fatty acids
are moved into the mitochondrial matrix, their
high energy redox potential is traded in for a
substantial payout of ATP This energy conversion
process of fatty acid --gt ATP involves oxidation
of fatty acids by sequential degradation of C2
units leading to the generation FADH2, NADH, and
acetyl CoA. The subsequent oxidation of these
reaction products by the citrate cycle and
oxidative phosphorylation generates large amounts
of ATP.
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• ?-oxidation reactions

The ?-oxidation pathway occurs at the ? carbon of
the fatty acid, thereby releasing the C-1
carboxyl carbon and ? carbon as the acetate
component of acetyl CoA. In the first of four
reactions, the enzyme acyl CoA dehydrogenase
catalyzes a dehydrogenation reaction (oxidation)
that introduces a trans CC bond between the ?
and ? carbons of the fatty acyl-CoA molecule
using a mechanism that reduces an enzyme bound
FAD to form FADH2. Mitochondria contain three
isozymes of acyl CoA dehydrogenase which differ
in their specificity for hydrocarbon chains of
different lengths, long chain (C12 to C18),
medium chain (C4 to C14) and short chain (C4 to
C8 ) acyl CoA dehydrogenases.
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• ?-oxidation reactions

The second reaction in the ? oxidation pathway is
a hydration step catalyzed by the enzyme enoyl
CoA hydratase that adds H2O across the CC bond
to convert trans-?2-enoyl-CoA to
3-L-hydroxyacyl-CoA. The third reaction is
another dehydrogenation (oxidation) step in which
the enzyme ?-hydroxyacyl-CoA dehydrogenase
removes an electron pair from the substrate and
donates it to NAD to form NADH. Finally,
coenzyme A is used in thiolysis reaction
catalyzed by the enzyme acyl CoA
acetyltransferase (also called thiolase) that
releases a molecule of acetyl CoA and in the
process, results in the formation of an fatty
acyl CoA product that is two carbons shorter than
the starting substrate.
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• ?-oxidation reactions

The complete oxidation of palmitoyl-CoA (C16)
requires seven rounds of the ? oxidation pathway
to convert one molecule of palmitoyl CoA into
eight molecules of acetyl CoA in a net reaction
that can be written as Palmitoyl-CoA 7 CoA
7 FAD 7 NAD 7 H2O --gt 8 acetyl
CoA 7 FADH2 7 NADH 7 H
After seven rounds of ? oxidation, palmitoyl-CoA
yields 8 acetyl CoA, 7 NADH and 7 FADH2. The
oxidation of acetyl CoA by the citrate cycle then
generates 24 NADH, 8 FADH2 and 8 GTP (ATP).
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• ?-oxidation reactions

The combined reactions of the electron transport
system and oxidative phosphorylation converts
these 31 NADH (31 x 2.5 ATP) 77.5 ATP 15
FADH2 (15 x 1.5 ATP) 22.5 ATP For a
grand total 100 ATP After subtracting the 2
ATP required for fatty acyl CoA activation (AMP
--gt PPi) And adding the 8 ATP obtained from
eight turns of the citrate cycle The total
payout for the complete oxidation of palmitate
is 106 ATP
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• ?-oxidation is a chemical source of water for
  desert animals

Besides the payout of ATP that comes from fatty
acid oxidation, another benefit is the generation
of H2O that occurs when O2 is reduced by the
final reaction in the electron transport system,
as well as, the formation of H2O in the ATP
synthesis reaction of oxidative phosphorylation
as shown in the three reactions below 2
NADH 2 H O2 --gt 2 H2O 2 FADH2 O2 --gt
2 H2O ADP PO42- --gt ATP H2O The water
production that accompanies fatty oxidation
benefits animals that live in dry climates where
liquid water is scarce, for example, the desert
kangaroo rat and Arabian camel. Large animals
that hibernate over the winter, like the Alaskan
brown bear, also take advantage of fatty acid
oxidation in order to replace H2O that is lost by
respiration.
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Ketogenesis

• Acetyl-CoA derived from fatty acid oxidation
  enters the Citrate Cycle only if carbohydrate
  metabolism is properly balanced.
• When fatty acid oxidation produces more
  acetyl-CoA than can be combined with OAA to form
  citrate, then the "extra" acetyl-CoA is converted
  to acetoacetyl-CoA and ketone bodies, including
  acetone. Ketogenesis (synthesis of ketone bodies)
  takes place primarily in the liver.

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Ketogenesis

• Three mitochondrial reactions are required to
  convert two acetyl CoA molecules into
  acetoacetate which is then reduced to form
  D-?-hydroxybutyrate. Acyl-CoA acetyltransferase
  (thiolase) is the same enzyme that releases one
  molecule of acetyl CoA in reaction 4 of the ?
  oxidation pathway, however in this case, the
  reaction is driven toward condensation by the
  high concentration of acetyl CoA in the
  mitochondria under ketogenic conditions. In the
  next step, the enzyme HMG-CoA synthase fadds
  another acetyl CoA group to form the intermediate
  ?-hydroxy-?-methylglutaryl-CoA, abbreviated as
  HMG-CoA, and then the enzyme HMG-CoA lyase
  removes one of the original acetyl CoA groups to
  yield acetoacetate.

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Ketones are an energy source for tissues

• Acetoacetate and D-?-hydroxybutyrate are exported
  from the liver and used by other tissues such as
  skeletal and heart muscle to generate acetyl CoA
  for energy conversion reactions. Even the brain
  which prefers glucose as an energy source, can
  adapt to using ketone bodies as chemical energy
  during times of extreme starvation.

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Ketogenesis occurs when glycogen stores are
depleted such as during fasting and in
undiagnosed diabetics

• Diabetes is a metabolic form of carbohydrate
  "starvation," and characterized by elevated
  concentrations of acetoacetate and
  D-?-hydroxybutyrate in the blood and urine.
  Diabetics can have high levels of acetone in
  their blood which can be detected on their breath
  as a fruity odor. Acetone is a spontaneous
  breakdown product of acetoacetate
  (decarboxylation), or is formed by enzymatic
  cleavage of acetoacetate by the enzyme
  acetoacetate decarboxylase