Lecture 1

Lipid metabolism
Learning goals:
● Explain how fats are mobilized and transported in tissues
● Describe the composition and function of lipoproteins
● Describe trafficking and metabolism of cholesterol
● Explain the biosynthesis of cholesterol
● Discuss regulation and role of cholesterol in human disease

Transport and storage of lipids.

Lipids Fulfill a Variety of Biological Functions
● Energy storage
● Constituents of membranes
● Anchors for membrane proteins
● Cofactors for enzymes
● Signaling molecules; which includes hormones
● Pigments
● Detergents
● Transporters
● Antioxidants

Dietary Fatty Acids Are Absorbed in the Vertebrate Small Intestine

Every food that is ingested is hydrolyzed to its monomers in the small intestines, where
absorption of these given monomers also takes place. In our case, triacylglycerols will be
digested into monoacylglycerol and free fatty acids which then are absorbed into the
intestinal epithelium. There they are reformed back to its original form of triacylglycerols.
Similar process occurs for cholesterol and phospholipids. In the enterocytes (the cells of the
intestinal lining) apolipoproteins are added and particles called chylomicrons are formed.

Chylomicrons are one of the four lipoproteins, and their role is to transport these dietary
lipids to the given tissues where these fats can be metabolized. Chylomicrons travel through
the lymphatic system and the blood in order to reach their desired destination. Lipoprotein
lipase is the key enzyme which is found in the small blood vessels capillaries. It is attached to
the wall of the capillaries and there it helps to break down the triacylglycerols into their given
monomers for the reabsorption of primary myocytes (heart muscle) and adipocytes (adipose
tissue).
Lipids Are Transported in the Blood as Lipoproteins - Macromolecular Complexes
Lipids are carried through the plasma on spherical particles
Lipoproteins are necessary as fats are water insoluble substances and therefore need
lipoprotein particles which are able to transport these fats into an environment primarily
made out of water, such as it is with blood and other fluids present in our body. Lipoproteins
are macromolecular complexes which at its core primarily consist of cholesteryl esters,
cholesterols and triacylglycerols to a large extent. They are coated and surrounded by
phospholipids which give them their hydrophilic shell and apolipoproteins which grant them
special abilities. Everything that makes up a lipoprotein is integral to its function as a
transport vessel for hydrophobic substances such as fats.
● Interior contains cholesterol, TAGs, and cholesteryl esters, which are more nonpolar
than cholesterol while exterior apolipoproteins and phospholipids
Four Major Classes of Lipoprotein Particles
● Named based on position of sedimentation (density) in centrifuge - Name based on
their density
● Composition varies between classes of lipoprotein - EX. Triacylglycerols percentage
differs from each lipoprotein. This could be said about all the given contents labeled
in the table below.
● Lipoproteins also transport fat soluble vitamins such as vitamin A and E

Electron Microscope Pictures of Lipoproteins

Density of the lipoprotein is determined by the amount of TAG:s present in the lipoprotein as
this given lipid takes up most of the physical space with their attached fatty acid tails being
very loose in nature. That is why the chylomicron is considered to be the lipoprotein with the
lowest density with the large concentration of TAG:s present, this is illustrated through the
large size commonly associated with chylomicrons.
Apolipoproteins Refer to the Protein Part of a Lipoproteins
Role of apolipoproteins: to ensure the transport of various lipids between organs
Apolipoproteins refer to the protein part of the liportein, and their role is to ensure the
transport of various lipids between the given organs as apolipoproteins are very versatile in
nature. For example, apolipoproteins can serve as the ligands compatible with tissue bound
receptors, they can also activate and inactive enzymes such as lipase which is responsible for
the further hydroxylation of the fats that these molecules are carrying around etc.
➔ Some apolipoproteins, such as apolipoprotein B (apoB-48 and apoB-100), are
embedded in the particle surface and determine interaction with cellular receptors.
➔ Others, such as apoC (apoC-2 and apoC-3), are only loosely bound and can be
exchanged between different lipoprotein classes, allowing this system to be a lot more
elastic.
Biological Roles of Lipoproteins in Trafficking Cholesterol and TAGs
Each class of lipoprotein has a specific function determined by its point of synthesis, lipid
composition and apolipoprotein content
Three different primary routes can be distinguished
1. Exogenous pathway ensures the transport of dietary lipids from intestines to the liver
2. Endogenous pathway ensures the transport of lipids synthesized in liver to periphery
3. Reverse cholesterol transport ensures the transport of excess cholesterol from the
periphery back to the liver

Biological Roles of Lipoproteins in Trafficking Cholesterol and TAGs

The illustration below shows the pathways in their own given color:
Exogenous pathway
Endogenous pathway
Reverse cholesterol transport

Exogenous Pathway
Chylomicrons in Charge
The exogenous pathway of the lipoproteins ensures the transportation of dietary lipids in the
body. Chylomicrons give plasma a milky appearance, and their half life is less than 1 hour
Process occurs in the following steps:
Step 1
● Chylomicrons, after they are formed in the enterocytes and released into the
lymphatic system, are able to enter the bloodstream via the left subclavian vein 1
Step 2:
● When these lipoprotein particles enter the bloodstream, the enzyme lipoprotein lipase
in the small capillaries2 is activated by the apolipoprotein apoC-2. The main function
of apoC-2 is to specifically activate lipoprotein lipase in order for the given enzyme to
be able to ensure the breakdown of the triacylglycerols found inside the chylomicrons.
The monomers of TAG:s, monoacylglycerol and free fatty acids, are released to the
following tissues; adipose, heart, skeletal muscle and lactating mammary tissues.
● These given tissues can utilize these monomers for the sake of either storage, energy
production or the synthesis of other important lipids for the milk production.
● The heart muscle utilizes the most out of fatty acids for its energy production as
glucose is its least preferred energy source from any of the known macronutrients.
Fats, such as free fatty acids, ketone bodies and even lactate, are the primary source of
energy for the heart muscle.
Step 3:
● When the chylomicron empties its large content of triacylglycerols and is released to a
large extent to these tissues, the particle itself decreases in size. The decreased size of
a chylomicron that still maintains its apolipoproteins is known as a chylomicron
remnant.
● The remnants still contain cholesterol, apoE and apoB-48 travel back to the liver via
the blood where it is able to perform endocytosis thanks to apoE.
● Apolipoprotein E3 ensures the endocytosis of lipoproteins into their desired tissues.
Step 4:
● When chylomicron remnants are picked up by the liver, free cholesterols are released
from the particles as they are broken down and degraded in the lysosomes. All
apolipoproteins present in the remnants are broken down to its amino acids, and all
the lipids are broken down into their monomers.

1
Location; right behind the clavicle bone which can be found in the neck region.
2
The enzyme lipoprotein lipase is the most abundant in the small capillaries of adipose tissue, muscle
tissue and the mammary glands.
3
apoE = Entrance into the cells through Endocytosis.
Endogenous pathway (I)
Step 5:
● Cholesterol and fatty acids ensure the formation of triacylglycerols and cholesteryl
esters in the liver, which can be packaged into very low density lipoproteins (VLDL)
along with a bunch of different apolipoproteins.
● Diet contains ↑ Cholesterol and Fatty acids than are needed → TAGs or cholesteryl
esters are formed and packed within Very low density lipoproteins (VLDL) along with
ApoB-100, ApoC-I, ApoC-II, ApoC-III and ApoE
● The formation of triacylglycerols and cholesteryl esters in the liver is not only the
result of a diet rich in fats, but also a diet rich in carbohydrates as it leads to the same
process formation of very low density proteins (VLDL). The reason for that has to do
with the fact that the eventuell oxidation of glucose, fructose and other carbohydrates
will lead to the formation of pyruvate. Pyruvate will later be converted into acetyl-
Coa, if acetyl-CoA as an energy source is not necessary then we can instead use it as a
substrate for the formation of fats like free fatty acids and cholesterol.
The use of the very low density lipoproteins is dependent on the state of the body
Step 6a - If Insulin ↑ [High in energy]
● The presence of insulin in the body suggests that we have recently eaten a meal and
therefore we have a lot of energy. In this case, the main route of very low density
lipoprotein involves the transportation of dietary lipids to adipose tissues for storage.
Step 6b - If Glucagon ↑ [Low in energy]
● The presence of glucagon in the body suggests that our glucose levels are low and that
we need energy. In this case, very low density lipoprotein primarily originates from
the fats that are stored in adipose tissue. The free fatty acids formed from the
breakdown of triacylglycerols performed by the adipose tissue will be transported to
the liver where it can be reformed back into triacylglycerols. These newly formed
triacylglycerols will be packed into very low density lipoproteins which are sent out
into the blood to target tissues in need such as the heart and skeletal muscle.

Endogenous pathway (II)

Step 6c
● It is important to note that the VLDL particles which are released into the bloodstream
are slowly changing into another form of lipoprotein during its travels through the
circulatory system. The reason for these changes has to do with the fact that the
lipoprotein lipase enzyme present in small capillaries will catch up to VLDL as it
passes the tissues in question. The activity of the lipoprotein lipase enzyme will
subsequently act on the triacylglycerols content and release them from its
confinenments, making the VLDL particle smaller the further it travels in the
circulatory system.
● As the size for VLDL becomes smaller, the particle in question will be known as
either very low density lipoprotein remnants or intermediate density lipoproteins
(IDL). The more triacylglycerols released, the smaller the particle becomes. By the
end of it, low density lipoprotein (LDL) is formed.
Step 7
● ApoB-100 is unique to Low density lipoproteins and is embedded into the structure.
As of now, the lipoprotein primarily contains cholesteryl esters which are carried to
tissues such as the muscle, adipose tissue and the adrenal glands in order to serve a
specific purpose to the given tissue. Adrenal glands, for example, is one of the tissues
which needs cholesteryl esters for he synthesis of cholesterol based hormones.
Step 8
● LDL also delivers cholesterol to our immune cells macrophages, they do afterwards
form foam cells.
Step 9
● Extra LDL that are not necessary at the moment for the tissues, are present in the
circulatory system to be recycled back and taken up by the LDL receptors in the liver
through endocytosis. Endocytosis is possible in this case with the presence of apoE.
Cholesterol Uptake by Receptor Mediated Endocytosis
Low density lipoproteins are able to perform endocytosis on the tissues which have LDL
receptors expressed on their cell membrane. In all of the given tissues, the corresponding
endocytosis will be ensured through the interaction between the apoB-100 and the LDL
receptors.
● ApoB 100 is also present in VLDL, but its receptor binding domain is not available
for binding to the LDL receptor. Conversion of VLDL to LDL exposes this part of
apolipoprotein.
● There is only one molecule of ApoB100 per each lipoprotein particle, thus the
measurement of the apoB in plasma reflects the sum of the VLDL, IDL, and LDL

Process of Endocytosis:
● Firstly, the cells synthesize the LDL receptors in the endoplasmic reticulum which
can later be delivered to the cells surface through the golgi complex.
● When the corresponding low density lipoprotein bypasses the cell, the exposed apoB-
100 will interact with the LDL receptors to ensure endocytosis and the whole particle
itself will be integrated into the given cell. Endosomes are formed around the
integrated particle and the segregation process is initiated. The segregation process
involves the removal of the LDL receptors and having them transported back to the
cell's surface.
● Once the space is only occupied by the low density lipoprotein, lysosomes will fuse
together to ensure the hydrolysis of the corresponding substances it is ingesting. That
includes the small amount of triacylglycerols and the large amount of cholesteryl
esters present, which are both broken down into their respective monomers. ApoB-
100 itself also ends up hydrolysed into its given amino acids. The cell receives pure
monomers from the whole LDL particle that has been ingested. Cholesteryl esters can
be further esterified to be stored into lipid droplets.
Reverse Cholesterol Transport
Step 1
● The initial formation of the nascent high density lipoprotein particles take place
primarily in the liver as well as to a smaller extent in the small intestines. The nascent
form of high density lipoprotein is basically a protein rich particle, small in size and
made up of the excess phospholipids shed from very low density lipoproteins while
they are hydrolyzed by the enzyme lipoprotein lipase. This ensures a large content of
phospholipids being carried by the nascent HDL particles. A nascent HDL particle is
not mature and therefore does not yet actually partake in the cholesterol transport.

Step 2
● Nascent HDL particles are released into the bloodstream and as they pass the
peripheral cells they are able to pick up free cholesterols with the help of the
corresponding apolipoprotein of apoA-1. ApoA-1 is unique to HDL as it ensures the
binding between nascent HDL to peripheral cells and further cholesterol transport by
the action of a membrane ATP binding cassette transporter A1. ABCA1 uses ATP as
a source of energy for the transportation of cholesterol from the peripheral cells to the
nascent HDL particles. The involvement of ATP in this process correspondingly
makes this the rate limiting step for the efflux of free cholesterol to apoA I. With this
process, the nascent HDL particle has obtained free cholesterol from peripheral cells.
Step 3
● The nascent HDL particle also contains the enzyme LCAT, short for Lecithin
cholesterol acyl transferase. This particular enzyme ensures the esterification of free
cholesterol, in simplified terms it adds fatty acids to the OH group of the cholesterol
making it less water soluble than the cholesterol molecule itself. We gain this fatty
acid from lecithin (phosphatidylcholine) which is a phospholipid found in very low
density lipoproteins. The esterification of cholesterol results in the nascent HDL
molecule becoming spherical in shape, transforming it into a mature HDL particle
which partakes in the reverse cholesterol transport.

Step 4
● Mature HDL particles can give away its cholesteryl esters to VLDL remnants which
further on are converted to low density lipoproteins. Keep in mind that the different
lipoproteins are not isolated from each other, but are constantly in the same space
with one another during their travel through the circulatory system. The constant
contact with one and other makes the transfer of cholesteryl esters possible. This
transfer is mediated by the enzyme CETP, short for Cholesteryl ester transfer protein.
CETP can either only deliver the cholesteryl esters to the IDL or it can simultaneously
deliver the cholesteryl esters to the given lipoprotein in exchange of triacylglycerols
from triglyceride rich lipoproteins (chylomicrons and VLDL) by lipoprotein lipase
enzyme. During this exchange, the size of the mature HDL particle slightly changes
as it becomes bigger.

Step 5
● The exchange described in the previous step changes our assumption that the HDL
particle is the only lipoprotein able to carry cholesterol back to the liver. Due to the
transfer of cholesteryl esters, IDL can also transport them back to the liver to be
further utilized. Therefore some of the cholesterol esters that are formed in HDL are
carried to the liver by VLDL remnants and chylomicron remnants. However, the main
route consists of the mature HDL particles themselves traveling back to the liver.

Step 6
● In the liver capillaries, the HDL particle will interact with another enzyme known as
HTGL which is short for hepatic TAG lipase. HTGL is a slightly different isoenzyme,
but it still has a similar function to lipoprotein lipase. HTGL hydrolyses the partial
release of triacylglycerols and phospholipids into their monomers of free fatty acids,
which are further transported to the liver. As the HDL particle continues on with its
travels it encounters corresponding scavenger receptors, class B type 1 (SR-B1). This
given receptor transfers the corresponding cholesteryl esters into the liver, and this
process occurs bidirectionally which converts large HDL particles, known as HDL-2,
back to the smaller HDL-3 (nascent).
Step 7
● As the process described above occurs bidirectionally, at the end the HDL particle
will return back to its nascent form. The hepatic TAG lipase enzyme ensures the
release of the remaining triacylglycerols and further on the release of the remaining
cholesteryl esters into the liver. Keep in mind that the cholesteryl esters are not
hydrolyzed but are transferred to the hepatocytes through the bidirectional carrier SR-
BI. Once the HDL particle has been depleted from the cholesteryl esters, it is
noticeably a lot smaller in size and dissacoties to recirculate in the bloodstream to
once again repeat the pathway of reverse cholesterol transport.

Reverse Cholesterol Transport involves three routes

1. ApoE mediated endocytosis of remnant particles which have obtained part of their
cholesteryl esters from HDL; This is in consequence of the HDL particle exchange
that was described in Step 4.
2. Direct transfer of cholesterol esters from HDL during lipolysis by HTGL, mediated
by SR-BI; Main route,
3. Endocytosis of large apoE coated HDL particles in the liver; In case the HDL particle
is too large and therefore needs to be taken up entirely by endocytosis. However, this
is a very rare route of this pathway.
Summary - Characteristics of Lipoproteins
Summary - Activation and Mobilization of Lipoprotein Contents

Functions of Cholesterol.
Sources of Cholesterol
Cholesterol can be found in food and it is estimated that we ingest approximately 30% of our
daily cholesterol which is utilized for our cell function. All cells in our body are able to
synthesize cholesterol on their own, with the liver as the primary site for cholesterol
synthesis. The body's capacity to synthesize cholesterol on their own in all cells makes
cholesterol intake from food obsolete as it is not technically mandatory from our diet. While
we could easily live without cholesterol, avoiding dietary cholesterol entirely would be very
difficult to achieve.

Functions of Cholesterol (I)

The belief that cholesterol is all about the bad stuff (with the relation with cardiovascular
diseases and other complications) is a false pretense, as without cholesterol we would not be
alive. Cholesterol is integrated into the cell membrane structure in a way where the tail part is
in the middle of the membrane and correspondingly binding to the fatty acid side chains of
the phospholipids. The only hydrophilic part of the cholesterol molecule, the hydroxyl group,
is facing the outer part of the cell membrane and it is correspondingly connected to the
phospholipid hydrophilic part which is the phosphorus head.

Part of the cell membrane which:

Prevents solidifying of Fatty acid tails thus...
● Adds firmness and integrity
● Maintains fluidity at different physiological temperatures
The incorption of cholesterol in between the phospholipids is important as it prevents the
solidifying of the fatty acid tails. The solidifying of the tails would involve the formation of a
clot from fatty acids sticking to each other. In that sense, cholesterol is an integral part of the
cell membrane as it grants the membrane its characteristic properties of elasticity and fluidity.
Reduces permeability to some solutes e.g.:
● Neutral solutes
● H+
● Na+
The incorporation of cholesterol is also important as it allows the necessary solutes to pass
through the cell membrane without any other complications.
Thus animal cells do not need cell wall and they can change the shape

Functions of Cholesterol (II)

Cholesterol is the precursor for steroid hormones
Cholesterol forms the 3 following groups of steroid hormones;
1. Sex hormones; EX. Testosteron, Estrogen
2. Mineralocorticoids; Aldosterone is the most important one as it regulates the
reabsorption of various ions in the kidney.
3. Glucocorticoid; Cortisol is the most important one as it affects the protein and
carbohydrate metabolism, suppresses inflammatory, allergic and immune response.
Cortisol is used in medicine during organ transplantation to lower the rejection rate
with the introduction of a new organ.

Functions of Cholesterol (III)

Precursor for bile acids
● Ensure emulsification of dietary lipids during digestion

Functions of Cholesterol (IV)

Cholesterol is the precursor for vitamin D. Vitamin D is not only a vitamin, but also acts as a
hormone with its capacity to trigger changes in gene transcription which further on takes
effect on cell proliferation and differentiation in apoptosis. Vitamin D is not all about the
calcium but also plays an important role in gene regulation.
● Vitamin D is a hormone, and in addition to its role in calcium homeostasis, it
influences genes involved in cell proliferation, differentiation, and apoptosis
● Deficiency of vitamin D produces rickets in children and osteomalacia in adults

Structure of cholesterol
Cholesterol is a 27 carbon complex molecule that consists of four ring structures which form
the steroid structure of the cholesterol. The acetate is part of the same Acetyl-CoA molecule
that built up the given cholesterol.

Biosynthesis of Cholesterol.
Cholesterol biosynthesis place of action
● Every cell of the human body, but primarily in liver
● Primarily in endoplasmic reticulum, but also in cytosol and peroxisomes. Varies from
reaction to reaction, keep in mind that all of them are important but most of them
happen to take place in ER.
Shuttle for transfer of acetyl groups from mitochondria to the cytosol needs ATP energy
Acetyl-CoA is produced in the matrix of the mitochondria, meanwhile the cholesterol
biosynthesis takes place in entirely different cell compartments. That is why we have this
transport system so it can transport Acetyl-CoA from the matrix into the cytosol and partake
in metabolic processes such as the biosynthesis of cholesterol or the fatty acids catabolism.

In the first step of the transport system, oxaloacetate is converted into citrate with the addition
of Acetyl-CoA which so happens to be identical to the first reaction of the citric acid cycle. In
the citric acid cycle, the concentration of citrate and the other intermediates are kept low.
Once citrate is formed more frequently and starts accumulating inside the cell, the citrate
transporter transports it out into the cytosol and correspondingly performs the reverse
reaction with the conversion into oxaloacetate. With the formation of oxaloacetate inside the
cytosol, 1 ATP is needed for the separation of the acetyl group.

In order to complete the cycle of this shuttle, the given molecules have to return back to the
matrix. This can be done in the 2 following ways:
1. ATP if malate aspartate shuttle is used [Dotted line - Optional route]
Malate is formed and can be transported back into the matrix through the malate/aspartate
shuttle in order to reform the molecule of oxaloacetate.
2. ATP if pyruvate transporter is used [Black line - Main route]
Malate is converted into pyruvate and the given pyruvate is brought back into the matrix of
mitochondria through the pyruvate transporter. This is the same transporter used in the case
of glucose oxidation. Pyruvate is further converted into the oxaloacetate molecule with the
help of the enzyme pyruvate carboxylase, this reaction is identical to a step in
gluconeogenesis. The conversion from malate into pyruvate is important as it forms the
cofactor NADPH + H+, which is the reduced form of the phosphorylated cofactor of
NADP+. NADPH + H+ is a cofactor necessary for the biosynthesis of fatty acids and
cholesterol. With this in mind, this given reaction hits 2 birds with 1 stone as we do not only
manage to transfer the acetyl-coa molecule (basic unit which initiates lipid synthesis) from
the matrix into the cytosol, but we also create the cofactor we need to use for the synthesis of
said lipids.
Overview of Eukaryotic Cholesterol Biosynthesis
4 major steps which ensure the biosynthesis of cholesterol:
1. Three acetates condense to form 6-C mevalonate
2. Mevalonate converts to phosphorylated 5-C isoprene
3. Six isoprenes polymerize to form the 30-C linear squalene
4. Squalene cyclizes to form the four rings that are modified to produce cholesterol

Step 1: Formation of Mevalonate from Acetyl-CoA

 ● Three acetyl-CoA are condensed to form HMG-CoA
● HMG-CoA is reduced to form mevalonate
- HMG-CoA reductase is a common target of cholesterol-lowering drugs
Reaction 1 [Cytosol]:
Enzyme: Thiolase (acetylCoA acetyltransferase)
Involves the enzyme thiolase transferring one Acetyl-CoA (2C) to another Acetyl-CoA (2C)
to form the molecule acetoacetate (4C).
Reaction 2 [Cytosol]:
Enzyme: HMG-CoA synthase
Involves the enzyme HMG-CoA synthase transferring another single Acetyl-CoA to
acetoacetate for the formation of HMG-CoA (β-Hydroxy β-methylglutaryl-CoA)4.
Reaction 3 [Endoplasmic reticulum]:
Enzyme: HMG-CoA reductase
Rate-limiting step
Involves the enzyme HMG-CoA reductase performing an oxidation reduction reaction and
using the reduced form of the phosphorylated NADP+. 2 NADPH + H+ cofactors are used
for this single reaction because once the bond is broken down and the CoA group is released,
you need to replace this particular bond with one of the H+ atoms. As we can see, the end of
the fifth carbon atom still has three more additional H+ atoms which explains why we need 2
NADPH + H+ cofactors in this case. In the end the molecule of mevalonate is formed.
Mevalonate is still an intermediate that has six carbons (6C) all together.

Step 2: Conversion of Mevalonate to Activated Isoprenes in peroxisomes

4
Nomenclature: Beta carbon is the third carbon atom and correspondly it tells you that a hydroxyl
group and a methyl group is attached to the third carbon atom.
Isoprene is a 5 carbon molecule, activated means that there are phosphorus groups attached to
them. The same phosphorylation occurs three times in a row.
● Three phosphates are transferred stepwise from ATP to mevalonate
● Decarboxylation and hydrolysis creates a diphosphorylated 5 C product (isoprene)
with a double bond
● Isomerization to a second isoprene
❖ (triangle shape)3 isopentenyl pyrophosphate (IPP)
❖ dimethylallylpyrophosphate (DMAPP)

Reaction 4 [Peroxisome]:
Enzyme: Mevalonate 5-phosphotransferase
Mevalanonate is converted into 5-phosphomevalonate with the addition of one phosphorus
group at the fifth carbon atom. This reaction is mediated by the enzyme mevalonate 5-
phosphotransferase.
Reaction 5 [Peroxisome]:
Enzyme: Phosphomevalonate kinase
The enzyme phosphomevalonate kinase is a transferease which ensures that another
phosphorus group is transferred. The attachment of 2 phosphorus groups is always called
pyrophosphate. That is why we call this newly formed molecule 5-pyrophosphomevalonate.
Reaction 6 + 7 [Peroxisome]:
Enzyme: Pyrophosphomevalonate decarboxylase
The enzyme pyrophosphomevalonate decarboxylase partakes in the two following reactions
as it ensures another phosphorylation is performed. In this case, it is the 3rd carbon atom
which is being phosphorylated as the previously attached phosphorus group is removed (3-
phospho-5-pyrophosphomevalonate) alongside the carboxyl group in the following step
(activated isoprenes).

This results in the formation of 2 activated isoprene molecules. 3-isopentenyl pyrophosphate

(IPP) is a molecule which is more commonly used in the synthesis of cholesterol.
Dimethylallylpyrophosphate (DMAPP) is an isomer with its change in the structure of the
given double bond which has been transferred. Dimethylallylpyrophosphate (DMAPP) is also
essential for the next set of reactions.

Step 3: Formation of Squalene in ER condensation

 ● Squalene has 30 carbons, 24 in the main chain and 6 in the form of methyl group
branches

Reaction 8 [ER]:
Enzyme: Dimethylallyl pyrophosphate prenyl transferase
The 2 formed isoprene units are now condensed with head to tail condensation. Head to tail
condensation involves that the head part of the molecule has a phosphorus group attached to
them while the tail part of the molecule does not have a phosphorus group attached to it. 2
phosphorus groups are released in the form of pyrophosphate, during which 3-isopentenyl
pyrophosphate (IPP) and the carbon skeleton of the dimethylallylpyrophosphate (DMAPP)
molecule are being condensed. These two 5 carbon atom molecules come together to form
one 10 carbon atom molecule called geranyl pyrophosphate.
Reaction 9 [ER]:
Enzyme: Prenyl transferase
Head to tail condensation takes place in this step, as one more 3-isopentenyl pyrophosphate
(IPP) is added to correspondingly form one 15 carbon atom molecule called farnesyl
pyrophosphate.
Reaction 10 [ER]:
Enzyme: Squalene synthase
Head to head condensation takes place in this step, therefore 2 pyrophosphate groups will be
released. Another farnesyl pyrophosphate is added alongside the previously created molecule
in our last together, all together this reaction yields one 30 carbon atom molecule called
squalene.
Step 4: Conversion of Squalene to Four Ring Steroid Nucleus
Squalene monooxygenase adds one oxygen to the end of the squalene chain
- forms squalene 2,3-epoxide

Reaction 11 [ER]:
Enzyme: Squalene monooxidase
The cyclization product in animals is called lanosterol, which is converted into cholesterol
with the help of the enzyme squalene monooxidase. This enzyme adds another oxygen atom
into the squalene structure which later on forms the only hydroxyl group characteristic for the
cholesterol molecule. This oxygenation of squalene usually involves slightly different
rearrangements of the double bonds, this facilitates the formation of rings in the squalene
molecule.
Reaction 12 - 20+ reactions [ER]:
Enzyme: Squalene cyclase
The enzyme squalene cyclase ensures the cyclization which is the characteristic formation
process in animals. With the finalization of the necessary ring structures, lanosterol is created.
Lanosterol is made up of 30 carbon atoms, so approximately 20 more reactions follow suit in
order to convert lanosterol into the final product of cholesterol. The primary thing to notice
here is that there is some loss and switch of double bonds, with three carbon atoms lost in the
first ring structure as well as the loss of one methyl group along the way. After 29 reactions,
the 27 carbon atom molecule of cholesterol is finally formed.
Cholesteryl Esters Formation and Use
● More hydrophobic form than cholesterol
● Prevents entering membranes
● Either packed in VLDL or stored in the liver in lipid droplets
Cholesterol in free form in our body and in our cells is not as common as the cholesteryl
esters. The reason for that has to do with the fact that free cholesterol can easily permeate the
cell membrane, changing its characteristic features. The esterification of cholesterol, in other
words the attachment of fatty acids to the hydroxyl group, prevents it from entering the
membrane. The cholesteryl esters are synthesized primarily in the liver, where they will be
packed into VLDL and will either be further transported to the peripheral tissues or stored in
lipid droplets present in the liver for safekeeping.
Five Modes of Regulation of Cholesterol Synthesis and Transport
Short term
1. Covalent modification of HMG-CoA reductase (rate limiting enzyme of cholesterol
synthesis)
Long term
2. Transcriptional regulation of HMG-CoA reductase gene
3. Proteolytic degradation of HMG CoA reductase; decrease cholesterol amount in
response to an overabundance of enzymes produced
4. Activation of acyl-CoA cholesterol acyl transferase (ACAT), which increases
esterification for storage
5. Transcriptional regulation of the LDL receptor; feedback regulation on general
synthesis of cholesterol

Regulation of Cholesterol Metabolism

HMG-CoA Reductase Is Most Active When Dephosphorylated
The synthesis of cholesterol is dependent on how much energy we have and how much
energy we can invest in the process. Cholesterol biosynthesis occurs only in an energy rich
state.
1. AMP-dependent protein kinase
Low concentration of ATP correspondingly means that there is an increase in adenosine
monophosphate (AMP). This in turn will lead to phosphorylation of the HMG-CoA by the
hands of the AMP-dependent protein kinase. This will result in the decreased production of
cholesterol as energy in the cell is not sufficient at the moment.
❖ when AMP rises, kinase phosphorylates the enzyme → ⬇ enzyme activity,
⬇ cholesterol synthesis
2. Glucagon, epinephrine
Glucagon and epinephrine, does the opposite as the cascade leads to the phosphorylation of
the given enzyme, lowering its activity. With these two hormones present the body is alerted
that we do not have sufficient enough energy.
❖ cascades lead to phosphorylation, ⬇ enzyme and synthesis activity
3. Insulin
Insulin and its cascade is triggered by the secondary messenger cascade, which will lead to
the activation of the enzyme HMG-CoA reductase through dephosphorylation. The activity of
this given enzyme will result in the increased activity of the cholesterol biosynthesis.
With insulin present, the body knows that we have a lot of energy, which can easily be used
for the synthesis of cholesterol.
❖ cascades lead to dephosphorylation, ⬆ activity
Cardiovascular Disease (CVD) Is Multifactorial
The cardiovascular disease is multifactorial, meaning that cholesterol is not the only molecule
which facilitates this problem, however it still plays a very big role in it.
Very high LDL5 cholesterol levels tend to correlate with atherosclerosis
- although many heart attack victims have normal cholesterol, and many people with
high cholesterol do not have heart attacks
Low HDL6 cholesterol levels are negatively associated with heart disease

Total cholesterol: 3.5 - 5 mmol/L

“Good cholesterol” HDL: ≥ 1.8 mmol/L
“Bad cholesterol” LDL: ≤ 3.5 mmol/L

Plaque Formation
How does plaque formation take place?
In addition, plaque formation is facilitated by low grade chronic inflammation, calcification
and fibrin cup formation.
Low grade chronic inflammation is the consequence of bad lifestyle choices. In the case of
adiposity7, adipocytes secrete inflammatory markers and these signaling molecules circulate
in the bloodstream. There is always the slight risk for it to cause micro inflammation of the
inner lining of the vascular system. The endothelium cells are not as tight together with the
cell conjunctions which increases the risk for low density lipoproteins to sneak in through the
endothelial cells.

5
Travels from the liver to the periphery
6
Travels back to the liver for the excretion of cholesterol formed in the periphery of the body
7
The fact or condition of having much or too much fatty tissue in the body; obesity: Adult weight
gain and adiposity in early adulthood were associated with marked increases in the risk of major
chronic diseases in middle-aged and older men.
Over time they will start accumulating between the outer layer of the smooth muscle and
between the endothelial cells. Usually these low density lipoprotein particles are also already
modified which makes them into oxidized low density lipoproteins, they are typically a lot
smaller in size which facilitates their incorporation and accumulation. The accumulation of
LDL of the extracellular matrix initiates the inflammation, and white blood cells (monocytes)
will arrive at the site in response. These monocytes will differentiate into macrophages, their
role is to pick up the oxidized LDL particles to form foam cells. Foam cells got their name
due to their foamy look in the microscope.

The formation of foam cells and the corresponding accumulation of cholesterol in the area
further stimulates the inflammation which results in the tissue getting a lack of oxygen. The
lack of oxygen of the given tissue will lead to apoptosis and eventual necrosis of said tissue.
Smooth muscle cells are now also growing on the other side of the plaque, in an attempt to
ensure it does not break off. The fibrin cup is also starting to form, which makes the plaque
more stiff and less elastic. On top of the plaque, calcification occurs to harden the plaque.

Everything will eventually lead to the entering bloodstream tearing itself apart and the
support formed for the plaque disintegrates from the blood vessel wall. The disintegration of
the plaque will form a thromb, which can travel all the way to the heart and brain, which will
correspondly cause a myocardial infarction or a stroke. These consequences can be very
severe, or even lethal in some cases.

Five Hyperlipoproteinemia Phenotypes

Familial Hypercholesterolemia
1. Due to genetic mutation in LDL receptor
2. Impairs receptor mediated uptake of cholesterol from LDL
3. Cholesterol accumulates in the blood and in foam cells
4. Regulation mechanisms based on cholesterol sensing inside the cell does not work
5. Homozygous individuals can experience severe CVD as youths
OBS. Keep in mind that all cells are capable of synthesizing their own cholesterol, so there is
a double increase in cholesterol in the body in response to cells not receiving the cholesterol
carried by the LDL molecules.

Symptoms:
● Atherosclerosis
● Corneal arcus (highly specific <45 years of age)
● Xanthelasmata (low
● Tendon xanthoma (higly specific)

Statin Drugs Inhibit HMG CoA Reductase to Lower Cholesterol Synthesis

Statins resemble mevalonate → competitive inhibitors of HMG CoA reductase
First statin, lovastatin, found in fungi
- Lowers serum cholesterol by as much as 30% in individuals with
hypercholesterolemia
The combination of these drugs, which lower the cholesterol synthesis and facilitates the
excretion of cholesterol from our body, is good for the prevention of disease.
● Also reported to improve circulation, stabilize plaques by removing cholesterol from
them, and reduce vascular inflammation
● Good in combination with edible resin that binds bile acids and prevents their
reabsorption from the intestines
OBS. The formation of the bile is the only way we can release cholesterol from our body.

Reverse Cholesterol Transport by HDL Explains Why HDL Is Cardioprotective

Very rare, less than 100 families worldwide with Tangier disease are known
The Tangier disease is characteristic for the lack of ABCA1 transporter. It is found on the
peripheral cells from where free cholesterol is released to be further transported back to the
liver. The lack of this ABCA1 transporter prevents the return of cholesterol back to the liver.
● HDL picks up cholesterol from non liver tissues, including foam cells at growing
plaques
● HDL carries cholesterol back to the liver

Summary of Cholesterol Transport

● Lipoproteins transport hydrophobic lipids between organs and tissues
● Chylomicrons mediate transport of dietary triacylglycerols
● VLDL mediate the transport of endogenously synthesized triacylglycerols
● Chylomicrons , VLDL, and remnant lipoproteins are part of the organism's fuel
distribution network
● LDL are cholesterol rich lipoproteins generated from the VLDL remnants. Similar to
the remnant particles, they are small enough to enter the arterial wall
● HDL mediate reverse cholesterol transport (e.g., removal of cholesterol) from the
peripheral cells and its transport to the liver

Summary of Cholesterol Biosynthesis

● Cholesterol is an essential constituent of cell membranes and the precursor molecule
for bile acids, steroid hormones, and vitamin D
● Cholesterol is both supplied with the diet and synthesized de novo from acetyl-CoA in
each cell of the human body
● The rate limiting enzyme in the cholesterol synthesis pathway is HMG-CoA reductase
● Production of isoprene for cholesterol biosynthesis occurs via the mevalonate
pathway and starts with 3 acetyl-CoA
● Short term regulation is mediated with hormones (e.g. Insulin and glucagon)
● Long term regulation includes increased expression and proteolysis of HMG-CoA
reductase
● Statins are competitive inhibitors of HMG CoA reductase and reduce the formation of
atherosclerotic plaques
Lecture 2a
Lipid metabolism
Learning goals:
● Describe the pathway for activation and transport of fatty acids to the mitochondria for
catabolism
● Outline the sequence of reactions involved in the oxidation of fatty acids in the mitochondria ;
view differences between β , α , and ω oxidation
● Describe the general features of pathways for the oxidation of unsaturated, odd chain, and
branched chain fatty acids
● Explain the rationale for the pathway of ketogenesis , and identify the major intermediates
and products of this pathway

Fatty acid catabolism

Sources of fatty acids
● Fats consumed in the diet
● Fats stored in cells as lipid droplets
● Fats synthesized in one organ for export to another; for humans the liver converts
excess carbohydrates into the fatty acids
● Fats obtained by autophagy in case of extreme stress or during starvation, where the
cells degrades its own organelles in order to supply the cell functions and manage to
gain some ATP

In industrialized countries, the dietary TAGs are approximately more than 40 percent of the
energy requirement during the day despite the fact that the nutritional guidelines suggest that
we consume only 30 percent of the fats daily.

For the skeletal muscle, triacylglycerides give more than 50 percent of its energy.
For the heart and liver, most of the energy requirement comes from triacylglycerides.
Fats Provide Efficient Fuel Storage
The advantage of fats over polysaccharides:
● Fatty acids carry more energy per carbon because they are more reduced
Stored fats are an efficient source of energy due to the fact that they carry more energy per 1
carbon. Fats carry more energy in comparison to carbohydrates because they are more
reduced. In the case of carbohydrates, 1 carbon atom has correspondingly one OH and H
group attached. If we look at the fatty acids, 1 carbon atom has correspondly only one H
group attached and they all form bonds with each other. This structure makes fats more
reduced in nature.
● Fatty acids complex carry less water because they are nonpolar
In addition to this, fats are a great energy source because of their non-polar properties. In
comparison to carbohydrates, their non-polar properties make them lighter as an energy
storage form. Thus it is beneficial for us to store large amounts of fats in the body as a good
potential source of energy.

Carbohydrates are for short term energy needs and quick delivery.
Fats are for long term (months) energy needs, good storage, and slow delivery.

Fat Storage in White Adipose Tissue

Fat droplets stored in adipocytes are so huge that they squeeze the cell's nucleus next to the
plasma membrane. Fat droplets basically take up all the space in an adipocyte cell.
Lipolysis place of action
Process in which triacylglycerides are broken down into fatty acids to be further oxidized.
● TAGs are stored in adipocytes (in steroid synthesizing cells of the adrenal cortex,
ovary, and testis, liver, muscle and other cells)
● Cytosol, in lipid droplets

Hormones (glucagon; epinephrine) - Trigger Mobilization of Stored TAGs

The mobilization of stored TAGs are triggered by the hormones glucagon and epinephrine.
Either of these hormones binds to the G-coupled receptor located on the adipocyte in order to
initiate the secondary messenger cascade. The activation of adenylyl cyclase in turn converts
ATP into cAMP (Cyclic adenosine monophosphate). The presence of cAMP allows for the
further activation of the enzyme Protein Kinase A.8

Protein kinase A has 2 jobs in this case; firstly it activates the enzyme hormone sensitive
lipase through phosphorylation, and secondly it phosphorylates the perilipins proteins.
Perilipins proteins are proteins which cover the lipid droplet. The lipid droplet itself contains
the lipids and is surrounded by a phospholipid monolayer, on the top of said layer are the
perilipins proteins which creates a coat around the lipid droplet. The phosphorylation of
perilipins proteins changes their structure and as this is happening, the perilipins proteins
release the previously bound CGI protein.

The CGI protein activates the enzyme adipose triacylglycerol lipase which initiates the
breakdown of triacylglycerol and converts it into diacylglycerol, with one subsequent fatty
acid released. At this point, the hormone sensitive lipase steps in to further break down the
triacylglyceride and converts it into monoacylglycerol, with the second fatty acids released.
The final breakdown of the traicylglyceride is done by the enzyme monoacylglycerol lipase,
converting it into glycerol and releasing the last fatty acid.

8
Protein kinase A is also an important contributor to the breakdown of certain carbohydrates, such as
it is with the breakdown of glycerol.
In this process alone, 3 different enzymes are needed for the breakdown of TAGs and the
release of the three fatty acids from the lipid droplet. With three separate fatty acids released
from the lipid droplet, they are transported out into the blood. In the blood we can find
albumin. Albumin is a protein which makes up half of the proteins present in the blood. Each
albumin molecule can non-covalently bind to 10 fatty acids. These fatty acids are bound to
the albumin protein and are transported primarily to the myocytes of the heart as well as the
renal cortex for further metabolization into energy. As albumin passes tissues, fatty acids are
picked up by specific fatty acids transporters located on the cell membrane of the given
tissues. These specific fatty acids transporters ensure the transport of fatty acids inside the
cell, for the corresponding catabolic processes and subsequent energy production.

The process described above is how tracylglycered are released from the adipocytes and
broken down into its monomers for the transportation to other tissues where its energy is
needed for cell function.

Important vocabulary:
Albumin ~1/2 of serum proteins [ Noncovalentyl binds ~ 10 FA ]
PKA (protein kinase A) - phosphorylation of HSL and perilipins
Perilipins - outer layer of lipid droplet, releases the CGI protein
CGI protein - activation of the ATGL enzyme
HSL (hormone sensitive lipase) - breakdown of diacylglycerol → monoacylglycerol + 1 fatty
acid
ATGL (adipose triacylglycerol lipase) - breakdown of triacylglycerol → diacylglycerol + 1
fatty acid
MGL (monoacylglycerol lipase) - breakdown of monoacylglycerol → glycerol + 1 fatty acid
Lipases Cleave Fatty Acids from Glycerol Backbone of Triacylglycerides

Glycerol metabolism place of action

What do we do with the glycerol backbone that is left behind in the previously spoken
process? Release 5% of TAG energy
● Primarily in liver, but enzymes expressed in all tissues
● Cytosol
Glycerol from Fats Enters Gluconeogenesis or Glycolysis
At the beginning of Glycerol metabolism, energy needs to be invested right away in order to
metabolize glycerol. However, we regain back the energy we invested in as soon as in the
second step, which makes this a worthwhile process.

How are glycerol molecules transported from the adipocytes (primarily released) to the liver;
They are able to get to the liver through aquaporins. Aquaporins ensures passive diffusion
from peripheral cells into the blood, and from the blood it can be delivered straight to the
liver. The glycerol transported to the liver is primarily used for the gluconeogenesis process.
If the glycerol backbone were to remain in the tissues from where it was released from, it
would join the glycolysis process.
Process:
● Glycerol kinase activates glycerol at the expense of ATP
● Subsequent reactions recover more than enough ATP to cover this cost
● Allows limited anaerobic catabolism of fats
● Passive diffusion through aquaporins ensure transport from peripheral cells to the
liver
Transport in Cell Requires Conversion to Fatty-Acyl CoA
Fatty acyl-CoA synthetase isoenzymes for:
● Short carbon chains
● Intermediate carbon chains
● Long carbon chains

In order for fatty acids to be transported inside the matrix of the mitochondria for further
oxidation, it needs to be activated in a sense where acyl-CoA is formed. A CoA group is
attached to the corresponding fatty acid, this is done by a group of isoenzymes. Fatty acyl-
CoA synthetase is different for the short, intermediate and long carbon chains of fatty acids.
The addition of a CoA group to the fatty acid requires energy in the form of ATP. However,
it is not a phosphate group that we attach but rather the Adenosine monophosphate (AMP)
group which is added to the fatty acid. Once this happens, CoA can perform a nucleophilic
attack to the corresponding fatty acid side chain which results in the formation of Acyl-CoA
and subsequent release of the AMP group.

From the previous steps, we have left overs in the shape of pyrophosphate groups as one
pyrophosphate molecule is released. Both of these reactions are considered to be exogenic, in
addition to the conversion from a pyrophosphate molecule into 2 inorganic pyrophosphate
molecules. This ensures that the corresponding reaction has actually taken pace and it is
within a high rate. Fatty acyl-CoA (Acyl-CoA for short) is used either for the oxidation of the
given fatty acid in the matrix of the mitochondria or for the synthesis of membrane lipids.
Keep in mind that this entire process described aboves takes place in the cytosol of the cell.
AcylCarnitine/Carnitine Transport
For fatty acids with > 12 C
Rate limiting step for fatty acid oxidation!
With the activation of fatty acids, they can proceed to the transport system which ensures that
the given Fatty Acyl-CoA are transported inside the matrix of the mitochondria. This
transport system is called the AcylCarnitine/Carnitine Transport. Carnitine is an amino acid
derivative which ensures the transportation of Acyl-CoA into the matrix of the mitochondria.

As the Acyl-CoA is synthesized in the cytosol, there is an enzyme located on the outer
mitochondrial membrane called carnitine acyltransferase 1. This particular enzyme performs
the transfer reaction which involves the removal of the CoA group and replacing it with
carnitine, this in turn forms the molecule of Acyl Carnitine. The acyl carnitine molecule is
able to be transported into the outer mitochondrial membrane and through the intermediate
space of the mitochondria. Acyl carnitine is further transported into the matrix of
mitochondria with the help of the AcylCarnitine/Carnitine Transporter. This enzyme is an
example of a translocase, which has the role of transporting specific molecules from point A
to point B.

While the molecule acyl carnitine is transported into the matrix of the mitochondria, an
antiport transports one unit of carnitine outside of the matrix of the mitochondria. This
exchange is possible with the reverse reaction mediated by the enzyme carnitine
acyltransferase 2, in which the attached carnitine molecule is removed in favor of adding
back the CoA group. The release of Acyl CoA into the matrix of the mitochondria enables the
next process of Beta-oxidation to occur.

With the simultaneous release of the carnitine group, we can exchange it with the following
acyl carnitine molecule already prepared by the enzyme carnitine acyltransferase 1. This way
we can ensure the continuous transport of the activated fatty acids into the matrix, with free
carnitine to pick up new fatty acids and bring them inside the matrix of the mitochondria.

This transport system is typical for fatty acids that are longer than 12 carbons, typically
starting from 14+ carbon atoms. The short and medium chains are actually able to diffuse
through the mitochondrial membrane. However, for the long and very long fatty acid chains
we need this specific transport system.
Dietary fats - Why this transport system matters
The carnitine shuttle plays a very important role in the oxidation of fats when you realize that
the majority of the dietary fats we consume through food or synthesise in our body are an
example of long or very long fatty acid carbon chains. Very long fatty acid chains start from
22 carbon atoms. This high percentage of long and very long fatty acid chains applies to most
meat and olive oil. This is why the AcylCarnitine/Carnitine Transport system is really
important for ensuring fatty acid oxidation and the resulting energy we gain from it.
The Beta-Oxidation place of action
Most common oxidation for saturated fatty acids
● Every cell with mitochondria, but with different rate
OBS. The brain has a high preference for glucose as an energy source. This is primarily due
to the fact that the fats need a lot more oxygen for their oxidation process. If we were to count
all the ATP and the values of phosphorus rich bonds created per 1 carbon, you need more
oxygen for metabolizing the fats. This also applies during the delivery of fats through the
blood-brain barrier, where oxygen is strictly limited for the brain. This is an important factor
to keep in mind for why the brain has a higher preference for glucose as an energy source.
There is also the fact that fat oxidation is related to a higher production of free radicals.
● Mitochondria

Stages of Fatty Acid Oxidation

Consists of the three following process;
Stage 1 - Beta oxidation
Involves the oxidative conversion of two carbon units into acetyl-CoA via B-oxidation with
concomitant generation of NADH +H and FADH 2
● involves oxidation of B-carbon to thioester of fatty acyl-CoA
Stage 2 - Citric acid cycle
Involves oxidation of acetyl-CoA into CO2 via citric acid cycle with concomitant generation
NADH +H and FADH2
Stage 3 - Electron transport chain
Involves the generation of ATP from NADH +H and FADH 2 via the respiratory chain
The Beta-Oxidation
Each pass removes one acetyl-moiety9 in the form of acetyl-CoA. The released acetyl-CoA
will go further on and activate the citric acid cycle where it will give us the most energy from
the given fatty acid (this is similar to how it is done with carbohydrates).
Why do we have Beta-oxidation;
Single bond between methylene ( CH2 --) is relatively stable in comparison to C-C between
two carbonyl carbons. This single bond makes them harder to break down which coincides
with the increased difficulty of releasing the Acetyl-CoA group. The C-C bond between the
carbonyl groups that we find in the last intermediate of the beta-oxidation process is a lot
easier to break and thus we can release the Acetyl-CoA with relative ease.

9
In organic chemistry, acetyl is a moiety, the acyl with chemical formula CH3CO.
Long Fatty Acid Oxidation is Performed by a Single Trifunctional Protein
Most of the fatty acids that we have in our body is performed by a single trifunctional
enzyme. The 4 steps of Beta-oxidation are taken care of by this single trifunctional enzyme.
The first enzyme is kinda separated from the rest, but is still attached to the inner
mitochondrial membrane. As for the rest of the 3 reactions, the corresponding enzymes are a
part of this single trifunctional protein which is bound to the inner mitochondrial membrane.
● Hetero-octamer (α4β4 subunits)
● May allow substrate channeling between enzymes
● Associated with inner-mitochondrial membrane
● Processes fatty acid chains with 12 or more carbons
● Shorter chains processed by soluble enzymes in the matrix; With short and medium
fatty acid chains, as they enter the matrix, free soluble enzymes catalyze the
conversion of these free fatty acids into the Acetyl-CoA.
Step 1: Dehydrogenation
Catalyzed by isoforms of acyl-CoA dehydrogenase (AD) on the inner-mitochondrial
membrane
● very-long-chain AD (12-18 carbons)
● medium-chain AD (4-14 carbons)
● short-chain AD (4-8 carbons)
The overlapping is intentional; however there is a particular preference towards larger fatty
acids over shorter fatty acids chains

The first step of Beta-oxidation consists of a dehydrogenation reaction. In this case, a double
bond is created between the Alpha and Beta carbon atom. This particular step is catalyzed by
the enzyme acyl-coa dehydrogenase and a trans double bond is formed. This is different from
naturally occuring unsatuurated fatty acids as they always have a cis double bond. This newly
formed trans double bond determines the breakdown of unsaturated fatty acids.

● Results in trans double bond, different from naturally occurring unsaturated fatty
acids
● Analogous to succinate dehydrogenase reaction in the citric acid cycle
● Electrons from bound FAD (prosthetic group) transferred directly to the electron
transport-chain via electron transferring flavoprotein (ETF)

OBS. In the first step, FADH2 is released. FADH2 is a membrane bound protein (in other
words a prosthetic group of the enzyme) and we need transferring flavoprotein for the
mobilization of FADH2. Transferring flavoprotein transport FADH2 to the electron transport
chain where it creates 1.5 ATP molecules per each FADH2 molecule created.
Step 2: Hydration
Catalyzed by two isoforms of enoyl-CoA hydratase:
● soluble short chain hydratase (crotonase)
● membrane bound long chain hydratase, as first part of trifunctional complex

The second step of Beta-oxidation consists of a hydration reaction. In this case we add a
water molecule, which is a common way of eliminating double bonds, creating secondary
alcohols in the process.

● Water adds across the double bond yielding alcohol on beta-carbon (typical reaction)
● Analogous to fumarase reaction in the citric acid cycle
● same stereospecificity

Step 3: Dehydrogenation
 ● Catalyzed by hydroxyacyl-CoA dehydrogenase
● The enzyme uses NAD cofactor as electron acceptor (always for secondary alcohol)
● Only L-isomers of hydroxyacyl CoA act as substrates
● Analogous to malate dehydrogenase reaction in the citric acid cycle

The third step of Beta-oxidation consists of a dehydrogenation reaction. In this case, the
hydroxyl group on the secondary alcohol is dehydrogenated with the help of NAD+ (Ex. we
see it be done when we convert pyruvate → lactate). The hydroxyl group is reduced to a
ketone group. We use NAD+ to get the reduced form of NADH + H+. The released cofactor
can activate the first complex of the Electron transport chain.

Step 4:
Transfer of Fatty Acid Chain and Release of Acetyl-CoA
The last step of Beta-oxidation involves the transfer of fatty acid chain and subsequent
release of Acetyl-CoA. In this case, the enzyme which catalyzes this process is called acyl-
CoA acetyltransferase10 (thiolase). This given enzyme will initially bind the substrate beta-
ketoacyl-CoA to the active site through a covalent bond. The active site will have a thiolate
group present and thus it will perform a nucleophilic attack which subsequently releases the
Acetyl-CoA. While the rest of the molecule still remains attached to the enzyme, another
nucleophilic attack will take place but this time from the Coenzyme A molecule itself. This
attack will result in the CoA group picking up the remaining part of the fatty acid structure
which has now been reduced by 2 carbon atoms.

Catalyzed by acyl-CoA acetyltransferase (thiolase) via covalent mechanism

● The carbonyl carbon in beta-ketoacyl-CoA is electrophilic
● Active site thiolate acts as a nucleophile and releases acetyl-CoA
● Terminal sulfur in CoA-SH acts as a nucleophile and picks up the fatty acid chain
from the enzyme
The net reaction is thiolysis of the carbon carbon bond

10
This is the enzyme which won't have different isoenzymes.
Each Round Produces an Acetyl-CoA and Shortens the Chain by Two Carbons
OBS. When the last round of beta-oxidation comes, 2 Acetyl-CoA molecules are released.
One of them is the one typically released, while the other one is the remaining acetyl-CoA.
The first Acetyl-CoA is released from the breakdown of a chain of 4 carbons (C4) while the
other Acetyl-CoA is simultaneously released from the remaining C2 chain. For that case,
beta-oxidation rounds are always needed one less than the number of the total Acetyl-CoA
molecules you create from the corresponding affty acid.

Calculation:
1. Number of carbons/2 = Number of Acetyl-CoA produced in total
2. Subtract 1 round from the Number of Acetyl-CoA = Answer is given with how many
rounds of beta-oxidation is needed.

NADH+H and FADH2 Serve as Sources of ATP

Similar Mechanisms Introduce Carbonyls in Other Metabolic Pathways

Genetic Defects in Fatty Acyl-CoA - Dehydrogenases Cause Serious Disease

Most common is the mutation in gene coding medium chain (6-12C) Acyl-CoA
dehydrogenase (first step of beta-oxidation)
● Carriers: 1 in 40
● Disease: 1 in 10’000
● Recurring episodes of syndrome include fat accumulation in liver, high blood levels
of octanoic acid, low blood glucose levels, sleepiness, vomiting and coma
● Episodes are very serious, despite no symptoms in between
● Mortality 25-60% in early childhood
● With early detection and careful management of the diet the prognosis is good
Medium chain fatty acids usually come from plant life. Short chain fatty acids are primarily
produced by our floral gut bacteria from the complex carbohydrates we are unable to digest.
Short chain fatty acids are important for a number of reasons, such as the role it plays in the
regulation of the cardiovascular system. If they can not be further metabolized, severe
consequences as seen in the given illness will occur.

Beta-Oxidation in Mitochondria versus Peroxisomes

Peroxisomal acyl-CoA dehydrogenase passes electrons directly to molecular oxygen
● energy released as heat
● hydrogen peroxide eliminated by catalase

The major difference between the beta-oxidation that occurs in the peroxisomes as opposed
to in the mitochondria is that energy is released as heat and the formation of free radicals in
the shape of hydrogen peroxide. Hydrogen peroxide is formed because the final acceptor of
the electrons from the FADH2 is oxygen, not the respiratory electron transport chain as it is
in the matrix. This in turn creates free radicals, for that reason the peroxisomes contain a lot
of catalase and other enzymes that eliminate free radicals.

Much more active on very long chain (≥22C ) fatty acids (such as
hexacosanoic acid (26C)) and branched chain fatty acids such as
phytanic acid and pristanic acid (obtained from dairy products, the
fat of ruminant animals, meat and fish). Such long chains of fatty
acids are quite rare in nature, but you can still find it in some
meat and fish.

X-linked adrenoleukodystrophy
XALD affects young boys before the age of 10 years (gender related pathology):
● Loss of vision, behavioral disturbances and death within a few years
Why does death occur:
The mutation occurs in the gene which codes for the transporter of very long fatty acid chains
which transports them to the peroxisomes to be oxidized. With the malfunctioning of the
transporter, these very long fatty acid chains can not be oxidized which in turn leads to
further accumulation over time. This in turn leads to inflammation which causes more free
radicals formation, the stress that is applied on the cell organelles will eventually result in cell
death. The brain is the most sensitive organ, making the consequences related to the failure of
oxidizing very long fatty acid chains a lot more lethal.

Oxidation of Unsaturated Fatty Acids

Naturally occurring unsaturated fatty acids contain cis double bonds
● are NOT a substrate for enoyl-CoA hydratase (2nd reaction of beta-oxidation)
Two additional enzymes are required:
● isomerase: converts cis double bonds starting at carbon 3 to trans double bonds
● reductase: reduces cis double bonds not at carbon 3
Monounsaturated fatty acids require the isomerase
Polyunsaturated fatty acids require both enzymes

Naturally occuring unsaturated fatty acids contain a cis double bond, which is not the
substrate for the second step of Beta-oxidation. During the second step, hydration occurs in
order to catalyze the trans double bond of the fatty acid. For this reason, 2 additional enzymes
are needed in the case of the oxidation of monounsaturated fatty acids and polyunsaturated
fatty acids. In both cases, we are left with 1 less FADH2 molecule from an entire beta-
oxidation cycle.

Oxidation of Monounsaturated Fatty Acids

At this point acyl-CoA dehydrogenase step is skipped, resulting in 1 FADH 2 less
The oxidation of monounsaturated fatty acids starts off with the regular beta-oxidation, where
we release the Acetyl-CoA one by one until we reach the point of the cis double bond. In this
particular case, the cis double bond is shifted with the help of the enzyme isomerase which in
turn forms it into a trans double bond. It should be noted that a shift also occurs between the
carbon atoms, as in the double bond went from being between the 3rd and 4th carbon atom to
the 2nd and 3rd carbon atom. Isomerase switches the double bond to the position which
ensures further beta-oxidation, however by doing so, 1 less FADH2 is gained from the entire
cycle of beta-oxidation of one monounsaturated fatty acid.

We yield 1 less FAHD2 in this because the first step of dehydrogenation does not occur.
Normally during dehydrogenation, a double bond is formed with the subsequent release of
one FADH2 molecule. With a pre-existing double bond formed by the enzyme isomerase, the
first step is unnecessary and is therefore skipped during the last round of beta-oxidation. With
one less FADH2 molecule produced per one whole fatty acid, 1.5 ATP is lost during the
oxidation of monounsaturated fatty acids.

Oxidation of Polyunsaturated Fatty Acids

At this point acyl-CoA dehydrogenase step is skipped, resulting in 1 FADH2 less.
NADPH+H reduces the remaining unsaturated bond, resulting in no further loss of FADH 2
The oxidation of polyunsaturated fatty acids starts off with the regular beta-oxidation, where
we release the Acetyl-CoA one by one until we reach the point of the cis double bond. In this
particular case, the cis double bond is shifted with the help of the enzyme isomerase which in
turn forms into a trans double bond. It should be noted that a shift also occurs between the
carbon atoms, as in the double bond went from being between the 3rd and 4th carbon atom to
the 2nd and 3rd carbon atom. Isomerase switches the double bond to the position which
ensures further beta-oxidation, however by doing so 1 less FADH2 is gained from the entire
cycle of beta-oxidation of one monounsaturated fatty acid.

In the next round of beta-oxidation, another double bond is formed with the enzyme Acyl-
Coa dehydrogenase. However as this double bond happens to be unsaturated, an additional
enzyme called Reductase has to step in to remove/saturate the corresponding double bonds.
The saturation of the double bond results in the double bond switching from being between
the 4th and 5th carbon atom to the 3rd and 4th carbon atom. NADPH + H will donate 2
hydrogen atoms for each of the given carbon atoms in order to translocate the double bond.
Isomerase will step in again to shift the double bond from being between the 3rd and 4th
carbon atom to the 2nd and 3rd carbon atom.

We yield 1 less FAHD2 in this because the first step of dehydrogenation does not occur.
Normally during dehydrogenation, a double bond is formed with the subsequent release of
one FADH2 molecule. With a pre-existing double bond formed by the enzyme isomerase, the
first step is unnecessary and is therefore skipped during the last round of beta-oxidation. It is
important to note that no further losses of FADH2 is noted as the NADPH+H reduces the
remaining unsaturated bond. With one less FADH2 molecule produced per one whole fatty
acid, 1.5 ATP is lost during the oxidation of polyunsaturated fatty acids.

Oxidation of Odd Numbered Fatty Acids

● Most dietary fatty acids are even numbered
● Many plants and some marine organisms also synthesize odd numbered fatty acids
● Propionyl CoA (3 carbon compound) forms during final cycle of beta-oxidation of
odd numbered fatty acids
● During fermentation the rumen of ruminants also produces propionyl-CoA from
carbohydrates

Naturally occurring odd numbered fatty acids can be found in some plants and marine
organisms which synthesize odd numbers of carbon atoms. Oxidation of uneven fatty acids
differs from the regular oxidation as in the end we have a Fatty acetyl-CoA with 5 carbons.
Once we have a 5C chain, the last round of beta-oxidation leaves us with one Acetyl-CoA
and one Propionyl-CoA molecule. Propionyl-CoA is made up of 3 carbons and no further
oxidation is possible as there is no way for the human body to utilize a single 1C atom
molecule.

For this reason, this molecule is further metabolized through the carboxylation of the CoA
group. During carboxylation, a carboxyl group is added and is mediated by the enzyme
Propionyl-CoA carboxylase. Keep in mind that Vitamin B7 catalyzes the addition of the
carboxyl groups to other molecules. With the creation of a 4 carbon chain sequence, 2
constituent reactions take place which catalyzes isomerization from D-methylmalonyl-CoA
to succinyl-CoA. Succinyl-CoA can enter the citric acid cycle, which involves further energy
production.

The ω oxidation of fatty acids in the endoplasmic reticulum

● In liver and kidney
● Preferred substrates are fatty acids with 10 or 12 C
● Important when β-oxidation is defective (e.g. In case of mutation or carnitine
deficiency)
● Product: fatty acid with carboxyl group at each end
● Either end can be attached to CoASH
● Molecule enter mitochondria for further β-oxidation
● The final 4C molecule (succinate) can enter CAC

Omega-oxidation takes place during very exceptional cases. This type of oxidation primarily
occurs in the liver and the kidney, their preferred substrate are fatty acid chains with 10-12
carbon atoms. The first reaction is catalyzed by mixed function oxygenase, which adds an
oxygen group to the omega side of the fatty acid chain. Further on in the cycle, a
dehydrogenation reaction occurs which introduces another carbonyl group on the other end of
the fatty acid. Both ends of the fatty acid contain the carboxyl group now, with either side
being available for the attachment of a CoA group. Once this happens, the molecule is
transported into the matrix of the mitochondria where it can go through a series of reactions
of Beta-oxidation. This leaves us with the product of succinate, which can further fuel the
citric acid cycle.

The α oxidation of a branched chain fatty acid (phytanic acid) in peroxisomes

● More pronounced in liver and brain
● Long chain fatty acid with methyl branches derived from phytol side chain of
chlorophyll
● Methyl group at β carbon
● Obtained from dairy products, the fat of ruminant animals; microorganisms in the
rumen produce phytanic acid as they digest plant chlorophyll
● Typical western diet includes 50-100 mg of phytanic acid per day
● Removes single C atom from carboxyl end

Alpha-oxidation is a very specific kind of oxygenation as it only oxidizes one type of fatty
acid chain, phytanic acid. Phytanic acid is a long chain fatty acid which has a methyl group
attached to its beta carbon, which prevents regular beta-oxidation from being useful in this
case. This branched fatty acid primarily comes from microorganisms catalyzed reactions that
release this phytanic acid. The idea of this given oxidation is to remove one carbon atom
from the phytanic acid from this corresponding carbonyl group attached to the side of this
molecule. Once this carbon atom is removed, a subset of reactions takes place which results
in the formation of pristanic acid which has its beta-carbon no longer blocked. The free beta-
carbon allows this fatty acid to resume the regular reactions of beta-oxidation. In the end, the
fatty acid will be broken down until it leaves us with the product of propionyl-CoA.

Ketogenesis
Ketogenesis - place of action
Acetyl-CoA usage during deficiency in carbohydrates, Ketone bodies are formed as a result.
● Liver
● Mitochondria
Ketone (Bodies) - historical artifact
Entry of acetyl-CoA into citric acid cycle requires oxaloacetate
When oxaloacetate is depleted, acetyl-CoA is converted into ketone bodies
● frees coenzyme A for continued beta-oxidation
Ketone bodies represent the three molecules which are created during ketogenesis; acetone,
acetoacetate, and beta-hydroxybutyrate. Keep in mind that only two of them are ketones,
beta-hydroxybutyrate is not a ketone.

Ketone bodies are formed in case when there is no oxaloacetate present, as the acetyl-CoA
which would normally enter the citric acid cycle insead is used as a substrate for the
formation of ketone bodies. Formation of ketone bodies releases the CoA group from the
Acetyl-CoA, which correspondingly has it proceed to beta-oxidation.

Ketone Bodies are Water Soluble

● Volatilized through lungs
● Energy for skeletal and heart muscle, brain , renal cortex
● Most prominent
Ketone bodies are good because they are water soluble, which gives these molecules the
ability to travel from the liver to the peripheral tissues via the blood without needing a
specific transport system. In the peripheral tissues, ketone bodies can be reconverted back
into Acetyl-CoA.

Ketogenesis and ketone utilization in extrahepatic tissues functions almost like a transport
system as the energy localized in the liver as fatty acids can be relocated to the tissues where
energy is needed. This given energy is very accessible as it also is in soluble form. Energy is
very vital for the skeletal muscles, heart, and especially the brain in case of hypoglycemia.
The most prominent ketone body present in our body is beta-hydroxybutyrate as its
concentration in the blood is normally the highest out of the three bodies.
Formation of Ketone Bodies: Generating Free CoA-SH
Together, two CoA SH are freed from three acetyl CoA
Step 1:
Thiolase
The first step is reverse of the last step in the beta-oxidation: thiolase reaction joins two
acetate units. The first step of ketogenesis is reversible and is mediated by the enzyme
thiolase. As the 2 acetyl-CoA join together to form the molecule Acetoacetyl-Coa, a CoA
group will be released. The released CoA group can be further used in beta-oxidation for the
continuous breakdown of fatty acids.
Step 2:
HMG-CoA synthase
A third acetyl CoA is incorporated in the second step. In the second step, another acetyl-CoA
is added to form the molecule of HMG-CoA. This step is mediated by the enzyme HMG-
CoA synthase.

OBS. The first 2 steps of ketogenesis overlap identically with the cholesterol biosynthesis.
The only difference between these two cases is their place of action; ketogenesis takes place
in the matrix of the mitochondria while the cholesterol biosynthesis takes place in the cytosol.

Formation of Ketone Bodies: Degradation of HMG-CoA

● In order to traffic to other tissues, CoA-SH must be removed. Acetone, acetoacetate,
and hydroxybutyrate can then travel through the blood
● Acetone is removed as a gas and exhaled, but acetoacetate and beta-hydroxybutyrate
can traffic to the brain for use in energy production

Step 3:
HMG-CoA lyase
During the third step, the enzyme HMG-CoA lyases removes an acetyl-CoA. This leaves
with a new molecule consisting of four carbons, acetoacetate. This step is important as the
previously attached CoA group prevents the molecule itself from traveling in the blood. The
removal of the CoA group makes the molecule soluble, enabling it to travel via blood.
Step 4a:
Acetoacetate decarboxylase → Acetone
The ketone body acetone is produced first from the substrate of acetoacetate. This step is
mediated by the enzyme acetoacetate decarboxylase. This reaction can happen non-
enzymatically (spontaneously). Acetone is a metabolite that the body can not further oxidize
nor use for any other metabolic processes. The only way for acetone to leave our body is as a
gas evaporated through our lungs. This information helps us determine a patient's status in
regards to ketoacidosis. If a patient were to have ketoacidosis, their breath would smell of
acetone.

Step 4b:
Beta-hydroxybutyrate dehydrogenase → Beta-hydroxybutyrate
The ketone body beta-hydroxybutyrate is produced afterwards from the substrate of
acetoacetate. This step is mediated by the enzyme beta-hydroxybutyrate dehydrogenase. The
cofactor NADH + H+ is used as an input of energy for the ketone bodies synthesis, leaving us
with the cofactor of NAD+. However, once this molecule goes to the extrahepatic tissues a
reverse reaction will occur which leaves us with more energy being created afterwards.

Utilization of Ketone Bodies: Fuel in extrahepatic tissues

Note: it is not the reversible process of synthesis
Liver produces ketone bodies, while the extrahepatic tissues use them as an energy source.
The reaction which occurs in reverse in the given tissues are similar, but not identical to the
steps of ketogenesis. During ketone utilization, one step is skipped. Instead of the formation
of HMG-CoA, the enzyme beta-ketoacyl-CoA transferase adds an CoA group to the
acetoacetate to form acetoacetate-CoA.
The additional CoA group was taken from succinyl-CoA (found in the citric acid cycle)
which in turn converts it into succinate. Succinate can proceed to the reaction of the citric
acid cycle. The formed acetoacetate-CoA is recreated into 2 seperate acetyl-CoA groups
through the addition of another CoA group. The formation of 2 new acetyl-CoA molecules in
the end leads to the continuous cycle of producing ketone bodies and the citric acid cycle
intermediate of succinate. This system relocates energy without any energy loss taking place.
OBS. The reason why the liver can only produce ketone bodies and not utilize them is due to
the fact that it lacks the enzyme beta-ketoacyl transferase.

The Liver Is the Source of Ketone Bodies

Ketogenesis and the utilization of ketone bodies occurs when the body is low on
carbohydrates and does not have enough energy in tissues, such as the brain, that prefers
glucose as their energy source. This is typically seen in diabetics, where the lack or absence
of insulin causes the hormone glucagon to be released into the blood in high amounts. The
subsequent release of this hormone triggers the mobilization of the triacylglycerols stored in
adipocytes. TAGs are broken down into fatty acids and then into glycerol. The glycerol
backbone can be used for gluconeogenesis (in the liver) in order for more glucose molecules
to form. The fatty acids transported into the liver goes through beta-oxidation for the release
of acetyl-CoA molecules. These released Acetyl-CoA will be used in ketogenesis for the
generation of ketone bodies.

Keep in mind that the insulin insufficiency will also take effect on muscle cells, which will in
response release glucogenic (facilitates glucose synthesis) and ketogenic amino acids
(facilitates fats synthesis). Under normal circumstances, the formed acetyl-CoA would
proceed into the citric acid cycle. However, we lack the intermediate oxaloacetate as it has
been used for the synthesis of glucose during gluconeogenesis and as fuel for other tissues
such as the brain. This leads to a shift where Acetyl-CoA will instead partake in the synthesis
of ketone bodies. This process allows us to continuously release the CoA group and reduce it
again for the fatty acid oxidation. The excessive breakdown of fatty acids leads to an
excessive generation of ketone bodies in the body.

In diabetics, the issue lies with the absence of insulin. Carbohydrates that are already present
in the body are unable to enter the tissue cells without insulin as its key signaling molecule.
This in turn leaves cells in need experiencing the sensation of starvation, even as there are
plenty of glucose molecules accumulating in the blood outside. Thus even more ketone
bodies are created in addition to the absence of glucose.
Diabetic Ketoacidosis vs Hyperosmolar hyperglycemic state
Diabetic Ketoacidosis - DBA
DKA is characterized by ketoacidosis and hyperglycemia
Biomarker: High ketone levels in your urine and blood serum, High blood sugar level
Symptoms: Excessive thirst, Frequent urination, Nausea and vomiting, Stomach pain,
Weakness or fatigue, Shortness of breath, Fruity-scented breath (Acetone), Confusion

Hyperosmolar hyperglycemic state - HHS

HHS usually has more severe hyperglycemia but no ketoacidosis.
Biomarker: Elevation of blood glucose, Elevation of hyperosmolarity 11.
Symptoms: Excessive thirst, Dry mouth, Increased urination, Warm, dry skin, Fever,
Drowsiness, confusion, Hallucinations, Vision loss, Convulsions, Coma

11
Hyperosmolarity: a condition in which the blood has a high concentration of salt (sodium),
glucose, and other substances.
Summary of lipid catabolism
● Unlike carbohydrate fuels, which enter the body primarily as glucose or sugars that
are converted to glucose, lipid fuels are heterogeneous with respect to chain length,
branching, and unsaturation
● The catabolism of fats is primarily a mitochondrial process but also occurs in
peroxisomes (both beta and alpha oxidation) and omega oxidation in ER
● During peroxisomal oxidation, fats can be oxidized to generate heat however, also
produces H2O2
● Using a variety of chain length specific transport processes and catabolic enzymes,
the primary pathways of catabolism of fatty acids involve their oxidative degradation
in two carbon units, a process known as β-oxidation, which produces acetyl-CoA
● In the process, a lot of NADH +H and FADH 2 forms these can yield a lot of ATP in
the electron transport chain

Summary of lipid catabolism

● In most tissues, the acetyl CoA units are oxidized further and used for ATP
production in the mitochondrion
● In liver, acetyl-CoA is catabolized to ketone bodies, primarily acetoacetate and β-
hydroxybutyrate by a mitochondrial pathway termed ketogenesis
● The ketone bodies are exported from the liver for energy metabolism in peripheral
tissue
● Ketonemia and ketonuria develop gradually during fasting, whereas ketoacidosis may
develop during poorly controlled diabetes, when fat metabolism is increased to high
levels for support of gluconeogenesis

Lecture 2b
Lipid biosynthesis
Learning goals:
 ● Describe the pathway of fatty acid synthesis, particularly the roles of acetyl-CoA carboxylase
and the multifunctional enzyme fatty acid synthase
● Explain the concepts of elongation and desaturation of the fatty acid chain
● Explain regulation of fatty acid synthesis
● Describe the synthesis of triacylglycerides
● Describe the synthesis of phospholipids

Fatty acids biosynthesis

Catabolism and Anabolism of Fatty Acids Proceed via Different Pathways
Catabolism of fatty acids
● produces acetyl-CoA
● produces reducing power (NADH +H ++, FADH2)
● takes place in the mitochondria

Anabolism of fatty acids

● requires acetyl-CoA and malonyl-CoA
● requires reducing power from NADPH +H
● takes place in cytosol in animals, chloroplast in plants

Fatty acid biosynthesis - place of action

● Every cell with mitochondria (as that is where the acetyl-CoA is also being
synetshsied), but with different rate
● Cytosol

Fatty Acid Synthesis Occurs in Cell Compartments Where NADPH +H Levels Are High
Cytosol for animals, yeast
Sources of NADPH +H:
● in adipocytes: pentose phosphate pathway and malic enzyme
❖ NADPH +H made as malate converts to pyruvate + CO2
● in hepatocytes and mammary gland: pentose phosphate pathway
❖ NADPH +H made as glucose-6-phosphate converts to ribulose-5-phosphate
OBS. The pentose phosphate pathway is one of the major producers of NADPH +H.
Acetyl-CoA Is Transported into the Cytosol for Fatty Acid Synthesis
In non-photosynthetic eukaryotes
● Acetyl-CoA is made in the mitochondria, however, fatty acids are made in the
cytosol. Acetyl-CoA is transported into the cytosol with a cost of 2 ATPs
➔ 1 ATP used for the conversion of citrate into oxaloacetate in the mitochondria
➔ 1 ATP used for the conversion of pyruvate into oxaloacetate in the cytosol
● It is far more likely for the malate in the cytosol to return back into the matrix of
mitochondria in the form of pyruvate. This is considered to be the main pathway, as
during the conversion of malate to pyruvate the reducing factor of NADPH + H is
produced. This single step reaction is ensured by malic enzyme.
● Therefore, the total cost of FA synthesis is 3 ATPs per 2C unit (1 acetyl-CoA)

Overview of Fatty Acid Synthesis

● Each time the fatty acid is elongated, it occurs with the addition of 2 carbon atoms, or
in other words one acetate unit at a time.
● The acetate unit comes from activated malonate in the form of malonyl-CoA. It is
important to note that this is done as the acetate unit has to go through a
transformation of some sort to be eligible for the eventual addition to the fatty acid
chain.
● Each pass involves reduction of a carbonyl carbon to a methylene carbon
The Acetyl-CoA Carboxylase Reaction:
Committed step
The committed step of the fatty acid biosynthesis pathway is ensured by a complex enzyme
called acetyl-CoA carboxylase. Once the malonyl-CoA is formed, it can be used for the fatty
acid synthesis.

Process:
In the middle of the enzyme, there is a biotin carrier protein as well as vitamin biotin attached
to it through the lysine side chain. Vitamin biotin (B7) performs the carboxylation reaction,
which involves the addition of CO2. CO2 is initially added as a bicarbonate ion, which is its
soluble form. 1 ATP is needed for each 2 carbon atom unit addition for 1 fatty acid.
In total, 3 ATP is used for Fatty acid biosynthesis;
➔ 2 ATP - used for the transport of acetyl-CoA from the matrix of mitochondria to the
cytosol
➔ 1 ATP - used for the malonyl-CoA formation.

In this first reaction, bicarbonate ion is added to form a carboxyl group to the biotin carrier
protein (Vitamin B7). This kind of reaction requires additional energy input. With the
addition of bicarbonate to the biotin carrier protein, it is now a lot easier to add the carboxyl
group to the acetyl-CoA. The first part of the enzyme (biotin carboxylase) ensures the
addition of the carboxyl group to the corresponding Vitamin B7. The carboxyl group end of
the molecule flips over to the other side of the enzyme's active site (transcarboxylase). Here
acetyl-CoA can be added on to the growing molecule. The addition of the carboxyl group to
the acetyl-CoA allows it to become more reactive. This is important as the CH3 group is
inert, which means it is stable as all 4 covalent bonds are not really willing to interact with
other molecules. Once we have added the carboxyl group to acetyl-CoA, it is a lot more
reactive due to the electron distribution around these given atoms.
Synthesis of Fatty Acids Is Catalyzed by Fatty Acid Synthase (FAS)
Once the malonyl-CoA has been formed, we can start with the synthesis of fatty acids
FAS I:
Fatty acid synthase 1
● Single polypeptide chain with 7 active sites in vertebrates.
➔ Each of these enzyme active sites are located in their seperate domain.
● Leads to the formation of a single product: palmitate 16:0
➔ Palmite; Saturated fatty acid, with 16 carbon atoms.
OBS. All of the active sites in the mammalian system are located in different domains within a single
large polypeptide chain. The different enzymatic activities are β ketoacyl ACP synthase (KS),
malonyl /acetyl CoA ACP transferase (MAT), β hydroxyacyl ACP dehydratase (DH), enoyl ACP
reductase (ER), and β ketoacyl ACP reductase (KR). ACP is the acyl carrier protein.

FAS II:
Fatty acid synthase 2
● Separate enzymes in plants and bacteria
● Leads to the formation of multiple different products; saturated, unsaturated,
branched etc.

Fatty Acid Synthesis

Overall goal: to attach acetate unit (2C) from malonyl-CoA to a growing chain and then
reduce the fatty acid chain
Reaction involves cycles of four enzyme catalyzed steps:
These reactions are the reverse of the beta-oxidation
1. Condensation of the growing chain with activated acetate
2. Reduction of carbonyl to hydroxyl
3. Dehydration of alcohol to trans alkene
4. Reduction of alkene to alkane
OBS. The growing chain is initially attached to the enzyme via a thioester linkage

Acyl Carrier Protein (ACP) Serves as a Shuttle in Fatty Acid Synthesis

Acyl Carrier Protein (ACP) is attached to the fatty acid synthase.
Contains a covalently attached prosthetic group 4-phosphopantetheine
● Flexible arm tether acyl chain while carrying intermediates from one enzyme subunit
to the next. Part of this arm consists of pantothenic acid (Vitamin B5). The role of this
flexible arm is to transfer the fatty acid from one active site to another in this large
fatty acid synthase complex, thus ensuring the proceeding reactions without releasing
any intermediates in between.
ACP also delivers the basic substrates of acetate (in the first step) or malonate (in all the next
steps) to the fatty acid synthase

Charging:
Addition of Acetyl-CoA to ACP and further transfer to SH of KS in synthesis Denovo
Acetyl-coa synthase needs to be charged up before it can start to work. Charging up involves
the addition of acetyl-CoA and malonyl-CoA so they could be condensed.
In this case, fatty acids biosynthesis occurs according to these 2 following pathways;
1. Synthesis Denovo describes the process in which we start from scratch as there are no
previously existing fatty acids.
2. The same process can take place in the exact same sequence in case of the elongation
of fatty acids
Firstly, we start off with the fatty acid synthesis Denovo as it is from scratch. In this case, the
ACP binds to the acetyl-CoA with the subsequent release of the CoA group. The ACP shifts
and shuttles the acetate group further on to the enzyme beta-ketoacyl-acp synthase that has
the thiol group attached to it. The first thioester bond is between the acetate group and the
thiol group of beta-ketoacyl-acp synthase. We have now added on one of the units necessary
for the fatty acids synthesis.

Charging: addition of Malonyl-CoA to ACP

● By using activated malonyl groups in the synthesis of fatty acids and activated acetate
in their degradation, the cell makes both processes energetically favorable, although
one is effectively the reversal of the other
Malonyl-CoA is the unit that is actually used for the elongation of fatty acids. Before each
fatty acid synthesis cycle starts, the Malonyl-CoA must be added on to the ACP. By using the
activated Malonyl-CoA groups in the fatty acid synthesis, at the same time, we also have this
activated acetate group created in the fatty acid breakdown. These two activated molecules
ensure that both of the processes are energetically favorable, despite the fact that they are the
opposite of eachother.
Condensation by β ketoacyl ACP synthase
● Coupling condensation to decarboxylation of Malonyl-CoA makes the reaction
energetically favorable
Once Malonyl-CoA has been added to the ACP, the elongation of the fatty acids can start.

First step - Condensation

Enzyme; beta-ketoacyl-acp synthase
Product: β-ketoacyl-ACP
The carboxyl group that was previously added during the formation of Malonyl-CoA is
removed as there is a free atom that is able to react and perform the condensation reaction
with the added acetate unit. The removal of carbon dioxide subsequently opens up a space
where the condensation reaction can occur.
These two carbon atoms and the acetate unit are transferred to either of these 2 molecules;
● Fatty acid - in case of normal fatty acid biosynthesis
● Acetate group - in case of fatty acid de novo (from scratch)
Now the molecule has been elongated by two more carbon atoms. Thus the product of
β-ketobutyryl-ACP is formed. The name is derived from the fact that the molecule consists of
4 carbon atoms.
OBS. β-ketoacyl-ACP is the name for the general intermediate of the fatty acid biosynthesis,
no matter how many carbon atoms it has. So stick by that name in the future!
Reduction by β-ketoacyl ACP reductase
This step is different from Beta-oxidation.
● The intermediate of L-beta hydroxybutyrate is formed in the beta-oxidation.
● The intermediate in fatty acid synthesis is the stereoisomer of the beta-oxidation
intermediate.
Second step - Reduction
Enzyme; β-ketoacyl-ACP reductase
Product: d-beta- hydroxyacyl-acp.
In this step, a hydroxyl group is used. This step requires the reducing cofactor of NADPH +
H, as it gives the two hydrogen atoms to the core, providing us a keto group and we are able
to form the secondary alcohol group (hydroxyl group). The intermediate created is d-beta-
hydroxyacyl-acp.

Dehydration by hydroxyacyl ACP dehydratase

Third step - Dehydration
Enzyme; β-hydroxyacyl ACP dehydratase
Product: trans-∆2-enoyl-acp
The water molecule is removed and thus reenforms the double bond
between the alpha and beta carbon atoms. As a result, we gain the
intermediate trans-∆2-enoyl-acp. Keep in mind that this is the
same intermediate of the first step in beta-oxidation.
Reduction by Enoyl ACP reductase
Fourth step - Reduction
Enzyme; Enoyl-ACP reductase
Product: acyl-ACP
Reducing factor of NADPH + H is used in this step. We are now saturating the fatty acid and
the corresponding carbon atoms of alpha and beta positions. The methyl groups typical for
the fatty acid are formed here. Saturated fatty acids are primarily the ones synthesized in our
body.

Translocation of extended chain to thiol group of Ketoacyl-ACP synthase domain

The extended chain of fatty acid is relocated to the Ketoacyl-ACP synthase domain. This
frees up ACP and it is able to bind to the next malonyl-CoA.
Recharging ACP for another round of synthesis by malonyl /acetyl CoA ACP
transferase
With the freed ACP, we are once recharging the ACP so the next round of fatty acid synthesis
can take place. Basically with the same type of reactions and sequences as previously
described. But now instead of the acetate group, we have the 4 carbon atom group and
correspondingly the next malonyl-CoA that could be used with the addition of 2 additional
carbon atoms.

Overall Palmitate (16C) Synthesis

For every round of fatty acid biosynthesis, the malonyl-CoA is used. After decarboxylation,
the acetate group is added to the existing fatty acid side chain (normal) or to the acetate
(denovo). Each time we are adding 2 carbon atoms and elongating the fatty acid chain. These
cycles repeat until the product of Palmitate is finally produced.
Fatty Acid Synthesis Is Tightly Regulated via Acetyl-CoA Carboxylase
Acetyl-CoA carboxylase (ACC) catalyzes both the committed step and the rate limiting step
● ACC is feedback inhibited by palmitoyl-CoA
● ACC is activated by citrate
➔ Citrate is made from acetyl-CoA in mitochondria
➔ Citrate signals excess energy to be converted to fat while inhibits glycolysis
(PFK 1)
● Citrate ensures the feed-forward regulation through allosteric regulations. Citrate
availability in the cytosol, suggests plenty of energy in the cell.
● Palmitoyl-CoA, as the end product, ensures negative feed-back regulation with similar
allosteric mechanisms
OBS. This type of regulation is on a short term basis and has a quick response time.

Acetyl-CoA Carboxylase Is Also Regulated by Covalent Modification

Inhibited when energy is needed
Glucagon and epinephrine:
● reduce sensitivity of citrate activation
● lead to phosphorylation and inactivation of ACC
➔ ACC is inactive as phosphorylated monomers
 ➔ When dephosphorylated, ACC polymerizes into long active filaments
➔ Phosphorylation reverses the polymerization

Reciprocal regulation by Malonyl CoA

Benefit for segregating synthetic and degradative pathways in different cellular
compartments
The malonyl-CoA formation automatically stops the breakdown of fatty acids as it interrupts
the beta-oxidation. More precisely speaking, it does not interrupt beta-oxidation itself but
rather the transport of fatty acid into the matrix of mitochondria. (acetyl-carnitine carnitine
shuttle). Malonyl-CoA, in case there is a lot of energy available, facilitates fatty acid
accumulation through cholesterol synthesis. Here primarily dietary carbohydrates as well as
dietary lipids are used. Malonyl-CoA will simply inhibit fatty acid transport into the matrix of
mitochondria in order to further inhibit the fatty acid breakdown. However, it is worth noting
that this facilitates further breakdown of glucose (glycolysis) as we are unable to store as
much glucose as we can with fats.

Palmitate Can Be Lengthened to Longer Chain Fatty Acids

Read up on the difference between saturated and unsaturated fats.
● Elongation systems in the endoplasmic reticulum and mitochondria create longer fatty
acids (CoASH used, otherwise identical)
● Palmitate can be elongated to Stearate (18:0), the most common product
● We are able to desaturate these saturated fatty acids by the enzyme acyl-coa
desaturase, this results in the formation of product oleate.
● We only have enzymes for double formation at these given carbon atom locations;
∆4 , ∆5 , ∆6 , and ∆9 desaturases but cannot desaturate
beyond ∆9 , thus cannot produce Polyunsaturated fatty acids (
PUFAs), such as omega 3 and omega 6 families, which help
control membrane fluidity and are precursors to eicosanoids
 ➔ All of the numbers mentioned above relate to the portistion of carbon atoms
(4th, 5th carbon atom etc).
● Linoleate and α-Linolenate (through food) are essential fatty acids for mammals.
Ensure membrane fluidity.
● In plants and bacteria PUFAs ensure membrane fluidity at reduced temperature

TAGs biosynthesis
Synthesis of Fat (Triacylglycerides) and Phospholipids
Animals and plants store fat for fuel
● plants: in seeds, nuts and fruits
● typical 70 kg human has ~15 kg fat
➔ Energy enough to last 12 weeks, compare with 12 hours worth of glycogen in
liver and muscle
● Animals and plants and bacteria make phospholipids for cell membranes
● Both molecules contain glycerol (or sphingosine) backbone and 2 (e.g.,
phospholipids) or 3 (e.g., triacylglycerides) fatty acids attached
Synthesis of Fat (Triacylglycerides) and Phospholipids
Phospholipids; used for growth (new organellen/cell formation)
Triacylglycerides; used for energy storage

Synthesis of Backbone of TAGs and Phospholipids

The synthesis of the glycerol backbone can be achieved in these 2 following ways;
1. The majority of glycerol 3-phosphate comes from siphoning off dihydroxyacetone
phosphate (DHAP) from glycolysis
➔ via glycerol-3-phosphate dehydrogenase
1. Some glycerol-3-phosphate is made from glycerol
➔ via glycerol kinase
➔ minor pathway compared to the previously mentioned pathway
➔ glycerol kinase absent in adipocytes, glycerol kinase can only be found in the
liver and kidneys
Synthesis of Phosphatidic Acid Occurs Before Synthesis TAGs
With the glycerol backbone ready, we need the addition of 2 fatty acids
Phosphatidic acid is the precursor for TAGs and phospholipids
● Before fatty acids can be attached, they need to be activated by the enzyme acyl-CoA
synthetase. 1 ATP is used for this activation step.
● The attachment of the fatty acids to the glycerol backbone are ensured by the enzyme
acyl-transferases. This attachment step releases CoA-SH
● These two simultaneous reactions will generate the phosphatidic acid (diacylglycerol
3-phosphate)
Advantage of making phosphatidic acid
● Phosphatidic acid is the precursor for both the triacylglycerol and phospholipid
synthesis pathways.
Phosphatidic Acid Can Be Modified to Form Phospholipids or TAGs
The generation of triacylglycerol, in case when our body wants to store extra energy
Phosphatidic acid phosphatase (lipin) removes the 3-phosphate from the phosphatidic acid
➔ The dephosphorylation of phosphatidic acid is necessary as this is the position where
the third fatty acid is attached
➔ yields the intermediate of 1,2 diacylglycerol
The third carbon is then acylated with a third fatty acid
➔ This step is ensured by the enzyme acyl-transferase
➔ yields the final product of triacylglycerol
Regulation of Triacylglycerol Synthesis by Insulin
Insulin results in stimulation of triacylglycerol synthesis as it suggests we have lots of energy
Lack of insulin results in:
Ex. in case of diabetes
● We are unable to use these dietary carbohydrates and proteins to synthesize the fatty
acids from them. The lack of insulin will go in the opposite direction, if the substrates
are available to use for the pathway of fatty acid synthesis.
● The lack of insulin will push for pathways in which energy is broken down. There
will be an increase in lipolysis (release of lots of fatty acids) as well as increased fatty
acid oxidation
➔ In case if citric acid cycle intermediates (oxaloacetate) that react with acetyl
CoA are depleted, ketone bodies will be produced in order to generate more of
those citric acid cycle intermediates. In this way, the gluconeogenesis pathway
is able to keep on going.
● In case of untreated diabetes; increased rates of fat oxidation, therefore loss of weight
The Triacylglycerol Cycle
Summary: The breakdown of TAGs through lipolysis in adipose tissue. The release of
glycerol and fatty acids straight away will undergo reesterification into the triacylglycerol in
the liver. These fats are brought back to the adipose through lipoproteins and with the help of
lipoprotein lipase, are released again as fatty acids for reformation of TAGs.
● Seventy five percent of free fatty acids (FFAs) released by lipolysis are reesterified to
form TAGs, rather than be used for fuel under all metabolic conditions (even in case
of starvation)
● Some recycling occurs in adipose tissue
● Some FFAs from adipose cells are transported to the liver, remade into TAG, and
redeposited in adipose cells
Hypothesis: The availability of TAGs in blood could be representing the energy that would
be needed in case of urgent fight or flight response.
Question: Where does glycerol-3-phosphate come from? Remember, the glycerol kinase is
only specific to the liver and kidneys. It is not present in the adipose tissue.
Glyceroneogenesis Makes DHAP for Glycerol 3 Phosphate Generation During TAG
Cycle
Glycerol-3-phosphate can also be formed from the Glyceroneogenesis
During lipolysis (stimulated by glucagon or epinephrine), glycolysis in adipocytes is inhibited
● With the inhibition of glycolysis, for this reason the intermediates of that given
pathway can not be used for the synthesis of Glycerol-3-phosphate and
reesterification in the triacylglycerol cycle.
● Adipocytes use this alternative way for the formation of Glycerol-3-phosphate which
involves a part of gluconeogenesis. The sequence up to dihydroxyacetone phosphate
which further on is used for the Glycero-3-phosphate formation is identical to
gluconeogenesis.
● So DHAP is not readily available to make glycerol-3-phosphate
● And adipose cells do not have glycerol kinase to make glycerol 3 phosphate on site
Glyceroneogenesis contains some of the same steps of gluconeogenesis
● The primary substrate of Glyceroneogenesis would be pyruvate (which could be
derived from alanine).
● Adipocytes ensure sufficient amounts of Glycerol-3-phosphate are stored to perform
the ressteirfican process. With 75% of the fatty acid reesterification in the
triacylglycerol (back to the triacylglycerol cycle pathway)
● Converts pyruvate → DHAP
● Basically , an abbreviated version of gluconeogenesis in the liver and adipose tissue
● Uses pyruvate, alanine, glutamine or any substances from the Citric acid cycle as
precursors for glycerol 3 phosphate
Thiazolidinediones Treat Type 2 Diabetes by Increasing Glyceroneogenesis
High levels of free fatty acids in blood interfere with glucose utilization in muscle and
promote insulin resistance that lead to type 2 diabetes
● Thiazolidinediones promote the synthesis of PEP carboxykinase
● Increase the rate of resynthesis of TAGs
The facilitation of the Triacylglycerol cycle activity is used in the treatment of Diabetes Type
2. These drugs upregulates the synthesis of PEP carboxykinase, which is involved in one of
the steps of both the Gluconeogenesis and Glyceroneogenesis in the adipocytes. Upregulation
of PEP carboxykinase will bind more of the fatty acids, which in turn forms more TAGs. The
formation of more TAGs will reduce the availability of free fatty acids in the blood,
preventing them from interfering with the glucose utilization in the muscle tissue and thus
promoting insulin resistance.

Phospholipids biosynthesis
Biosynthesis of Membrane Phospholipids
All the biosynthetic pathways of different phospholipid species follow a few basic patterns:
● Synthesis of the backbone molecule (glycerol or sphingosine)
● Attachment of fatty acid(s) to the backbone either through an ester or amide linkage
(depending on the backbone it is attached to)
● Addition of a hydrophilic (polar) head group to the backbone through a
phosphodiester linkage
● In some cases, alteration or exchange of the polar head group to yield the final
phospholipid product

Phospholipid biosynthesis - place of action

● Every cell; as every cell has a need to build up its cell membrane, or membrane of
various organelles
● Primarily surface of the smooth ER and the inner mitochondrial membrane where
these phospholipids are synetshsied,
Transport to other cellular locations not fully understood
Biosynthesis of Membrane Phospholipids
Begins with phosphatidic acid (diacylglycerol)
Attach head group to C-3 OH group
● C-3 has OH; head group has OH
● new phospho head group created when phosphoric acid condenses with these two
alcohols
● eliminates two H2O
The reaction between the alcohol of diacylglycerol and the phosphoric acid wouldn't be
necessary if phosphoric acid was available. Only one condensation reaction with the
elimination of the water molecule between the phosphorus group and head group would be
necessary for the formation of the phosphodiester bond.

Attaching Phospholipid Head Group Requires Activation by Cytidine diphosphate

 ● Either one of the alcohols is activated by attaching to CDP (cytidine diphosphate)
● The free (not bound to CDP) alcohol then does a nucleophilic attack on the CDP
activated phosphate

Attaching the head group to the phospholipid requires activation with the cytidine
diphosphate. This could be achieved in the 2 following reactions;
1. Diacylglycerol becomes activated with the cytidine diphosphate and thus further on
the headgroup is able to perform the nucleophilic attack while simultaneously
releasing the cytidine monophosphate, forming the final glycerophospholipid.
2. The head group is the one that is initially charged up with the cytidine diphosphate
and thus the 1,2 diacylglycerol and the free alcohol group would be the one that
would perform the nucleophilic attack on this head group that is bound to the cytidine
diphosphate. In that case cytidine monophosphate would be released, forming the
final product glycerophospholipid.

Example: Phospholipid
Synthesis in E. coli
Two main pathways:
● Phosphatidylserine is synthesized and can be decarboxylated to
phosphatidylethanolamine
● Phosphatidylglycerol is synthesized by addition of a Glycerol 3 phosphate
➔ Further modification to cardiolipin can be achieved by replacement of the
glycerol head group with another phospholipid
Synthesis of Phosphatidylserine and Phosphatidylcholine in Mammals Constitutes a
Salvage Pathway
● Phosphatidylserine is made backwards from phosphatidyl ethanolamine or
phosphatidylcholine via head group exchange reactions
➔ catalyzed by specific synthases
Summary of Phospholipid Biosynthesis Pathways in Eukaryotes
Similar Pathways Used for synthesis of:
● Ether Lipids
● Plasmolagens
● Sphingolipids
Phospholipids are not only an important constituent of the cell or organelle membrane, but
also ensure the signal transduction, various anti-inflammatory properties, endocytosis,
phagocytosis and macropinocytosis etc.

Subcellular Localization of Lipid metabolism

Black color: Catabolic processes
Pink color: Anabolic processes
Lecture 3
Protein, Amino Acid Metabolism and the Production of Urea
Learning goals:
● Describe protein degradation processes
● Describe different amino acid sources in the human body
● Explain how amino acids are oxidized for energy
● Describe how urea is made and excreted
● Explain significance of certain enzyme activity in blood serum
● Give insight about amino acid degradation pathways
● Give insight about defects in amino acid degradation pathways

Protein Catabolism
The Use of Amino Acids as Fuel Varies Greatly by Organism
● In the case of standard diet, carbohydrates and lipids are main energy sources for the
human body. Amino acids of protein breakdown are used primarily for protein and
Nitrogen containing compound synthesis.
● About 90% of the energy needs of carnivores can be met by amino acids immediately
after a meal.
● Microorganisms scavenge amino acids from their environment for fuel when needed.
● Only a small fraction of energy needs of herbivores are met by amino acids.
● Plants do not use amino acids as a fuel source but can degrade amino acids to form
other metabolites.

The use of amino acids for energy varies for different organisms. Humans obtain energy from
the nutrients in food, in the shape of carbohydrates, fats and proteins. At different ages the
recommended amount of protein for a balanced diet varies. From the ages of 7 and upwards,
10-20% of protein is recommended, which is the same amount for the daily calorie intake of
an adult person.12 The intake of proteins is a lot less in comparison to the other two
macromolecules. Proportionally proteins are ingested in smaller amounts, therefore most of
our energy is utilized from carbohydrates and fats. Proteins and unsaturated fats, on the other
hand, are used for the regeneration of our cells.
❖ Carnivores obtain energy primarily from amino acids, up to 90% of their diet consists
of protein.
❖ Herbivores obtain a very small amount of energy from amino acids.
❖ Plants generally do not use amino acids for energy, however they do use amino acids
for the synthesis of other molecules.

12
Physiology calculations
Metabolic Circumstances of Amino Acid Oxidation
Amino acids can be used to obtain energy in various metabolic convidions:
Leftover amino acids from normal protein turnover
Protein degradation (proteolysis) and resynthesis. The body has a constant turnover of
protein, because the life expectancy of different proteins differs. The body is not a perfect
system, and defected proteins are quickly broken down to amino acids. Pre amino acids are
used for the synthesis of new proteins and other nitrogen-containing molecules. This
includes; DNA, RNA, Heme etc. Remaining amino acids not used in the mentioned processes
are oxidized for energy.

Proteins in the body can be broken down to supply amino acids for energy when
carbohydrates are scarce (starvation, diabetes mellitus).
The second option includes amino acids being used for energy during fasting or conditions
such as it is with diabetes. The blood glucose is high, but cells are stravring as no glucose can
enter the cell. Therefore the cells are unable to proceed with glycolysis. Under these
conditions, cellular proteins are broken down to the monomers of amino acids.

Dietary amino acids that exceed the body's protein synthesis needs.
In the case where we consume more dietary protein than necessary for the synthesis of new
proteins and nitrogen-containing molecules. After the breakdown of dietary proteins, the
excess amino acids can not be stored for later like it is with triacylglycerols and glucose (in
the form of glycogen). Excess amino acids are instead oxidized for energy. They initially lose
their amine group and are then converted into alpha-ketoacids carbon skeletons. All 3-4
carbon units can be converted into glucose through the process of gluconeogenesis. Further
on, the stored glycogen and triacylglycerols can be replenished if intake of proteins exceeds
the body's need for protein synthesis.
❖ Under mentioned circumstances amino acids “are losing” amino group and get
converted into α- keto acids – carbon skeletons.
❖ Carbon skeletons undergo oxidation to CO2 and H2O or provide 3-4 C units for
gluconeogenesis pathway – synthesis of glucose.

Protein degradation I
Lysosomal Proteolysis
The sources of free amino acids in the body can be different.
Protein degradation prevents abnormal protein accumulation in the cell and ensures normal
protein turnover. Defected proteins are degraded rapidly. Depending on the protein's function,
they have different half-lifes. Half life of proteins can range from seconds, minutes, to several
days or even years. Most poreins, however, have short half lifes in comparison to the lifetime
of a cell. Regulatory enzymes are able to recognize defective proteins by their amino acid
sequence. All cells in the body can degrade proteins using the proteolytic systems. One of the
systems involves the degradation of proteins in the lysosomes.
Lysosomes are membrane bound cellular organelles that contain enzymes such as proteases.
Proteases are able to cleave peptide bonds. The membrane in this case protects the cell from
premature degradation as it prevents the lysosomal enzymes from leaking out into the
cytosol. The lysosomal enzymes are unable to differentiate between molecules of which they
should attack or leave alone. Lysosomes degrade proteins with long or medium survivals,
such as it is with the cell membrane proteins and extracellular proteins. Lysosomal proteases
do not require energy in the form of ATP. In this process, the lysosomes are able to engulf the
proteins through autophagy.
● Lysosomes contain enzymes – proteases
● Lysosomal proteolysis do not require ATP
Non selective degradation:
Involves the degeneration of proteins with a long half life such as cell organelles.
● Lysosomes take up cellular, extracellular proteins, cell organelles and proteins with
long half – lives
Selective degradation:
Enforced in the case of starvation, where degradation of specific proteins with a certain
amino acid sequence is possible as it is recognized by specific enzymes.
● Under starvation conditions, proteins are sacrificed to provide amino acids and energy

Illustration:
When the mitochondria is enclosed by the membrane, formed from the endoplasmic
reticulum, autophagosomes are formed. The autophagosomes fuse together with lysosomes,
forming phagolysosomes. Mitochondria is then degraded by several enzymes which have the
ability to break down various bonds. In the case of proteins, these given enzymes break down
their bonds and we are left with amino acids.
Protein degradation II
The Ubiquitin-Proteasome pathway
One cycle consists of these 3 given steps, and ensures the addition of 1 ubiquitin. If there is
need for additional ubiquitin, this cycle has to be repeated.
The Ubiquitin-Proteasome pathway breaks down proteins with short half-lifes as well as
defective or damaged proteins. Unlike in the process of lysosomal proteolysis, this pathway
requires energy as it is an ATP dependent pathway. Proteins are labeled with the specific
marker of ubiquitin. Ubiquitin is a polypeptide consisting of 76 amino acids. The action of
labeling target proteins with ubiquitin promotes rapid protein degradation. This pathway
plays an important role in the degradation of defective proteins, by their rapid degradation we
prevent the accumulation of proteins in cells so they are enabled to perform their normal
functions.
Step 1:
The amino acid glycine and the carboxyl terminus of ubiquitin is activated by ATP. Enzyme
1 is attached to the ubiquitin which in turn forms a thioester bond. This reaction subsequently
released an AMP molecule and a pyrophosphate group.
Step 2:
Ubuiqitin is transferred from Enzyme 1 to Enzyme 2. Now the ubiquitin is attached to the
Ubiquitin-conjugating enzymes (E2).
Step 3:
Enzyme 3 ligase catalyzes the transfer of ubiquitin from Enzyme 2 to the target protein. An
amide bond is formed between the ubiquitin and the lysine side chain of the target protein.
● ATP dependant system
● Pathway uses ubiquitin as a marker to target cytosolic and nuclear proteins to rapid
proteolysis
● Ubiquitin is a 76-aminoacid polypeptide that is highly conserved in all eukaryotes
(yeasts, animals, and plants).
● Defective proteins and those slated for rapid turnover are generally degraded by an
ATPdependent system.
Protein degradation III
The Ubiquitin-Proteasome pathway
Ubiquitin proteins are degraded by a large complex of proteasomes. Proteasomes consist of
two identical parts, each part consists of at least 32 different subunits. The regulatory unit
forms a cap-like shape on both sides of the core unit. The regulatory particle binds to the
ubiquitin proteins and unfolds the globular proteins until the first structure. After the structure
changes the protein, it is transported to the core. The core consists of four rings. Inside the
core, the protein is broken down to peptides consisting of 3 - 25 amino acids.
These peptide fragments are then broken down to amino acids in the cytosol. Amino acids
will then be used for the synthesis of new proteins while the released ubiquitin can be used
again to label new proteins for the degrational process.
● Polyubiquitinated proteins are recognized and degraded by a large, multisubunit
protease complex – proteasome.
● 19S regulatory particle on each end of the core contains approx.18 subunits.
● 19S Regulatory particle recognizes and binds to the ubiquitinated proteins.
● There are 6 subunits which unfold the protein and translocate unfolded protein into
the core particle.
● In 20S core particle protein is degraded to peptides of 3-25 amino acid residues.

Dietary Proteins Are Enzymatically Hydrolyzed into Amino Acids

Another source of amino acids are dietary proteins, which are hydrolyzed into amino acids in
the gastrointestinal tract. The digestion of proteins starts in the stomach, with the entry of the
protein rich food mixture stimulating the gastric mucosa to secrete the hormone gastrin.
Gastrin in turn stimulates the secretion of hydrochloric acid (HCL -parietal cells) and
pepsinogen (Chief cells) the gastric glands. The acidic gastric juice works both as an
antiseptic (attacks foreign substances) and a denaturing agent. The unfolding of globular
proteins allows the enzyme to break down the peptide bonds. Pepsinogen is converted into
active pepsin by the enzymatic action by pepsin itself. In the stomach, pepsin hydrolyses the
ingested proteins into shorter peptides.
The acidic stomach content passes into the small intestines, and the low pH triggers the
secretion of the hormone secretin into the blood. Secretin stimulates the pancreas to secrete
bicarbonate into the small intestines to neutralize the HCL to ensure a pH of around 7. All
pancreatic secretions pass into the small intestines through the pancreatic duct. The addition
of proteins continues into the small intestine, so the arrival of peptides in duodenum causes
the release of the hormone cholecystokinin into the blood. Cholecystokinin stimulates the
secretion of several pancreatic enzymes, which are also in inactive form. The zymogens
become an active enzyme inside the small intestines.

The synthesis of inactive enzymes protects the endocrine cells of the pancreas from a
destructive proteolytic attack. In the small intestines, peptides are hydrolyzed into pre amino
acids and then pre amino acids are transported into the epithelial cells lining the small
intestine. Further on the amino acids enter the blood capillaries in the villi to travel to the
liver.
● Dietary proteins are enzymatically degraded into amino acids through digestive tract
● Digestion of proteins begins in stomach by proteolytic enzyme pepsin
● Degradation of proteins continues in the small intestine by trypsin, chymotrypsin,
elastase, carboxypeptidase A and B.
● Free amino acids enter blood capillaries through the villi to travel to the liver.
!Digestion and absorption of proteins was discussed in detail!
[In Semester Week 9]
Amino Acid Oxidation
Overview of Amino Acid Catabolism
Once broken down to amino acid, all types of protein are treated the same way dependent on
the organism’s energy needs:
1. Recycled into new proteins
2. Oxidized for energy
➔ removal of amino group (urea cycle)
➔ entry into central metabolism (glycolysis, citric acid cycle)
Amino acids cannot be stored!

As soon as proteins are broken down into amino acids, regardless of their source, can be used
in the same metabolic pathways.
Anabolic processes: amino acids are used for the synthesis of new molecules. They can
synthesize new proteins and nitrogen-containing molecules.
Catabolic processes: amino acids are oxidized for energy. They are oxidized for energy by
undergoing several changes.
Step 1:
Key step
Amino group is separated from amino acids through a transamination reaction, releasing
carbon skeleton. There are several fates for separated amino groups. It can undergo amino
group metabolization with the synthesis of new amino acids. It can be used as a substrate for
the urea cycle in order to excrete excess nitrogen in the form of urea. The carbon skeleton,
meanwhile, is converted into intermediates of the citric acid cycle.
Fates of Nitrogen in Organisms
Plants conserve almost all the nitrogen, while many aquatic vertebrates release ammonia to
their environment.
● passive diffusion from epithelial cells
● active transport via gills
Many terrestrial vertebrates and sharks excrete nitrogen in the form of urea.
● Urea is far less toxic than ammonia.
● Urea has very high solubility.
Some animals such as birds and reptiles excrete nitrogen as uric acid.
● Uric acid is rather insoluble.
● Excretion as paste allows the animals to conserve water.
Humans and great apes excrete both urea (from amino acids) and uric acid (from purines).

Excess nitrogen can be excreted in different forms depending on the organism. Plants
preserve nitrogen, while aquatic vertebrates release ammonia into the aquatic environment.
Birds excrete uric acid in the form of a paste, it is highly concentrated to help birds and
reptiles keep fluid in their body. Humans excrete excess nitrogen in several forms; urea, uric
acid, or ammonium ion.

Amino acid catabolism is the resulting substrate for the urea cycle. The end product of urea is
non-toxic and is polar, making it very soluble in water. Humans also excrete uric acid which
is the end product of the purine catabolism. Purines are heterocyclic compounds consisting of
two rings. Heterocyclic means that the rings also contain another atom besides a carbon and
hydrogen. Purines, like adenosine and guanine, form nitrogen bases for RNA, DNA, GTP and
ATP molecules. Another form we can excrete nitrogen is in the form of ammonium ion. It
can be found in urine within the normal range. Ammonia (NH3) passes into the urine from
the kidney tubule cells and decreases the acidity of urine by binding protons. Ammonium
binding to protons will form NH4 ions.
Step 1: Removal of the Amino Group
● Release of free ammonia is toxic.
● Ammonia is captured by a series of transaminations.
● Transaminations allow transfer of an amine to a common metabolite (e.g., α-
ketoglutarate) and generate a trafficable amino acid (e.g., glutamate).
● Four amino acids play central roles in nitrogen metabolism:
Easy to convert (reversible reaction)
- Glutamate (added amino group) → α-ketoglutarate (no amino group)
- Glutamine (added amino group) → α-ketoglutarate (no amino group)
- Alanine (added amino group) → Pyruvate (no amino group)
- Aspartate (added amino group) → Oxaloacetate (no amino group)

The process of carbohydrates and lipid catabolism does not produce waste products unlike in
the case of amino acid catabolism. Amino group has to be removed from the amino acid to
form the carbon skeleton. This is ensured by the transamination reaction. The removed amino
group from the amino acid is transferred to the ketoacid, which is the acceptor of the amino
group. Alpha-ketoglutarate is the most commonly used amino group acceptor.
Transamination reaction have two substrates, the amino acid and the amino group acceptor.
The two following products are formed, the new amino acid and the new ketoacid, which is
the carbon skeleton of our amino acid substrate.

The substrate amino acid used in the transamination reaction is deaminated as it loses an
amino group, but alpha-ketoglutarate accepts this amino group and gets aminated. The
substrate amino acid when deaminated loses its amino group and becomes the new ketoacid
(product).

The illustration below depicts the liver cell. Most of the amino acids are metabolized in the
liver. If this transamination takes place in the liver, glutamate is formed with the added amino
group from the amino acid of alpha-ketoglutarate. It does not need to be transported outside
the liver cell. Glutamate is later on deaminated, the removed amino group can be used in
various biosynthesis pathways. Excess ammonia in extrahepatic tissue must be transferred to
the liver where it will be converted into non-toxic molecules. Urea is the product of the urea
cycle and the cycle takes place in the liver cells. From the muscles, excess amino groups are
transported in the form of alanine. From other tissues, including muscles, excess amino
groups are transported in the form of glutamine.
Enzymatic Transamination
● Catalyzed by aminotransferases
● Aminotransferase = transaminase
● Uses the pyridoxal phosphate cofactor (B6 – pyridoxine)
● Typically, a-ketoglutarate accepts amino groups.
- Transfer of one amine to a-ketoglutarate results in synthesis of glutamate (e.g.,
transamination).
- Transfer of a second amine results in synthesis of glutamine (e.g., glutamine
synthetase).

Transamination reactions are catalyzed by the enzyme aminotransferase, which is also known
as transaminase. There is no loss of an amino group, it is only transferred from the amino
acid to the keto-acid which results in the formation of a new amino acid and a new ketoacid.
The purpose of transamination reactions is to transfer an amino group from various amino
acids to alpha-ketoglutarate to form glutamate. Glutamate functions as an amino group donor
in biosynthesis pathways or excretion pathways. This leads to the elimination of nitrogen as
base product. The enzyme aminotransferases is named according to the amino acid which
loses its amino group to the ketoacid. For example, if the amino acid was alanine the enzyme
would be named alanine aminotransferase. All aminotransferases have the same prosthetic
group of pyridoxal phosphate. Pyridoxal phosphate is derived from vitamin B6 - pyridoxine.

Structure of Pyridoxal Phosphate and Pyridoxamine Phosphate

● Intermediate, enzyme-bound carrier of amino groups
● PLP is enzyme’s prosthetic group
● Aldehyde form can react reversibly with amino groups
● Aminated form can react reversibly with carbonyl groups

Pyridoxal phosphate is converted into pyridoxamine phosphate by amination. Pyridoxal

phosphate functions as the intermediate carrier between the amino group and the active site of
the aminotransferase. The amino group from pyridoxamine phosphate is then transferred to
the ketoacid which in most cases is alpha-ketoglutarate. The aldehyde form of pyridoxal
phosphate can react reversibly with the amino group. The aminated form of the pyridoxal
phosphate is pyridoxamine phosphate. Pyridoxamine phosphate reacts reversibly with the
carbonyl group of the ketoacid, which gives away the amino group to the ketoacid.

Pyridoxal Phosphate Is Prosthetic Group of the Aminotransferase Enzyme

● PLP is noncovalently linked to the enzyme aminotransferase by an internal aldimine
(Schiff base).
● Internal aldimine is formed between amino and carbonyl groups.
● The linkage is made via a nucleophilic attack of the amino group of an active-site
lysine.

Pyridoxal phosphate is a prosthetic group of the enzyme and there is an internal aldimine
bond formed between the amino group lysine side chain of the enzyme and the carbonyl
group of the prosthetic group. Aminotransferases are typical examples of ping pong reactions.
Transamination reactions have two substrates and two products. As soon as the first substrate
amino acid reacts with the pyridoxal phosphate, it gives away the amino group. The newly
formed ketoacid product must leave the active site of the enzyme, as soon as it leaves the
second substrate (most likely being alpha-ketoglutarate) binds to the pyridoxal phosphate to
take over the amino group. It leaves the active site as a new amino acid (most likely
glutamate).
Mechanism of Transamination
Main thing to keep in mind is that the pyridoxal phosphate overtakes an amino group from an
amino acid and then transfers it to the ketoacid.

PLP Also Catalyzes Decarboxylation of Amino Acids

● The PLP-dependent decarboxylation of some amino acids yields important biological
amines, including neurotransmitters.
Pyridoxal phosphate is not only a prosthetic group for aminotransferases, but also plays an
important role in amino acid decarboxylation reactions. In these reactions, important
neurotransmitters and hormones are synthesized. These include a single decarboxylation
reaction of glutamate for the synthesis of GABA.13

13
Synthesis of biogenic amides.
Ammonia Is Safely Transported in the Bloodstream as Glutamine
● Excess ammonia is added to glutamate in extrahepatic tissues forming glutamine.
● Glutamine is safely transported to the liver.
● L-Glutamine acts as a temporary storage of nitrogen.
● L-Glutamine can donate the amino group when needed for amino acid biosynthesis.

In many tissues, we use the carbon skeleton of excess amino acids for energy.
Transamination reactions must take place in order for us to obtain the carbon skeleton. When
the amino group of an amino acid is transferred to alpha-ketoglutarate, a new ketoacid and a
new amino acid of glutamate is formed. The ketoacid will be converted into the intermediate
of the citric acid cycle after a series of reactions. In many tissues, such as it is in the brain, the
catabolism of nucleotides also releases free ammonia. The conversion from toxic ammonia to
a non-toxic compound occurs in the liver cells. To ensure safe transport of ammonia to the
liver, free ammonia is combined with the glutamate molecule to yield glutamine. This given
reaction consists of two steps and is catalyzed by the enzyme glutamine synthetase, the
reaction takes place in the cytosol.
Step 1:
Glutamate is activated by the ATP molecule and one phosphate group of ATP is added to the
carboxyl group of the glutamate This forms the intermediate glutamyl phosphate. In the first
step, one ADP molecule is released.
Step 2:
Glutamyl phosphate reacts with the ammonium, forming the glutamine molecule and
inorganic phosphate is subsequently released.
Glutamine is now ready for the transfer of non-toxic ammonia from extrahepatic tissues to
the liver. Compared to other amino acids, glutamine is found in high concentrations in the
blood as it also serves as an amino group donor in many biosynthesis reactions.
Glutamine is transported to the mitochondria of liver cells, where it is deaminated in a
reaction catalyzed by the enzyme glutaminase. Glutaminase belongs to the hydrolase class,
and it ensures the release of ammonia with the help of water. The ammonia released in the
mitochondria will be used as the substrate for the urea cycle, where it will be converted into a
non-toxic molecule that can be excreted out with the urine.

Ammonia Collected in Glutamate Is Removed by Glutamate Dehydrogenase

● Oxidative deamination occurs within the mitochondrial matrix.
● Can use either NAD+ or NADP+ as electron acceptor
● Ammonia is processed into urea for excretion.
● Pathway for ammonia excretion; transdeamination = transamination + oxidative
deamination.

The cleavage of ammonia from glutamate occurs in the reaction of oxidative deamination.
Oxidative deamination involves the coordination of the glutamate molecule, which requires
cofactor NAD(P) [phosphorylated form]. We start with the substrate of glutamate, and it
becomes oxidized by the cofactor NAD(P)+. The cofactor becomes reduced by accepting 2
protons. In the next step, the oxidized intermediate molecule [molecule in brackets] will be
deaminated, an amino group will be cleaved with a water molecule.
The reaction produces the products of alpha-ketoglutarate and ammonia which serves as the
substrate for the urea cycle in liver cells, The molecules providing the substrate for the urea
cycle are predominantly glutamate and glutamine. The pathways for ammonium removal
involve both transamination and oxidative deamination reactions. Both reactions are
important for the excretion of ammonium from the organism.

The Glucose-Alanine Cycle

● Vigorously working muscles operate nearly anaerobically and rely on glycolysis for
energy.
● Glycolysis yields pyruvate.
● During intensive training muscle protein is degraded to amino acids for fuel.
● Pyruvate can be converted to alanine for transport into the liver.

Alanine is one of the four amino acids that play an important role in amino acid metabolism.
Alanine, like glutamine, is involved in the non-toxic transport of amino groups from the
muscle to the liver. Muscle protein is degraded into amino acids to use them for energy.
Transamination reactions take place in order to obtain the carbon skeleton of these amino
acids. Amino acids are deaminated and the alpha-ketoglutarate gets aminated by accepting
the amino group. The new product of glutamate is formed. In the muscle cells, another
transamination reaction takes place. This reaction consists of the two substrates of glutamate
and pyruvate, the reaction is catalyzed by the enzyme alanine aminotransferase. Glutamate
donates an amino group to pyruvate, and the amino acid product of alanine is formed from an
aminated pyruvate. The ketoacid product of alpha-ketoglutarate is formed from a deaminated
glutamate. Alanine is transported from the muscle to the cytosol of the liver cells. The
enzyme alanine aminotransferase catalyzes the transfer of the amino group from alanine to
alpha-ketoglutarate in the cytosol.
The products of pyruvate and glutamate are formed. Glutamate is then able to cross the inner
mitochondrial membrane of hepatocytes, where it undergoes oxidative deamination. It
provides the substrate for the urea cycle. In turn, the pyruvate in hepatocytes is converted into
glucose by the gluconeogenesis pathway. The produced glucose is able to enter the
bloodstream and then be reuptaken by the muscle cells.

The glucose-alanine cycle is an example of the body's internal economy. For example, during
exercising, the muscle acts anaerobically which procedures pyruvate, lactate and ammonia as
a result of muscle protein breakdown. Lactate and ammonia reach the liver where they
undergo several reactions. Pyruvate in the liver is converted into glucose by gluconeogenesis,
which continues its way to the muscle cells in the cori cycle and glucose-alanine cycle. The
energetic burden of the gluconeogenesis is imposed on the liver rather than on the muscle
cells as all available energy in the form of ATP in muscle tissue is devoted to muscle
contractions. This again demonstrates how economically these systems work in our body.

Transport of ammonia to the liver

Summary
Overall picture shows the transport of ammonia in the form of glutamine from extrahepatic
tissues (muscle) and the transport of alanine from the muscle cells.

Toxicity of ammonia
● Impacts blood pH (NH3 + H = NH4)
● Deplets α –ketogluterate (energy loss)
● Deplets glutamate stores (impairs neurotransmission)
Glutamate is an important neurotransmitter present in over 90% of all brain synapses and is a
naturally occurring molecule that nerve cells use to send signals to other cells in the central
nervous system.
!Leads to reduced energy production and neurotransmission
Ammonia is toxic and it is the reason why it has to be converted into a non-toxic compound
of either urea or uric acid. The brain is particularly sensitive to ammonia, which causes
cognitive function epilepsy, seizures or in more severe cases cerebral edema which could lead
to death. Approximately 98% of ammonia in the blood is in the protonated form NH4.

The protonated form is not able to cross the plasma membrane, however the remaining 2% is
in the unprotonated form of NH3 which can cross all the membranes. Once in the brain cells,
ammonia uptakes the protons and accumulates in the cytosol of the brain cells. In order to get
rid of the accumulating ammonium, the enzyme glutamate dehydrogenase catalyzes the
addition of ammonia to alpha-ketoglutarate to form glutamate. In another reaction, the
conversion of glutamate to glutamine is ensured by the enzyme glutamine synthetase and this
given reaction also helps to get rid of the free ammonium. However, the consumption of the
citric acid cycle intermediate of alpha-ketoglutarate and glutamate (substrate for important
neurotransmitters) does not bode well for the pathways described.

Increased ammonium alters the capacity of the astrocytes to maintain potassium homeostasis
across the membrane. NH4 competes with the potassium ions for the transport of the cell via
the proton pump of adenosine triphosphatase. This results in the increased extracellular
potassium concentration. Excess potassium ions enter the neurons through another transport
system in the shape of the sodium/potassium/chloride simport. This transport brings 1 sodium
and 2 chloride ions into the cell alongside the potassium ions. When too many chloride ions
enter the cell alongside the excess potassium ions, a disruption of the normal processes of
neurotransmitters occurs. Meanwhile, if ammonium ions remain in high concentrations
outside the cell, it promotes edema which can eventually lead to death.

Excess Glutamate Is Metabolized in the Mitochondria of Hepatocytes

Ammonia Is Recaptured via Synthesis of Carbamoyl Phosphate

● The first nitrogen-acquiring reaction of the urea cycle requires 2 ATP
● Takes place in mitochondria of liver cells
● Substrate of the reaction: Bicarbonate + Ammonia
● Product of the reaction: Carbamoyl phosphate
● Enzyme: Carbamoyl phosphate synthetase I – regulatory enzyme
The capture of nitrogen begins with the reaction in the mitochondria, which is catalyzed by
the enzyme Carbamoyl phosphate synthetase I.14
Step 1:
The substrate of bicarbonate is phosphorylated by ATP in order to activate it for the next step
with ammonia.
Step 2:
The phosphate group is replaced with the ammonium which forms the intermediate molecule
of carabamate. Inorganic phosphate is subsequently released here.
Step 3:
The carbamate is again phosphorylated and we can see one more ATP molecule is used. The
phosphorylation of carbamate results in the formation of the end product of Carbamoyl
phosphate.

In total, 2 ATP molecules are needed for the activation of the Carbamoyl phosphate for the
next set of reactions in the urea cycle.
❖ 1 ATP is needed for the phosphorylation of bicarbonate, so it is able to react with
ammonium
❖ 1 ATP needed for the phosphorylation of carbamate, so it can form the end product of
Carbamoyl phosphate.

Production of Urea
The Reactions in the Urea Cycle I
● Cycle takes place almost exclusively in the liver.
● The enzymes catalyzing these reactions are distributed between the mitochondrial
matrix and the cytosol.
● The majority of reactions within the urea cycle occur within the cytosol.
● Nitrogen from Carbamoyl phosphate enters the Urea cycle
The enzymes of the urea cycle are mainly localized in the cytosol. Some reactions also occur
in the mitochondria. Carbamoyl phosphate is synthesized in the mitochondria, and this
molecule is now ready to further incorporate nitrogen into the urea cycle by reacting with the
amino acid ornithine.

14
This reaction can be considered to be the first official step in the urea cycle, However, in many
literary sources they refer to this reaction as the set of steps which incorporate nitrogen into a
molecule that will activate the urea cycle.
The Reactions in the Urea Cycle II
1. Formation of citrulline from ornithine and carbamoyl phosphate by enzyme
ornithinetranscarbomoylase (entry of the first amino group); the citrulline passes into
the cytosol.
2. Formation of argininosuccinate through a citrullyl-AMP intermediate by enzyme
argininosuccinate synthetase (entry of the second amino group).

The urea cycle consists of 4 enzymatic reactions:

Enzymes are highlighted in blue color, while metabolites are highlighted in purple color.
First reaction
Carbamoyl phosphate gives away the carbonyl group to ornithine to form citrulline.
Cleavage of the high energy phosphate group provides the energy necessary for this reaction.
This step is catalyzed by the enzyme ornithine transcarbamylase. Citrulline is transported
from the mitochondria to the cytosol, in exchange with cytosolic ornithine molecule. This
exchange is made possible via the ornithine/citrulline transporter.

Entry of Aspartate into the Urea Cycle

● This is the second nitrogen-acquiring reaction.
● In the cytosol, citrulline reacts with ATP to produce citrullyl-AMP.
● AMP acts as a good leaving group, as aspartate attracts the imide carbon to produce
argininosuccinate.
Second reaction
Takes place in the cytosol which is catalyzed by the enzyme argininosuccinate synthetase.
This reaction takes place in 2 steps:
1. The first part of the reaction requires an ATP molecule, with the addition of AMP to
the citrulline molecule and subsequent release of pyrophosphate. The addition of the
AMP molecule forms the intermediate of citrullyl-AMP.
2. The second part of the reaction involves the combination between citrullyl-AMP and
the amino acid aspartate. Here, the AMP molecule is released and the product of
arginine succinate is formed.
OBS. The amino acid aspartate brings in the second nitrogen into the urea cycle.
Aspartate is obtained in the mitochondria of hepatocytes by transamination reaction. The
reaction is catalyzed by aspartate aminotransferase. When the amino group from glutamate is
transferred to oxaloacetate, the products of aspartate and alpha-ketoglutarate are formed.

The Reactions in the Urea Cycle IIi

3. Formation of arginine from argininosuccinate by enzyme argininosuccinase; this
reaction releases fumarate, which enters the citric acid cycle.
4. Enzyme arginase cleaves both nitrogens added in the urea cycle from arginine,
resulting in free urea. Ornithine is able to serve as a substrate for the next round of the
cycle.
Third reaction
The enzyme argininosuccinase cleaves arginine succinate into fumarate and arginine. The
intermediate of arginine continues onto the last reaction of the urea cycle, while the carbon
skeleton of aspartate is released as fumarate.

There is no specific transporter for fumarate of the inner mitochondrial membrane, so it is

converted into malate in the cytosol for it to be able to cross the inner mitochondrial
membrane. In this way, malate is able to join the citric acid cycle. In this citric acid cycle, the
oxidation of malate to oxaloacetate is ensured by the enzyme of malate synthase. This
reaction generates NADH + H molecules, which gives us 2.5 ATP for each reducing factor.
The 3 ATP used in the urea cycle are partially compensated with the subsequent release of
fumarate.
➔ 2 ATP; used for the phosphorylation of bicarbonate so it would be able to react with
ammonia, and the phosphorylation of carbamate so it can form the end product of
Carbamoyl phosphate.
➔ 1 ATP; used in the first part of the 2nd reaction of the urea cycle, where we form
argininosuccinate
Fourth reaction
The enzyme arginase cleaves arginine with the help of a water molecule, which in turn
releases both the urea molecule and amino acid ornithine. Ornithine can then be transported
back into the mitochondria in exchange to the mitochondrial citrulline (via the
ornithine/citrulline transporter). The urea molecule released in the last step is considered to
be a non-toxic molecule, which makes it eligible to enter the bloodstream. It will travel to the
kidneys where it can finally be excreted out with the urine.

Aspartate–Argininosuccinate Shunt Links Urea Cycle and Citric Acid Cycle

● The interconnected cycles have been called the “Krebs bicycle.”
● The pathways linking the citric acid and urea cycles are known as the aspartate-
argininosuccinate shunt.
● These different cycles and processes rely on a limited number of transporters in the
inner mitochondrial membrane.

The linkage between the citric acid cycle and the urea cycle.
Both the urea cycle and the citric acid cycle can operate independently, but communication
between these two cycles are based on the transport of key intermediates from the cytosol to
the mitochondria and vice versa. The following transporters are involved; malate/alpha-
ketoglutarate transporter, glutamate/aspartate transporter etc.

In the urea cycle, 2 nitrogen donors are needed to create the urea molecule:
 ➔ The 1st nitrogen is obtained from either glutamine or glutamate, which further on is
incorporated into the formed intermediate of carbamoyl phosphate.
➔ The 2nd nitrogen is obtained from aspartate, which is incorporated into the second
part of the second reaction of the urea cycle. Aspartate is therefore the 2nd nitrogen
donor

Synthesis of aspartate consists of a transamination reaction which takes place in the

mitochondria. When glutamate donates its amino group to oxaloacetate, the products of
aspartate and alpha-ketoglutarate are formed. Aspartate then crosses the mitochondrial
membrane to enter the cytosol, where it joins in the urea cycle. This transamination reaction
and transport of aspartate to the cytosol are all part of the malate/aspartate shuttle. In the third
reaction of the urea cycle, arginosuccinase is cleaved into arginine and fumarate.

Fumarate is then converted into malate in the cytosol by the cytosolic enzyme fumarase.
Malate is transported into the mitochondria where it joins the citric acid cycle. Keep in mind
that the transport of malate from cytosol to the mitochondria is also part of the
malate/aspartate shuttle. All these reactions together form the Aspartate–Argininosuccinate
Shunt. The connection between these two cycles and interconnecting cycles is called Krebs
bicycle.

Regulation of the Urea Cycle

 ● Carbamoyl phosphate synthase I is activated by N-acetylglutamate.
● Formed by N-acetylglutamate synthase:
- when glutatmate and acetyl-CoA concentrations are high
- activated by arginine
● Expression of urea cycle enzymes increases when needed.
- high-protein diet
- starvation

Urea cycle is regulated in two levels:

The cycle intensity is increased by availability of the substrate of ammonia.
In case of high protein diet, with the case of carnivores or prolonged starvation period,
expression of urea cycle enzymes increases in order to more efficiently obtain energy of
carbon skeletons as well as convert toxic ammonia to harmless urea molecules. Urea cycle
enzymes and carbamoyl phosphate synthetase 1 enzyme are synthesized at a higher rate. This
is an example of a long-term regulation mechanism.

The regulatory enzyme carbamoyl phosphate synthetase 1 is regulated allosterically. Its

allosteric activator is the molecule n-acetylglutamate, which is synthesized from acetyl-CoA
and glutamate. The enzyme which catalyzes the combination between acetyl-CoA and
glutamate to form the product of n-actylglutamate, is the enzyme n-acetylglutamate synthase.
This enzyme is also regulated allosterically, and it is activated by arginine (an intermediate of
urea cycle). This is an example of a short-term regulation mechanism.

Genetic Defects in the Urea Cycle

 ● People with genetic defects in any enzyme involved in urea formation
cannot tolerate protein-rich diets. Toxic ammonia cannot be converted into the urea
inside the liver.
● Enzyme deficiency can result in hyperammonaemia or build up of one or more urea
cycle intermediates.
● Protein-free diet is not a treatment option!
● Deficiency of N-Acetylglutamate synthase is treated by administering carbamoyl
glutamate, an analogue of N-acetylglutamate, which is allosteric effector of
carbamoyl phosphate synthetase I.
● Phenylbutyrate is used in treatment for deficiencies in any urea cycle
enzymes as it collects excess ammonia in the form of Phenylacetylglutamine and is
excreted.

People who lack any urea cycle enzymes or regulatory enzymes of the urea cycle, do not
tolerate dietary proteins. If protein intake is greater than necessary for new protein synthesis,
then the excess amino acids are deaminated for ammonia which can not be further converted
into urea and be excreted out alongside the urine. Definicny of the first enzyme (Carbamoyl
phosphate synthetase I) which catalyzes the combination of bicarbonate and ammonia to
form carbamoyl phosphate molecule, will have an increased level of ammonia in the blood.
This condition is known as hyperammonemia.

If any other enzymes of the urea cycle are missing, there will be a build up of specific
intermediates. This could help us to identify the missing enzymes in correspondence to the
elevation of specific intermediates. For example; elevation of the intermediate arginine in the
blood would lead us to believe that the person lacks the enzyme arginase, the enzyme which
breaks down arginine into the urea molecule and ornithine.

Depending on the stage of the genetic defect, the appropriate treatment must be applied.
For example; if there is a defincity of the enzyme n-acetylglutamate synthase, 15 then the N-
acetylglutamate analog (carbamoyl glutamate) is used in therapy.

In most cases where there is a deficiency in urea cycle enzymes, phenylbutyrate is

administered to the diet. Phenylbutyrate is a three reaction step that is combined with the
amino acid glutamine to form the compound phenylacetyl glutamine. This molecule is
excreted via the urine, so the body gets rid of the 2 nitrogens.

OBS. Protein-free diet is not a treatment option, as a person must have essential amino acids.

Essential vs. Nonessential and Conditionally Essential Amino Acids

15
N-acetylglutamate synthase catalyzes the combination of acetyl-coa and glutamate into the
molecule of n-acetylglutamate. It also serves as an allosteric activator for the enzyme carbamoyl
phosphate synthetase 1.
 ● Essential amino acids must be obtained as dietary protein. About half of the 20 amino
acids are essential, and can only be provided through diet. Consumption of a variety
of foods supplies all the essential amino acids.
● Non-essential amino acids can be synthesized in the body through transamination
reactions. This could be illustrated through the amination of the oxaloacetate which
gives aspartate, or the amination of pyruvate which gives alanine. Nonessential amino
acids are easily made from central metabolites.
● Conditionally essential amino acids are classified as non-essential, but in certain
conditions the requirements for those amino acids of the given organism exceeds the
synthesis. It could be in case of pathologies or in growth periods. This could also be in
the case of premature babies, as these amino acids are not synthesized in the required
amounts.

End Products of Amino Acid Degradation

● Intermediates of the central metabolic pathway
● Some amino acids result in more than one intermediate.
● Ketogenic amino acids can be converted to ketone bodies.

● Glucogenic amino acids can be converted to glucose.

In catabolic processes, the carbon skeleton of amino acids are converted into citric acid cycle
intermediates. Depending on the intermediate, the carbon skeleton is converted into either
ketogenic or glucogenic amino acids. In the liver, ketone bodies can be synthesized from the
ketogenic amino acids while glucose can be synthesized from the glucogenic amino acids.
OBS. Some amino acids can fall into both groups.

Summary of Amino Acid Catabolism

The reason why phenylalanine is considered to be both ketogenic and glucogenic has to do
with the fact that it can be converted into these 2 following products. The degradation of
phenylalanine amino acid results in the formation of two citric acid cycle intermediates. One
part of the phenylalanine carbons is incorporated into fumarate, while the other part is
converted into acetoacetyl-coa. Acetoacetyl-coa is an intermediate of ketogenesis, it is a
molecule which was formed from the combination of two acetyl-Coa molecules.

Some amino acids are exclusively ketogenic or glucogenic. For example, methionine is
converted into succinyl-CoA, which is a molecule that joins the citric acid cycle and
continues its way through the cycle as oxaloacetate. Oxaloacetate is an intermediate of
gluconeogenesis (the pathway in which glucose is synthesized), that's the reason why
methionine is considered to be a glucogenic amino acid.

Degradation of Ketogenic Amino Acids

OBS. Know which amino acids fall into which class, and also why they are classified as such.
Degradation Intermediates of Tryptophan Are to Synthesize Other Molecules
The intermediate synthesized during the degradation of tryptophan can be used for the
synthesis of important molecules. For example, tryptophan is the precursor for niacin. Niacin
can be converted into its main metabolically active form, the coenzyme nicotinamide adenine
dinucleotide (NAD). NAD+ molecules participate in glycolysis or beta-oxidation as an
oxidizing cofactor. From tryptophan we can also derive well-being hormones or
neurotransmitters such as serotonin, which regulates mood, memory, cognitive function etc.
Serotonin itself is the precursor for the sleep hormone melatonin, which is responsible for the
regulation of the sleep-wake cycle.

Genetic Defects in Many Steps of Phe Degradation Lead to Disease

Most amino acids act as neurotransmitters or antagonists16. Neurotransmitters are derived
from amino acids. Defects in different stages of amino acid catabolism lead to nervous
system disorders and mental retardation. Deficiency in any enzyme in the degradation of
phenylalanine leads to serious diseases.

Phenylketonuria Is Caused by a Defect in the First Step of Phe Degradation

● A buildup of phenylalanine and phenylpyruvate.
● Impairs neurological development leading to intellectual deficits.
● Controlled by limiting dietary intake of Phe.

Phenylketonuria; hereditary disorder of amino acid metabolism

Develops when the first carbatolic reaction of phenylalanine is disrupted due to deficiency or
decreased activity of the enzyme phenylalanine hydroxylase. Phenylalanine is not converted
to amino acid tyrosine and blood levels of phenylalanine would increase while levels of
tyrosine would decrease. The amino acid tyrosine is the precursor for the synthesis of
melatonin, dopamine, adrenaline as well as thyroid hormones, which are vital for the normal
mental and physical development of a person. The increase in amino acid phenylalanine in
the blood will lead to it competing with other important amino acid transporters in the blood
brain barrier.

16
A receptor antagonist is a type of receptor ligand or drug that blocks or dampens a biological
response by binding to and blocking a receptor rather than activating it like an agonist. Antagonist
drugs interfere in the natural operation of receptor proteins.
This will result in further deficiency of other amino acids in the brain, which interferes with
normal brain development. In alternative pathways, phenylalanine is broken down to
phenylpyruvate. Both metabolites accumulate in the blood and some are excreted out via the
urine. Part of phenylpyruvate is either decarboxylated to phenylacetate or is reduced to
phenyllactate. These two molecules are responsible for the unique odor of urine associated
with phenylketonuria patients. This specific smell sometimes makes medical staff suspicious
of the diagnosis in a newborn.

The goal of therapy is to ensure acceptable levels of phenylalanine and tyrosine in the blood.
The treatment of phenylketonuria is based on a low protein diet supplemented with a mixture
of amino acids that lack phenylalanine. These specific amino acids instead contain other
amino acids, vitamins, and minerals that are needed for the body. Patients must avoid
artificial sweeteners aspartame as it consists of amino acids that are later broken down to
phenylalanine and aspartate in our body.

Degradation of Amino Acids to Pyruvate

Pyruvate can either be converted into acetyl-CoA or be used as a substrate for
gluconeogenesis in the liver cells.
Degradation of Amino Acids to α-Ketoglutarate
● Proline, arginine, histidine, and glutamine are all converted to glutamate.
● Glutamate is deaminated to α- ketoglutarate.
● Arginine degradation is part of the urea cycle.
Amino acids which are degraded into alpha-ketoglutarate are considered to be glucogenic.
This is due to the fact that alpha-ketoglutarate is formed in the beginning of the citric acid,
therefore it can synthesize glucose further on as oxaloacetate. With this in mind, the carbons
of glucogenic amino acids, such as histidine or proline, will enter gluconeogenesis as
oxaloacetate and these same carbons will form the molecule of glucose.

Degradation of Branched-Chain Amino Acids Does Not Occur in the Liver

 ● Leucine, isoleucine, and valine are oxidized for fuel in muscle, adipose tissue, the
kidneys, and the brain.
● Deficiency of enzyme branched – chain α-keto acid dehydrogenase complex results in
Maple syrup urine disease.

Despite the fact that the majority of amino acids are catabolized in the liver, branched-chain
amino acids are oxidized for energy mainly in skeletal muscles, adipose tissues, and the
brain. Branched-chain amino acids Leucine, isoleucine, and valine got their name from their
branched side chains. Oxidation of branched-chain amino acids is impossible in hepatocytes
as they lack the enzyme branched – chain α-keto acid dehydrogenase complex. This enzyme
ensures the synthesis of the carbon skeletons of these amino acids. This enzyme further
catalyzes the oxidative decarboxylation of all these given carbon skeletons, this in turn will
form acyl-CoA derivatives. In case of deficiency of the enzyme branched – chain α-keto acid
dehydrogenase complex, the maple syrup urine disease develops. As you can hear from its
name, it is characterized by the smell of maple syrup in the urine.

Degradation of Branched-Chain Amino Acids Does Not Occur in the Liver

● Branched-chain amino acids are degraded to succinyl- CoA, an important citric acid
cycle intermediate.
Two of the branched-chain amino acids, valine and isoleucine, are converted into succinyl-
CoA (citric acid cycle intermediate). At the point where succinyl-CoA enters the cycle, we
can produce 1 GTP, 1 FADH2 and 1 NADH + H. The addition of succinyl-CoA in the citric
acid cycle will subsequently increase the concentration of oxaloacetate.
Degradation of Asn and Asp to Oxaloacetate
The degradation of the glucogenic amino acids of asparagine and aspartate will form
oxaloacetate. Asparagine loses its amino group and the reaction is catalyzed by enzyme
asparaginase, which is a hydrolase. The amino group is cleaved with the help of water
molecules and the molecule of aspartate is formed. A transamination reaction occurs between
the substrate of aspartate and alpha-ketoglutarate, where the amino group of aspartate is
transferred to alpha-ketoglutarate. Glutamate leaves the reaction and the loss of an amino
group from aspartate will form the final product of oxolalacte. This reaction is catalyzed by
the enzyme aspartate aminotransferase, this step requires the prosthetic group of pyridoxal
phosphate (PLP) for successful aminotransferase reaction.
Fasting vs Fed state
A. Fed state
Dietary protein is hydrolyzed into amino acids
Amino acids are used:
● Protein synthesis
● Nitrogen containing compound synthesis (amino acids, nucleotides, biogenic amines)
Carbon skeletons of excess amino acids are used:
● Fulfill energy storage in form of glycogen and triacylglycerols
B. Fasting state
Muscle protein is degraded into amino acids
● Carbon skeletons are used as fuel
● Ammonia is excreted in form of urea in the urine

Absorption phase
During the consumption of a meal, the only energy available is the form of food
After a meal, dietary proteins are broken down into amino acids. Amino acids are absorbed
into the endothelium of small intestines and further on delivered to the liver via the portal
vein. During the absorption phase, the dominant hormone is insulin and most of the metabolic
reactions are composed of anabolic processes. In the liver, amino acids are used for the
synthesis of new proteins and other nitrogen containing compounds. Amino are transported
from the liver to other cells they can be used for protein synthesis. The carbon skeletons of
excess amino acids in the liver are converted to either glucose or triacylglycerides. If the
carbon skeleton comes from glucogenic amino acid, it is converted into an intermediate of the
citric acid cycle for further conversion into glucose. Glucose is used either to maintain the
blood sugar levels or to fulfill glycogen storage. If the carbon skeleton comes from ketogenic
amino acids, it is converted into acetyl-CoA for further usage in the fatty acid synthesis.
Acetyl-Coa is the substrate for fatty acid biosynthesis and the esterification of glycerol. With
the addition of three fatty acids, hepatocytes are able to synthesize triacylglycerols via the
lipogenesis pathway. Triacylglycerols in the liver will be packed into very low lipoproteins
(VLDL) where it can enter the bloodstream for the transfer of TAGs to all cells around the
body (muscle cells, adipocytes etc).

Post-absorptive phase
Nothing is consumed, the only energy available at the moment is stored in the body
When all food has been digested and energy reserves are replenished. During the post-
absorptive phase, the dominant hormone is glucagon and most of the metabolic reactions are
composed of catabolic processes. This could occur overnight, with a skipped meal, or during
starvation. In this case, the body relies on the stored energy. Muscle proteins are broken down
into amino acids and the following transamination reaction steps occur. The glutamine and
alanine from various tissues serve as non-toxic ammonia transport molecules. Their carbon
skeletons in muscles are oxidized in the citric acid cycle and energy is obtained. The released
amino acids from the muscle protein are transported directly to the liver where amino acids
carbon skeletons are oxidized. In the liver, ketones are synthesized from carbon skeletons of
ketogenic amino acids, and glucose is synetshsied from glucogenic amino acids. Both ketone
and glucose are used for energy in other tissues. For example, erythrocytes will only be able
to use glucose for energy as they lack the organelle (mitochondria) necessary for the
conversion of ketone bodies into acetyl-CoA, therefore they can not initiate the citric acid
cycle. In turn, ammonium is converted into urea molecules in the urea cycle and is excreted
out via the urine. If glutamine enters the kidneys, the enzyme glutaminases catalyzes the
release of ammonia NH3. NH3 helps regulate the pH of urine as it accepts protons and in turn
becomes NH4 ammonium ion, making urine more basic.

Nitrogen balance=Nitrogen intake-Nitrogen loss

Nitrogen balance; the difference between nitrogen intake and nitrogen loss. It reflects gain or
loss of total body protein.
● If nitrogen ingested via the diet is greater than the nitrogen excreted out (urea, uric
acid, ammonium ions) via the urine, then there is a positive nitrogen balance.
● If nitrogen excreted out is greater than the nitrogen ingested, then there is a negative
nitrogen balance.
Positive nitrogen balance; promotes synthesis of new proteins, amino acids and other nitrogen
containing compounds. Positive nitrogen balance can be seen in pregnancies, because the
increased activity of protein synthesis ensures normal development of the fetus. It can also be
seen during lactation, as breast milk provides all the important proteins (immunoglobulins) to
the baby. Positive nitrogen balance is also prevalent during the child's growth period, as
protein synthesis is more active than the degradation of protein.
Negative nitrogen balance; promotes degradation of proteins, amino acids and other nitrogen-
containing compounds. Negative nitrogen balance can be seen as the person ages, with body
proteins being more actively broken down and resynthesized into smaller amounts. During
the aging process, muscle mass decreases and the waste product of protein breakdown
(nitrogen) is removed from the body. It can also be seen in the case of malnutrition or in
general when the dietary protein intake is not sufficient.

Amino acids are mobilized from the body's proteins to use carbon skeleton for energy, even if
the diet is poor and lacks essential amino acids. If the synthesis of new proteins in the body is
imparied, then the amino acids are deaminated and amino groups from used amino acids will
be excreted out via the urine in its non-toxic form. In case of acute injuries or burns (body
tissue is damaged), the damaged proteins which have been degraded to nitrogen have to be
removed from the body. It is worth noting that during recovery periods the nitrogen balance
is positive as the active tissue regeneration involves synthesis of new protein. In a healthy
adult, the nitrogen balance should be either positive or negative, the ingested amount of
nitrogen should be equal to the excreted amount of nitrogen.

Clinical Significance of ALAT, ASAT

Blood serum enzymes can be divided into serum specific and non-specific serum enzymes.
● Serum specific enzymes in serum have a specific function, such as it is with
lipoprotein lipase. This enzyme is located on the endothelial surface of the blood
vessels. This serum enzyme breaks down triglycerides into fatty acids and glycerol.
● Non-specific serum enzymes do not have a specific function.

A high concentration of enzymes in blood signals damage of cells or tissues. Therefore the
detection of specific markers via a blood test is very valuable in the diagnosis of various
diseases. ALT, AST are the enzymes which play an important role in amino acid catabolism.
Naturally speaking, enzymes are present inside the cell and only a small amount enters the
blood. With certain diseases, where the cell membrane is damaged, will the cell content spill
into the blood. The released enzymes can then be detected via a blood test. The blood test
result in combination with the patient's symptoms, can help us determine the final diagnosis.
↑ALT (GPT)
Alanine aminotransferase
(Glutamate-pyruvate transaminase)
ALT is considered as a specific marker of liver disease.
↑ ↑ Acute hepatitis (>10% of the upper limit of normal range)
↑ Myocardial infarction (<5% of the upper limit of normal range)
↑ Hepatotoxical medication (ex. antibiotics, anabolic steroids)
↑ Chronical hepatitis (5-10% of the upper limit of normal range)
↑ Liver metastasis
↑ Skeletal muscle diseases
ALT; considered to be a specific indicator of liver disease. Reference range is dependent on
age and gender. ALT has been reported to increase in patients with hepatic metastasis and
skeletal muscle diseases. In order to interpret the blood results test, we need to compare it
with other markers and take the patient's specific symptoms into consideration.

↑AST (GOT)
Aspartate aminotransferase
(Glutamate-oxaloacetate transaminase)
AST in greater concentrations is found in liver, heart, skeletal muscle and kidney tissue.
↑ ↑ Acute hepatitis (AST > 10–50 times, rarely – 100 times. ALT value is the same as AST or
higher)
↑ Myocardial infarction (AST increases 6–8h after MI, reaches max.in 8–24h. On the 4th,
5th day returns to normal range)
↑ Hepatotoxical medication
↑ Chronical hepatitis (moderately elevated value)
↑ Liver metastasis
↑ Skeletal muscle diseases, hemolysis
AST; 2 isoenzymes can be detected, one being cytoplasmic and the other mitochondrial.
Cytoplasmic enzymes enter the blood easily, even with a minor membrane damage. While
the activity of mitochondrial enzymes in the blood indicates even more severe cell damage.

Clinical Significance of Creatine kinase

↑CK
Creatine kinase
There are isoenzymes of CK found in:
- Skeletal mucle - CKMM
- Brain – CKBB
- Heart muscle - CKMB
↑ Mechhanical muscle demage
↑ Myocardial infarction(4-6h; norm: 72h)
↑ Muscle overload, intramascular injections
↑ Skeletal muscle diseases
↑ Brain damage
CK; other markers are taken into account to make a precise diagnosis. CK catalyzes the
reverse phosphorylation of creatinine, which produces energy in the form of phosphocreatine.
Phosphocreatine is phosphorylated creatine and regenerates ATP (energy). It provides the
muscle, heart and brain with immediate energy. For example, fast energy is required for
sprinting or weight lifting. The highest creatine kinase activity is observed in skeletal muscles
as well as the nervous system and heart cells. There is zero activity in the liver. The CK
marker helps make an accurate diagnosis. For example, the combined increased activity of
ALT, AST and CK in blood serum would most likely not be associated with liver disease. If
plasma creatinine kinase activity is high, three different isoenzymes are taken into account for
precise diagnosis. The activity of particular enzymes help pinpointing the location of the
damage as it could either be in the skeletal muscle, brain or the heart cells etc.
Lecture 4
Biosynthesis of Amino Acids, Nucleotides and Related molecules
Learning goals:
● Understand why nitrogen fixation in microorganisms is essential for sustaining life on Earth
● Understand the general mechanisms of ammonia incorporation into biomolecules
● Understand general principles of (not required to know all the pathways by heart)
- Biosynthesis of amino acids
- Biosynthesis and degradation of heme
- Biosynthesis of nucleotides
- Catabolism of purines

Biosynthesis of Amino Acids

Importance of nitrogen
Nitrogen 4th most widespread element in living systems (behind C, H and O)
Most of it bound in amino acids and nucleotides
Also found in:
● Several cofactors (NAD, FAD, biotin etc.)
● Many small hormones (epinephrine)
● Many neurotransmitters (serotonin)
● Many pigments (chlorophyll)
● Many defense chemicals (amanitin)

Biochemistry of molecular nitrogen

The biochemistry of nitrogen affects the general metabolism of biologically available
nitrogen and its tight regulation.
● Atmosphere comprises of 78% N2 , but most of it can’t be used by organisms because
molecular nitrogen is chemically inert
➢ N2 chemically inert
➢ NH3 - biologically available form
- [ N2 + 3 H 2 → 2 NH 3 ]
In order for us to use it, it is converted into the biologically available form of ammonia NH3.
● Reduction of N2 to NH 3 has G = -33.5 kJ/mol. Reaction is exogeneric, releases
energy and has negative Free Gibbs energy, but the stable triple bond makes the
activation energy for this reaction extremely high.
● Some organisms have developed the ability to carry out this conversion in an
enzymatic way, but overall, biologically available nitrogen in nature is scarce.

The Nitrogen Cycle

Maintains balance between N2 and biological forms of nitrogen.
1. Fixation - reduction of N2 by nitrogen fixing bacteria, yielding ammonia
- [ N2 → NH 4 + or NH 3 ]
2. Nitrification - soil bacteria convert ammonia first to nitrite and then to nitrate.
- [ NH4+ or NH 3 → NO 2- → NO 3- ]
3. Assimilation
 a. plants and many microorganisms reduce nitrate and nitrite using reductases.
b. Plants then assimilate ammonia into amino acids
c. Animals use plants as source for amino acids for protein production
d. After death, organisms decompose, returning NH 3 to soil, where it is again converted
to nitrate or nitrite
4. Denitrification - bacteria convert NO3 to N2 under anaerobic conditions to maintain
the balance
➢ NO 3 - is the ultimate electron acceptor instead of NO2

The Nitrogen Cycle

Relationship between the molecular nitrogen (N2) and various kinds of biologically available
nitrogen (NH3) forms.
The main role of the nitrogen cycle, besides allowing the circulation of biologically available
forms of nitrogen, is to also maintain the balance between molecular nitrogen (inert form -
N2) in the atmosphere and the biological forms (NH3).

First step
Fixation of nitrogen
Nitrogen fixing organisms are mainly those of bacteria and archaea. They can perform the
reduction of molecular nitrogen, yielding ammonia (NH3). These nitrogen fixing bacteria use
energy in the form of ATP to overcome the high activation energy of this reaction. They
carry out this process by using a highly conserved protein complex called the nitrogenase
complex. These nitrogen fixing organisms can fix more nitrogen than what is necessary for
the cells, so they release the excess into their surrounding environment.

Second step
Nitrification
After the release of excess ammonia into the surrounding environment, it is picked up by the
nitrifying soil bacteria and archaea. These organisms convert ammonia [NH3] first to nitrite
[NO2], before further on converting nitrite [NO2] into nitrate [NO3]. They use these
reactions to gain energy. Almost all of the nitrogen that reaches the soil is oxidized because
of how many organisms there are, and it is in the soil that we get nitrate [NO3].

Third step
Assimilation
This step is backwards to the nitrification step. Plants and many other microorganisms can
readily pick up reduced nitrate and nitrite to further on reduce them back to ammonia [NH3].
Ammonia [NH3] is the biologically usable form. These plants will then assimilate the
ammonia [NH3] into amino acids, which animals ingest as their source of amino acids for
protein production. When an animal dies, their corpse will decompose and the present
ammonia will spill back into the soil where it is converted into nitrate [NO3]. This process
continues on and the biologically forms circulate through various types of organisms.
Fourth step
Denitrification
Denitrifying bacteria convert nitrate or nitrite back into its molecular form of nitrogen [N2] in
order to maintain the balance between these two forms of nitrogen. These given bacteria use
nitrate as their ultimate electron acceptor instead of oxygen, so they end up using these nitrate
and nitrate molecules to generate transmembrane protein gradients for ATP production.

Additional step
Anammox bacteria
Another small step involves anammox bacteria. They were recently discovered and they have
the ability to replicate the nitrogen cycle without the denitrification step.

OBS.
N2; inert nitrogen in the atmosphere
NH3 [Ammonia]; biologically available form of ammonia, known as toxic free ammonia
NH4 [Ammonium]; the non-toxic ammonium ion
Ammonia is incorporated in biomolecules through glutamate and glutamine
● Glutamate is the source of amino groups for most other amino acids, derived from
alpha-ketoglutarate and is the source of amino groups for most other amino acids.
● Glutamine is involved in a wide range of biosynthetic processes, provides amino
groups from this amide group (circled in the illustration), this amide group provides
the amino acids to a wide range of biosynthesis processes.
● In most types of cells Glu and Gln are present at significantly higher concentrations
than other amino acids.

Biosynthesis of amino acids

The catabolism of amino acids is necessary as ammonia is first incorporated into amino acids
and then usually into other biomolecules (including nucleotides).
The critical entrypoint of ammonia into our metabolism is provided by the two following
amino acids: glutamate and glutamine.

The biosynthesis pathways involving glutamate and glutamine are simple and are partially
found in all organisms. In humans, glutamate can be formed in various ways. It could be
formed either through transamination reactions of alpha-ketoglutarate during amino acid
catabolism. Or it could be in some part of a direct reaction between alpha-ketoglutarate and
free ammonia (presented in the illustration below).

Glutamine has the important ability to incorporate free ammonia for transport in the body.
The amide group of glutamine also serves as an important amide group donor. With the
involvement of glutamine, the toxicity of free ammonia is neutralized alongside the group
being available for many other biosynthetic processes.

Overview of amino acid synthesis

● In total 20 amino acids used for building peptides and proteins
● All of these amino acids that we use are derived from intermediates of:
- Glycolysis
- Citric acid cycle
- Pentose phosphate pathway
● Organisms vary in ability to synthesize amino acids mammals can synthesize only
half
Illustration portrays the amino acid biosynthesis in bacteria, of all 20 amino acids. Bacteria
are able to synthesize all 20 on their own. While mammals can only synthesize half of them,
others must be taken up through diet.

Categorization of amino acids

Non-essential amino acids; we can synthesize (simple structure)
Essential amino acids; can not synthesize on our own (complex structure)
Nonessential amino acids and their precursors

Amino acids deriving from α ketoglutarate

Biosynthesis of proline from glutamate

● Synthesis from glutamate carried out by three different enzymes
● Involves non enzymatic cyclization
● Happens in mitochondrial matrix
● Final reduction step yields proline

Proline can be synthesized from either L-glutamate or argine in our body. This synthesis is
carried out by three different enzymes in three successive steps. The following process occurs
in the mitochondria.
First step
Glutamate kinase uses ATP to phosphorylate the glutamate molecule, yielding the
intermediate of gamma-glutamyl-phosphate
Second step
Glutamyl phosphate reductase uses NAD(P)H + H to reduce the gamma-glutamyl-phosphate
molecule and induce a non-enzymatic cyclization. This produces the intermediate of 1-
pyrroline-5-carboxylate (P5C).
Third step
After another reduction reaction ensured by pyrroline carboxylate reductase, another
NAD(P)H + H is used and the final product of proline is formed.

The important molecule we need to look at here is the first molecule that contains this cyclic
structure, the 1-pyrroline-5-carboxylate (P5C). This reaction is formed through the non-
enzymatic cyclization step. Upon the first formation of P5C, it contains a double bond in the
cycle and so the second reduction is needed to remove the double bond and form the amide
group NH2.

Biosynthesis of proline from arginine

● Ornithine is derived from the urea cycle or degradation of arginine.
● Ornithine aminotransferase converts ornithine to glutamate γ semialdehyde that
cyclizes and converts to Pro.
Proline synthesized from arginine
This reaction is derived from ornithine, the molecule that is produced in the urea cycle,
during degradation of arginine. In this reaction the ornithine delta-aminotransferase uses one
alpha-ketoglutarate molecule to form glutamate-gamma-semialdehyde, which again
continuously cyclyze into the P5C molecule. This molecule can then go through the second
reduction, in which it is converted into proline.

Arginine is the last molecule derived from alpha-ketoglutarate and glutamate. Although this
reaction's equilibrium favors the formation of pyrroline-carboxylate molecule (P5C), the
reverse reaction is the only mammalian pathway for the synthesis of ornithine. This is
necessary for arginine synthesis, as when arginine levels are insufficient we could at least
somewhat compensate for the arginine that is necessary for our body.

Arginine is synthesized from ornithine in animals

● Ornithine comes from the urea cycle.
● Ornithine aminotransferase can operate in direction of ornithine formation in
insufficient arginine conditions
● Considered an essential amino acid for children

After the reverse reaction of the delta-aminotransferase, the produced ornithine is converted
into citrulline and then eventually arginine. Arginine is sometimes called semi-essential
amino acid.

Although there are pathways in which arginine is produced, it is not the final product of these
cycles. This reaction is illustrated in a linear way in the illustration below, however arginine
is one of the intermediates of the urea cycle and not the final product. The arginine which is
synthesized as an intermediate of the urea cycle meets the arginine levels requirements for a
normal healthy adult. In growing children and in individuals recovering from trauma, it is
considered an essential amino acid which is needed to be taken up through the diet.

Below is the production of arginine described in detail:

Arginine is produced with the availability of the ornithine molecule. The enzyme ornithine
carboxyl transferase uses the carbamoyl phosphate molecule to form citrulline. 1 ATP is used
by the enzyme argininosuccinate synthase to form argininosuccinate. In the last reaction,
argininosuccinate releases the two products of arginine and fumarate.

Amino acids deriving from 3 phosphoglycerate

Serine synthesis from 3-phosphoglycerate

 ● Same pathway in all organisms
● Requires Glu as source of NH 2 group
Steps:
● Oxidation (using NAD+)
● Transamination from glutamate
● Dephosphorylation to yield serine

Serine synthesis
Glutamate is the source of amino groups in this case
From 3-phosphoglycerate is a pathway that is the same in all organisms, and it is relevantly
simple as it only has three reactions.
First step
Oxidation (using NAD+)
The oxidation of 3-phosphoglycerate is ensured by the enzyme phosphoglycerate
dehydrogenase. In this step, 1 NAD+ is used as the proton recipient, and the 3-
phosphoglycerate is converted into 3-phosphohydroxypyruvate. Here the double bond at the
second carbon is formed.
Second step
Transamination from glutamate
Glutamate acts as the donor of the amino group, so the phosphoserine aminotransferase
transfers the amino group from glutamate to the 3-phosphohydroxypyruvate molecule to form
3-phosphoserine. In this reaction alpha-ketoglutarate is released.
Third step
Dephosphorylation to yield serine
With the acquired amino group, the last step involves the removal of the phosphate group that
is not needed for serine. Phosphoserine phosphatase hydrolysis this bond between the
phosphate group and the rest of the molecule. In this reaction inorganic phosphate is released
and the final product of serine is formed.

Glycine biosynthesis from serine

 ● Removal of one carbon to go from serine (3C atoms) to glycine (2C atoms). Reaction
carried out by serine hydroxymethyltransferase. In this reaction, H4 folate
(tetrahydrofolate) is used as the molecule which takes up the methyl group. Also
water is released in this molecule,because together with this one carbon we also
remove the hydroxyl group.
● In this reaction we need a cofactor of pyridoxal phosphate (PLP) for the addition and
elimination of various groups. Upon the removal of this one carbon, adding this
molecule to tetrahydrofolate and also releasing water molecules we yield glycine.
● Reversible reaction, requires tetrahydrofolate and pyridoxal phosphate (PLP)

Serine gives rise to two other amino acids, one of them being glycine. Glycine has the most
simple structure of all amino acids as it does not have any side chains.

Cysteine in mammals synthesized from serine and methionine

Methionine is first converted to homocysteine through multiple reactions. Cystine is one of
the amino acids classified as conditionally-essential amino acids. Even though we can
synthesize cysteine, this process still requires an essential amino acid. Serine provides the
carbon skeleton of cystine. Methionine is an essential amino acid as it is necessary for this
pathway as it provides the thiol group for the formation of cystine.

First step
Homocysteine and serine are condensed by the enzyme cystathionine beta synthase. This
reaction needs the prosthetic group of PLP and in this step a water molecule is released. We
gain the intermediate cystathionine, which is a seven carbon molecule with sulfur in the
middle of the carbon chain.
Second step
The enzyme cystathionine-gamma-lyase ensures both the removal of the ammonia group
from the homocysteine section of the molecule as well as the cleavage of the cystathionine
molecule. The cleavage point of cystathionine leaves this group added to the serine carbon
skeleton, forming the two products of alpha-ketobutyrate and cystine.
Amino acids deriving from oxaloacetate
OBS. Only two amino acids derived from oxaloacetate can be produced in humans: aspartate
and asparagine.

Biosynthesis of aspartate and asparagine

Occurs in all organisms.
● Aspartate is produced by direct transamination of oxaloacetate by enzyme
transaminase
● Asparagine produced by amidation of aspartate (glutamate as NH 4 donor) by
asparagine synthetase. In this reaction we use glutamine instead of glutamate. The
amide group is transferred from glutamate. This reaction is carried out by the enzyme
asparagine synthetase. We use energy in the form of ATP and subsequently an
AMP+PP molecule is released.

Amino acids deriving from pyruvate

Alanine is synthesized by the transamination reaction of pyruvate. This reaction is ensured by

the enzyme alanine transaminase (alanine aminotransferase or ALT for short).
In this reaction, glutamate gives an amino group to pyruvate to form the product of alaine,
with keto-glutarate leaving the step.
Clinical significance of ALT: serves as a marker for liver disease.

Biosynthesis of tyrosine
In general, the biosynthesis of aromatic amino acids de novo happens in complex branched
pathways that occur in fungi and plants. In humans we do not possess the ability to form
these particular aromatic amino acids.

The only amino acid that can be in some way synthesized in our body is tyrosine. This can
only be done by using another aromatic amino acid, being phenylalanine. This reaction
occurs in the liver. If the dietary intake of tyrosine is too low, up to 50% of the ingested
phenylalanine can be converted into tyrosine. If we have enough dietary intake, the required
phenylalanine levels will decrease as not too much is needed to be converted into tyrosine.

● Tyrosine can be synthesized through hydroxylation of phenylalanine

● Place of action: liver
● Reaction happens when dietary intake of tyrosine is insufficient.
● Reaction catalyzed by phenylalanine hydroxylase which metabolizes excess
phenylalanine
● Reaction coenzyme tetrahydrobiopterin; has quite a complex role as it donates 2
protons to the hydroxyl group during the formation of tyrosine. The additional
hydroxyl group was taken from phenylalanine, which does not have any functional
groups attached to the aromatic ring.
● Tetrahydrobiopterin donates these 2 protons, allowing the hydroxyl group formation.
By losing 2 hydrogens, Tetrahydrobiopterin takes the form of D-hydrocarbon. The
cofactor itself is later reduced back to Tetrahydrobiopterin thanks to NAD, now it can
be reused again.
In this reaction, we need molecular oxygen for the formation of tyrosine. The source of
oxygen is needed for the formation of hydroxyl groups.

Important metabolites are derived from amino acids

 ● Porphyrin rings (e.g, heme)
● Phosphocreatine
● Glutathione
● Neurotransmitters and signaling molecules

Biosynthesis of hemes
● Porphyrin rings makes up the heme of hemoglobin , cytochromes , myoglobin
● In higher animals , porphyrin arises from reaction of glycine with succinyl CoA
● First important intermediate gamma-aminolevulinate
● From two molecules of gamma-aminolevulinate porphobilinogen is formed, which in
turn can form this porphyrin ring.

Hemes has the ability to bind to oxygen and ensure safe transport around the body.
Heme group can be found in enzymes as a prosthetic group (these are called heme enzymes).
Hemes also have a very high importance in redox reactions, and it is worth noting that heme
groups can also be bound to proteins without any enzymatic activity. For hemoglobin and
myoglobin, they do not have enzymatic activity but the heme group serves their role of
binding to oxygen.

Synthesis of δ aminolevulinate
Two molecules of δ aminolevulinate needed for synthesis of porphobilinogen
First step
In the first reaction, succinyl-CoA and glycine react together to form the intermediate alpha-
amino-beta-ketoadipate. In this step, the CoA-SH which was previously attached to succinyl-
CoA is released.

Second step
In the second step, the intermediate is decarboxylated which involves the removal of the CO2
group from glycine. This produces the final product of delta-aminolevulinate. The second
step was ensured by the enzyme δ-aminolevulinate synthase.

Synthesis of heme from δ-aminolevulinate

After the formation of δ-aminolevulinate, 2 molecules of δ-aminolevulinate are condensed to
form porphobilinogen. We need 4 molecules of porphobilinogen, that through a series of
complex reactions, are combined together to form the protoporphyrin. After the formation of
protoporphyrin, the iron ion is incorporated into the porphyrin ring. This step is catalyzed by
the enzyme called ferrochelatase, it allows for this coordination of the iron 2+ ion in the
center of the pyrophin ring. This incorporation of iron ions results in the formation of a heme
molecule. The porphyrin biosynthesis is regulated by the concentration of the heme product;
it is considered to be the feedback inhibitor in the early steps of the pathway.

Defects in heme biosynthesis

● Most animals synthesize their own heme.
● Mutations or misregulation of enzymes in the heme biosynthesis pathway can lead to
a group of diseases called porphyrias. 7 different diseases based on which step of
heme synthesis is defective.
➔ Defect in the heme synthesis results in precursors accumulating in red blood
cells, body fluids, and liver.
● Accumulation of precursor uroporphyrinogen I (one of the rarer porphyrias)
➔ Urine becomes discolored (pink to dark purple depending on light, heat
exposure).
➔ Teeth may show red fluorescence under UV light.
➔ Skin is sensitive to UV light.
➔ There is a craving for heme.
➔ Explored as possible biochemical basis for vampire myths

Heme is the source of bile pigments

Heme from erythrocyte breakdown is degraded to bilirubin in two steps:
● Heme oxygenase linearizes cyclic heme to create biliverdin , a green compound
● Biliverdin reductase uses NADPH + H as proton donor to convert biliverdin to
bilirubin , a yellow compound
➔ Bilirubin travels in the bloodstream as a complex with serum albumin as it is
largely insoluble.
➔ When bilirubin is transported to the liver, it is transformed into a soluble bile
pigment which gets secreted.
➔ In the liver, bilirubin gets converted into urobilinogen, which is further
degraded by intestinal microbiota to stercobilin.
➔ Stercobilin is excreted out with feces, and gives the distinct color of feces.
➔ Part of urobilinogen gets reabsorbed back into the blood and into the kidneys,
where it is converted into urobilin which gives rise to color of urine and
excreted out in urine.

Jaundice is caused by bilirubin accumulation

Jaundice - condition resulting in yellowing of skin and eyeballs.
Can be caused by:
● Impaired liver (liver cancer, hepatitis)
● Blocked bile secretion (gallstones, pancreatic cancer)
● Insufficient glucuronyl bilirubin transferase to process bilirubin (occurs in infants)
treated with UV to cause photochemical breakdown of bilirubin

Impairments cause the bile to leak into the blood, leading to yellow coloration

Biosynthesis of creatine and phosphocreatine

● Produced from Gly and Arg, with S adenosylmethionine as methyl group donor.
● Phosphocreatine is an important energy buffer in skeletal muscles, hydrolyzed for
energy (exergonic reaction)
● Creatine can be phosphorylated by ATP for use as a stored energy source.
First step
Arginine has two amide groups attached to it, both of them are transferred to glycine by
aminotransferase. This step produces the intermediate of guanidinoacetate.
Second step
Methionine, in the form of adenosyl-methionine, donates its one methyl group to
guanidinoacetate in order to yield the synthesis of creatine. This reaction is ensured by
methyltrasnferase.
Third step
The phosphorylation of creatine requires an ATP, this yields the final product of
phosphocreatine.

Phosphocreatine is used as an energy source for skeletal muscles. This molecule allows for
immediately accessible ATP as this atp can be regained from this one quick reaction. This
reaction is just the last step in reverse, where one ADP is used for the release of ATP which
gives us creatine. Phosphocreatine serves as an important energy source for skeletal muscles
as there is no need for transportation of energy. Phosphocreatine ensures a period of time
where necessary energy is generated to continue muscle activity.
Glutathione (GSH) derives from Glu, Cys and Gly
One of the roles of glutathione is the neutralization of the active oxygen species.
Because of its reducing properties, glutathione alternates between its reduced state (GSH)
and its oxidized state (GSSG).
● GSH is present in most cells at high amounts.
● Reducing agent/antioxidant
- keeps proteins, metal cations reduced
- keeps redox enzymes in reduced state
- removes toxic peroxides
Oxidized to a dimer using disulfide bond (GSSG)
First reaction
Condensation reaction between glutamate and cysteine. 1 ATP is used here, and we get the
intermediate gamma-Glutamate-Cysteine.
Second step
Adds a third amino acid, glycine, to the carbon chain. 1 ATP is used here to produce the
reduced form of glutathione.

In total, this process used 2 ATP molecules. This is an energy consuming process. The active
form Glutathione (GSH) with reducing properties is the final product of this pathway. After it
donates its protons, it becomes its oxidized dimer form Glutathione (GSSG). From the whole
glutathione molecule, the thiol group serves as a proton donor. After oxidation, a disulphide
bond is formed at the 2 sulfhydryl groups between the two oxidized glutathione molecules.
These two glutathione molecules are formed into this dimer. Later on when GSSG is reduced
by NADPH + H, it becomes its reduced monomer form of glutathione (GSH). The cycle
repeats.

Some neurotransmitters are derived from amino acids

Epinephrine, histamine, serotonin are all derived from essential amino acids. They could also
be derived from conditionally-essential amino acids in the case of tyrosine. Tyrosine gives
rise to a whole group of neurotransmitters called catecholamine. These molecules have a
catecholamine ring, which include the following neurotransmitters; dopamine,
norepinephrine, epinephrine. They can act as neutronstarmteers in the central and peripheral
neuronal system, as well as hormones in the endocrine system. These neurotransmitters are
all involved in a decarboxylation step which is catalyzed by PLP dependent enzymes. All of
these steps are highlighted in these reactions below.

Common for all pathways all are decarboxylated using PLP dependent enzymes

Examples of amino acids as effector molecules or precursors

Biosynthesis of Nucleotides
Role of nucleotides
Nucleotides serve various important roles in our body:
● Precursors of DNA and RNA
● Components of coenzymes (e.g ., NAD[H], NADP[H], FMN[H 2 ], and coenzyme A)
● Energy currency , driving anabolic processes (e.g ., ATP and GTP)
● Carriers in biosynthesis (e.g ., UDP for carbohydrates and CDP for lipids)
● Allosteric modulators of key enzymes in metabolism
● Second messengers in important signaling pathways e.g ., cAMP and cGMP

Biosynthesis of nucleotides
Two types of pathways lead to nucleotides:
● De novo synthesis; produces nucleotides from their metabolic precursors -amino
acids, ribose 5 phosphate, CO2 and NH3
● Salvage pathways; recycle free bases and nucleosides from RNA, DNA, and cofactor
degradation

Basic structure of nucleotides

Nucleotides are made up of the following components; 1-3 phosphate groups, pentose sugar,
nitrogen base which is divided into either purine (2 heterocyclic rings) or pyramides (1
heterocyclic ring).

The nitrogen base differs between RNA and DNA; thymine is in DNA, while uracil is in
RNA. The pentose sugar is also different, as deoxyribose is in DNA, while ribose is in RNA.

The nitrogen bases combined with 5 carbon sugar forms nucleodies.

When nucleosides are phosphorylated, in which one phosphate group is added, we produce
nucleotides.
OBS. Nucleosides (bottom) are made of a nitrogenous base, usually either a purine or
pyrimidine, and a five-carbon carbohydrate ribose. A nucleotide is simply a nucleoside with
an additional phosphate group or groups (blue); polynucleotides containing the carbohydrate
ribose are known as ribonucleotide or RNA.

De novo synthesis of nucleotides

 ● Occurs approximately the same in all organisms, but the demand varies greatly
between tissues. Ex. high in cells about to divide (active in growing tissues,
embryonic and fetal tissues for the fetus growth, actively proliferating cells such as
red blood cells and cancer cells), increased during wound healing and tissue
regeneration.
● Glutamine provides most amino groups; it serves as a donor for most amino groups
during nucleotide synthesis. involved in 5 different steps in this pathway.
● Glycine is precursor for purines
● Aspartate is precursor for pyrimidines
❖ Both de novo synthesis of purine and pyrimidine are energetically expensive
pathways. That is why these processes are effectively regulated. Nucleotide
pools (other than ATP) are kept low, so cells must continually synthesize
them.
❖ This can sometimes limit the rates of transcription and replication, simply
because not enough nucleotides are available.

Origin of ring atoms in purines

There are 5 different molecules involved in the formation of purine.
3 of those molecules are from glycine, which contributes to the purine ring formation.

Synthesis of inosinate (IMP) (1)

biosynthesis of both purines initially is the same, basically with the synthesis of IMP. From
IMP then either adenylate or guanylate is derived.
1. Amino group from Glutamine attached to the 1st carbon of PRPP
[Committed step]
2. The highly unstable molecule of PRPP reacts with Glycine, where the three atoms
from Gly are added to the given molecule. In this step, ATP is used in order to
activate the glycine carboxyl group for this condensation. This results in the formation
of the molecule glycinamide ribonucleotide (GAR).
3. After the addition of glycine, the glycine amino group is formulated [a formyl group
is created]. This is done with the help of formyl tetrahydrofolate, a molecule derived
from folic acid. This results in the formation of the molecule formylglycinamide
ribonucleotide (FGAR).
Synthesis of IMP (2)
4. A nitrogen molecule is contributed by Glutamine, this subsequently releases a
glutamate. ATP is used here.
5. The first ring is closed, this formylglycine ribonucleotide (FGAR) gets cyclized by
FGAM cyclase. This enzyme dehydrates this molecule and yields 5-aminoimidazole
ribonucleotide (AIR).
[First ring formed]

Synthesis of IMP (3)

At this point, 3 of the 6 atoms that are needed for the 2nd ring are already in place
Only in bacteria and fungi:
6. Carboxylation
7. Rearrangement of carboxylate
In higher eukaryotes:
6a. The molecule of AIR is carboxylated, to yield CAIR
8. Aspartame is involved, where it donates its amino grouP. In the first reaction, an amide
bond is formed between aspartate and CAIR.

Synthesis of IMP (4)

9. The elimination of Aspartate carbon skeleton happens, releasing fumarate.
10. The final carbon necessary for the second ring formation is contributed from another N10
formyltetrahydrofolate.
11. In the last step of cyclation, yields the inosinate molecule. So the first intermediate with a
complete purine ring.
[Second ring formed]

OBS. This whole process, formation of purine base happens already with the attachment of
ribose sugar.
Synthesis of AMP and GMP from IMP
ATP is used for GMP synthesis, while GTP is used for AMP synthesis

The formation of inosinate will not lead to its accumulation in the cell. As from inosinate
(IMP), either AMP or GMP is formed. Two enzymatic reactions are required in this case.

AMP synthesis
The insertion of an amino group from aspartate is needed. The principal is the same as before
for the second purine ring formation. The first aspartate is added with the help of GTP, which
forms the intermediate adenylosuccinate. Fumarate is released in the next step which is
ensured by the enzyme adenylosuccinate lyase, leaving only the amide group attached which
in turn forms the final product of adenylate (AMP).

GMP synthesis
IMP is first dehydrogenated into XMP in which a double bonded oxygen is introduced. In the
second step glutamine joins in.. Here this double bonded oxygen is substituted with an amide
group from glutamine, this step is ensured by the enzyme by XMP glutamine transferase. 1
ATP is also used here in order to form the final product of guanylate (GMP). The used up
ATP leaves the reaction in the form of AMP + pyrophosphate.
Important note for the synthesis of purine bases
The opposite triphosphates are used for the synthesis of AMP and GMP.
➔ AMP synthesis; 1 GTP is used by the enzyme adenylosuccinate synthetase.
➔ GMP synthesis; 1 ATP is used by the enzyme xmp-glutamine transferase.

De novo synthesis of pyrimidine molecules (1)

Contrary to purine synthesis, here in pyrimidine synthesis, the ring is formed first and then
later on attached to the ribose sugar.
● Requires carbamoyl phosphate (intermediate of urea cycle and aspartate).
❖ Carbamoyl phosphate is the intermediate of urea cycle and aspartate.
However, the carbamoyl phosphate used in urea synthesis is synetshsied in the
mitochondria by carbamoyl phosphate synthetase I. In this case, the carbamoyl
phosphate required for pyrimidine synthesis is produced in cytosol by
carbamoyl phosphate synthetase II
● Aspartate and carbamoyl phosphate provide the atoms for the pyrimidine ring
structure. Ring formation starts with formation of carbamoyl aspartate, with the
combination of these two molecules together. catalyzed by aspartate
transcarbamoylase.
● The ring structure is already formed in the second reaction, after formation of
carbamoyl aspartate a water molecule is removed. In this reaction, catalyzed by
dehydrooratase, the cyclic structure of dihydroorotate is formed. this pyrimidine ring
is already closed
● This dihydroorotate is oxidized by dihydroorotate dehydrogenase by using nadh as
electron acceptor, which yields orotate.
● In eukaryotes, the first 3 enzymes are involved in pyrimidine synthesis. carbamoyl
phosphate synthetase, aspartate transcarbamylase, dihydroorotate dehydrogenase all a
part of a single trifunctional protein called CAD.
De novo synthesis of pyrimidine molecules (2)
● Once orotate is formed, the ribose sugar is added. Addition of ribose 5-phosphate via
PRPP results in nucleotide orotidylate
● Orotidylate is decarboxylated to form uridylate (UMP), the first possible pyrimidine.
De novo synthesis of pyrimidine molecules (3)
Another difference between the purine and pyramide synthesis, is the fact that for purines the
formation of amp and gmp is diverted from this common intermediate of isonoate (imp).
for pyrimidines, these nucleotides derived from one and other.
● We first produce the first pyrimidine nucleotide called Uridylate (UMP). UMP is
twice phosphorylated by kinases to form UTP. so the full triphosphate molecule.
● After formation of UTP, the cytosine nucleotide CTP can be formed through the
action of cytidylate synthetase. with glutamine again serving as an amide group
donor. so this cytidine triphosphate is formed by amidation of UTP, forming CTP. So
from one nucleotide we can form the other nucleotide that can be found in rna.

Reduction of ribonucleotides
Ribonucleotides (Sugar ribose) is present in this given structure, however in DNA we have
deoxyribonucleotides (deoxyribose). In order for ribose to become deoxyribose, it has to lose
an oxygen at its 3rd carbon.

Reduction of ribonucleotides
Ribonucleotides are reduced by the enzyme ribonucleotide reductase.
● Adenine, guanine and uracil deoxyribonucleotides are synthesized from their
corresponding ribonucleotide diphosphates by direct reduction of the 2nd hydroxyl
group. This reaction is ensured by the enzyme ribonucleotide reductase.
 ● The removal of oxygen is done by combining the oxygen from the 3rd carbon with
hydrogen, forming water.
➔ This reaction is unusual by itself as it happens at a non-activated carbon of the
sugar.
● This reduction reaction occurs on the enzyme itself, in this case being ribonucleotide
reductase. This is done by directly reducing the 2nd carbon hydroxyl bond of the T-
ribose to simple hydrogen bonds. In short, the reduction involves the removing
hydroxyl group.
➔ Ribonucleotide reductase provides the best example of involvement of free
radicals in biochemical transformations.
➔ Ribonucleotide reductase has 2 thiol groups. The tyrosyl radical is derived
from the thiol group in its active site, which helps to catalyze this oxygen
removal.
● This reduction process needs additional proteins that can carry protons and transfer
this reducing energy. As the primary electron donor, NADPH is used in this process
to further on reduce the enzyme ribonucleotide reductase itself.

The 2 following parallel systems can be used for the reduction process:
A) Glutaredoxin pathway
Glutaredoxin is a protein which is closely related to thioredoxin. Glutathione is used here for
the transfer of reducing equivalents from NADPH, which are first given to the oxidized form
of glutathione (GSSG). Upon receiving these two protons, glutathione becomes reduced to 2
molecules of the reduced form of glutathione (GSH). They transfer these protons to
glutaredoxin, which also becomes reduced. The reduced glutaredoxin transfers the two
protons to the enzyme ribonucleotide reductase, which in turn becomes oxidized again. This
allows for the ribonucleotide reductase to reduce these ribonucleotides into
deoxyribonucleotides.

B) Thioredoxin pathway
Thioredoxin is a hydrogen carrying protein that contains a pair of sulfhydryl groups that is
able to carry the protons from NADPH to ribonucleotide reductase in order to reduce the
enzyme itself. Thioredoxin has FAD as its prosthetic group, which is involved in the transfer
of these protons from NADPH. Upon receiving the 2 protons, FAD becomes its reduced form
of FADH2. Then they transfer these two protons to thioredoxin and in turn thioredoxin
transfers them to ribonucleotide reductase, successfully reducing the enzyme itself. The
reduction of the enzyme is important as it allows it to reduce the ribonucleotides into
deoxyribonucleotides.

● Carried out by ribonucleotide reductase

● NADPH as the electron donor.
● Funneled through glutathione (a) or thioredoxin pathways (b)
● Best characterized example of free radical involvement in biochemical
transformations.
Regulation of ribonucleotide reductase
Has two types of regulatory sites:
Affecting activity
❖ ATP activates the ribonucleotide reductase, however dATP inhibits the enzyme
activity [Feedback inhibition regulation]
Affecting substrate specificity
with goal to maintain balanced nucleotide pools
❖ If high levels of ATP or dATP, there is less specificity for adenine, and more
specificity for other types of nucleotides such as UDP and CDP etc. To accomplish
such changes, the enzyme oligomerizes to accomplish conformational change

dTMP is produced from dUTP

We have the deoxyribonucleotides, but DNA is still missing thymine nucleotides.
● Conversion from uracil (deoxyuracil nucleotides) into thymine.
● Only deoxynucleotides are involved in thymine de novo synthesis.
● dTMP is formed from either cytidylate or uridylate. Immediate precursor is dUMP.
The formation of deoxynucleotides is done for these diphosphates. This reduction is
performed by uridine diphosphate yielding Deoxyuridine (dUDP).
● Phosphorylation of dUDP into dUTP, later on it is converted back into dUMP through
dephosphorylation (removal of 2 phosphate groups) which is ensured by dUTPase.
Another route would be using cytidylate, where CTP can be emaninated into dUTP.
 ● Thymidylate synthase converts dUMP to dTMP, by adding a methyl group from
tetrahydrofolate (this time, methylenetetrahydrofolate). This results in the formation
of thymildate in the form of monophosphate (dTMP) can be then phosphorylated to
Thymidine triphosphate (dTTP) and be incorporated into DNA.

An important step to mention is that this process of conversion from dUDP into dUMP needs
to happen quite quickly and efficiently so this dUTP does not accumulate in the cell and it is
not incorporated in the DNA.

Folic acid deficiency leads to reduce thymidylate synthesis

● Folic acid deficiency is widespread, especially in nutritionally poor populations.
● Reduced thymidylate synthesis causes uracil to be incorporated into DNA.
● Repair mechanisms remove the uracil by creating strand breaks that affect the
structure and function of DNA, as there is no thymidylate to replace the removed
uracil.
➢ Associated with cancer, heart disease, neurological impairment
Catabolism of purines
 AMP GMP

1. Removal of phosphate group by the 1. Removal of phosphate group by the

reaction of 5prime-nucleotidase. This reaction of 5prime-nucleotidase. This
reaction uses 1 water molecule and releases reaction uses 1 water molecule and releases
inorganic phosphate. inorganic phosphate.

2. Deaminated back into inosine (nitrogen 2. Hydrolyzed by nucleosidase to release

base of IMP). this ribose sugar which leaves just the
nitrogen base.

3. Hydrolyzed by nucleosidase to release 3. Deamination of guanine forms the

this ribose sugar which leaves just the molecule xanthine (keto form).
nitrogen base.

4. The hypoxanthine molecule, formed upon

the removal of the ribose sugar, gets
oxidized. This step releases hydroperoxide
(H2O2), with the subsequent formation of
xanthine (keto form).

In both cases, the enzyme xanthine oxidase oxidizes the molecule of xanthine into the final
product of uric acid. This step will also release H2O2.

Excess uric acid causes gout

Gout - a painful form of arthritis caused by elevated concentration of uric acid in blood and
tissues
● Affects joints, with the deposits of sodium urate crystals. This results in painful joints,
most prevalent in toes. Leading to inflammation and eventual deformation of joints.
● Also affects kidneys, with excess uric acid also being deposited in kidney tubules
● This disease is troublesome as its exact cause has yet not been pinpointed; possible
connection with issues with the uric acid synthesis.
● Primarily affects males
● Gout can be treated with avoidance of purine rich foods (seafood, liver) or avoidance
of fructose. It can also be treated with drugs to alleviate these symptoms of gout. Such
a drug would be allopurinol, which is a xanthine oxidase inhibitor. Xanthine oxidase
is the enzyme that directly produces uric acid from xanthine. Allopurinol irreversible
competitive inhibitor of Xanthine oxidase. It acts as substrate, but upon binding to the
enzyme active site it remains tightly bound to it and inactivates this enzyme,
preventing the continuation of the synthesis of uric acid.

Catabolism of pyrimidines
Pyrimidine catabolism is not a frequent source of pathologies
● Leads to NH 4 production and thus urea synthesis
● Pyrimidines are usually degraded into soluble compounds, which are readily
eliminated in the urine or reused in other metabolic pathways. The production of
usable intermediates of metabolic pathways can be seen in the degradation of thymine
yields the product of methylmalonyl-semialdehyde. This is an intermediate which
could take part in the valine synthesis, which would in the end generate the molecule
of succinyl-CoA (an important intermediate of the citric acid cycle).

Purine and pyrimidine bases are recycled by salvage pathways

 ● Free bases of purine and pyrimidine are constantly released in cells during
degradation of nucleotides. Most of them are mostly salvaged and reused for
nucleotide formation.
● Adenine recycling is one of the primary salvage pathways: Adenine reacts with PRPP
to form the adenine nucleotide AMP. A lot less energy consuming than the de novo
synthesis.

● Recycling of guanine and hypoxanthine is carried out the same way as adenine, so by
a shared enzyme hypoxanthine guanine phosphoribosyltransferase
● Pathways are much more simple than de novo synthesis, and it is a lot less energy
consuming.
● Problems with this pathway are connected with the pathology known as Lesch Nyhan
syndrome. The lack of hypoxanthine guanine phosphoribosyltransferase leads to
Lesch Nyhan syndrome
❖ Neurological impairment, poor coordination, mental retardation, hostile and
self destructive tendencies. Usually affected are males and seen in patients
already as young as 2 years old.
● This syndrome highlights the importance these salvage pathways are for the brain.
The brain is a high energy consuming organ, so it tries to find the most optimal
pathway in which the least amount of unnecessary energy is consumed.
● Salvage pathways can be used as targets for specific drugs.
❖ Ex. Anti-parsystic drugs; many parasites do not have this de novo synthesis
pathway, so they mostly have to rely on salvaging the free bases of purines
and pyrimidines from its host. If the salvage pathway is inhibited, parasites
can not continue to proliferate.

Many chemotherapeutic agents target nucleotide biosynthesis

Cancer cells have greater requirements for nucleotides, so they are more sensitive than
normal cells to inhibitors of nucleotide biosynthesis
Drugs developed against cancer cells:
Glutamine analogs: azaserine , acivicin
❖ Inhibit glutamine amidotransferases, not allowing the transfer of amide groups during
de novo synthesis.
Fluorouracil
❖ Converted by salvage pathway into FdUMP, which inhibits thymidylate synthase
Methotrexate and aminopterin
❖ Inhibit dihydrofolate reductase as competitive inhibitors.
Lecture 5
Hormones
Learning goals:
● Explain the difference between neuronal vs hormonal signaling
● Describe the hormone classification based on path from place of release to target
● Describe the hormone classification based on their structure and the corresponding
mechanisms of signal transduction in cell
● Outline the major functions of hormones
● Explain the Top down and Bottom up regulation of endocrine system

Classification and characteristics based on structure

Metabolism at the Organism Level
In previous lectures, we focused on metabolism primarily at the cellular level
● role and mechanism of specific enzymes
● flux of metabolites through pathways
● feedback regulation of metabolic pathways
● transport of metabolites across organelle membranes
In this lecture and through the rest of the course , we will focus on metabolism at the whole
organism level
● role and structure of specific tissues and organs
● flux of metabolites from organ to organ
● hormonal regulation of metabolism
● control of body mass

Challenges in Detection and Quantification of Hormones

Hormones are extremely potent and produced in small amounts, so it is difficult to purify in
appreciable quantity
● Chemical analysis of thyrotropin releasing hormone (TRH) from pigs required one
million hypothalamuses (1,000,000 pigs)

➔ The radioimmunoassay (RIA) for peptide hormones was developed to be a more

sensitive way to measure hormones using radiolabeled antibodies rapid, quantitative,
and specific measurement of hormones in minute amounts
➔ Modern equivalent the enzyme linked immunosorbent assay (ELISA) uses
photometry
OBS. The carboxyl and amino end of the protein and its charge has a huge impact on the
molecule's binding capacity.
Regulation of bodily functions
Nervous system: parasympathetic (rest and digest functions) and sympathetic (fight or flight)
division. Works quickly, short lived localized effect

Endocrine system: endocrine major glands. Adipocytes and myocytes are also considered to
be a part of the endocrine system as they can secrete signaling molecules. Works a lot slower,
long lasting general effect
Neuronal versus Hormonal Signaling
● In neuronal signaling, nerve cells release neurotransmitters that act on nearby cells;
distance may be small (<1 m)
Includes actual nerve impulse and its transduction to various cells. Nerve cells can transmit
signals to another nerve cell, facilitating the downstream effects. The nerve impulse can also
induce contraction of muscle cells. The third type of the cells that the nerve impulse will
activate are endocrine cells which release signaling molecules.
● In hormonal signaling, hormones are carried by the bloodstream to nearby cells or
other organs; distance may be great (1 m or more)
Hormones are carried within the blood, thus the place of secretion (ex. pancreas), will release
the hormones which can facilitate effects similar to the nervous cell. Action range is far wider
thanks to the bloodstream reaching more target cells.

Three Classes of Mammalian Hormones

This classification is based on path from place of release to target
● Autocrine: affect the cell where they are produced (but bind to surface receptors)
EX. cytokines
● Paracrine: released into extracellular space, diffuse to neighboring target cells and
influence it action
EX. eicosanoids
● Endocrine: released to blood, carried to far-away target cells and influence their
action
EX. insulin, glucagon
● Intracrine: signaling across gap junctions, cell targets a cell connected by gap
junctions
OBS. Hormones can have more than one mode of transformation. One example is insulin,
which can act in both paracrine and endocrine mode.

● Classical hormones: secreted by endocrine cell in the blood, effects the target cell
● Neurohormones: released by neuronal cells in the blood,effect the target cell
(EX. oxytocin released by hypothalamus).
● Local hormones: exerting primarily paracrine action , with the secreting cell
influencing nearby cells
● Neurotransmitters: released in the synaptic cleft by neurons
Difference between hormones and neurotransmitters:
Difference in release and travel mechanism
Hormone Neurotransmitters

Produced in endocrine glands and are Released by presynaptic nerve terminal into
secreted into the bloodstream the synapse

Transmitted via blood Transmitted across the synaptic cleft

Virtually Every Process in a Complex Organism is Regulated by One or More

Hormones
Some examples:
● Maintenance of blood pressure, volume and electrolyte balance
● Embryogenesis
● Sexual differentiation, development and reproduction (ex. steroid hormones)
● Hunger, eating behavior, digestion, and fuel allocation
Among all the others

Hormone-Receptor Interactions Are Specific and High Affinity

● Different types of cells have different sets of receptors, which define the range of
hormone responsiveness. In other words, not all hormones are able to influence all
cells present in our body, due to the high affinity of the different sets of receptors.
This also defines the action range hormone, as insulin has a very wide action range as
it binds to basically every single cell in our body.
● Different cells with the same receptor can have different intracellular targets, thus
different downstream effects to the same hormone. In other words, hormones that
bind to one type of cell will induce one type of change while for another cell another
type of change will be induced. Take insulin for example, the binding of liver cells
will activate glycogen synthesis while the binding of adipocytes will activate the
storage of triacylglycerols.
● Structurally similar hormones can bind different receptors
● Interactions are high affinity so that only low amounts of hormones are needed to
exert the particular effect.
● In the design of drugs it is necessary to know the relative specificity and affinity of
the drug and natural hormone

Five Types of Downstream Events Following Hormone Binding

1. A secondary messenger (cAMP; inositol triphosphate, IP3) is released inside the cell
➔ allosterically regulates enzymes
Metabotropic receptors; hormones which bind to the receptor on the membrane, introduce a
secondary messenger cascade. Alters activity of already present enzymes, changing
biochemical processes in the cell.
2. A receptor Tyr kinase is activated
3. A hormone gated ion channel is opened or closed
➔ resulting in changes in membrane potential
Ionotropic receptors; forms an ion channel pore which opens and closes in response to the
binding of a chemical messenger (ligand hormone). Ensures the flux of various ions,
changing the potential and correspondly the functions in the cell
4. An adhesion receptor sends information to the cytoskeleton
5. A steroid bound to receptor protein in the nucleus alters gene transcription
➔ resulting in changes in protein expression
Hormones that are lipid soluble, diffuse through the cell membrane and bind to receptors on
the nucleus. This complex of the nucleus receptor and hormone will further alter the
transcription of a given gene (down or up regulation of synthesis of the proteins).

Formation of Hormone Receptor Complex Results in Signal Transduction and

Amplification
Hormone action through membrane receptors (typical of water soluble such as the following;
peptide, amino acid derived hormones):
Faster response, and short lasting effect
 ● Rapid physiological or biochemical response (within seconds)
● Change of the activity of preexisting enzymes in the cell by allosteric or covalent
modification
Hormone action through nuclear receptors (typical of lipid soluble such as the following;
steroid or thyroid hormones):
Slower response, and long lasting effect
● Promote maximal responses in target tissues only after hours or days. The delayed
response is due to the fact that the change of gene expression takes significantly more
time.
● Change of gene expression results in up or down regulation of the regulated protein(s)
causes physiological and biochemical changes

As illustrated in the illustration below:

The following second messengers generated in this case;
cAMP - cyclic adenosine monophosphate
The binding of a neurotransmitter/hormone would activate G stimulatory protein (Gs), which
further activates adenylyl cyclase. Adenylyl cyclase is able to convert ATP into cAMP (cyclic
adenosine monophosphate) by the formation of cyclic nucleotides (derived from ATP). The
formation of cAMP will in turn activate protein kinase A which can change certain protein
activity through phosphorylation. → opening or closing of pump/channel, activation of
intracellular proteins.
[Ex.] Excitatory pathway of cAMP is achieved through the bonding between the
neurotransmitter epinephrine and the Beta 1 receptor (compatible). This activity can be found
in the heart, as this secondary messenger pathway leads to a stronger muscle contraction.
[Ex.] Inhibitory pathway of cAMP is achieved through the bonding between the
neurotransmitter acetylcholine and the M2 receptor (compatible). Heart activity is suppressed
as protein kinase A remains inactive (unable to carry out its functions)
Classification of Hormones
Based on Chemical Structure

Peptide Hormones
● Varies from small peptides (3 AA) to long chain proteins (>200 AA)
● Short half life (minutes) because once in the blood, there are enzymes present which
can degrade them. Typically peptide hormones have a wide action range.
● Pancreatic hormones (insulin, glucagon and somatostatin), parathyroid hormone and
calcitonin, all hormones of hypothalamus and pituitary
● Synthesized on ribosomes in the form of prohormones. Peptide hormones can thus be
stored in secretory vesicles and released on demand. Proteolytic cleavage ensures the
ractication of peptides.
● In many hormones terminal residues are modified as in thyroid hormone, in order to
form these mide bonds and shed the charges from the C-N terminals.
 ● Some peptide prohormones can yield multiple products, which leads to a more diverse
effect on the body;
- Pro Opiomelanocortin (POMC) has at least eight different cleavage sites , it produces
at least 10 different peptide hormones including: endorphin, melanocyte stimulating
hormones and others; mutations in the POMC gene are associated with obesity

Insulin is a Peptide Hormone

● Synthesized on ribosome of beta cells as preproinsulin and processed into the 5.8 kDa
active form
● Signal sequence directs its passage into secretory vesicles in beta cells for storage
● Mature insulin and C peptide released in blood by exocytosis

Insulin production [Steps]

1. Translation and translocation - takes place in ribosomes with the amino acid
sequencing [Preproinsulin]
2. Folding, oxidation and signal peptide cleavage - takes place in the lumen of
endoplasmic reticulum, the signal sequence cleavage generates the inactive form of
insulin. Keep in mind that the given signal sequence directs proinsulin to the vesicles
it will eventually be stored in. [Proinsulin]
3. Endoplasmic reticulum, Golgi transport, Vesicle packaging - thanks to the signal
sequence, proinsulin is able to be stored in the pancreatic beta cells [Proinsulin]
4. Protease cleavage liberates C-peptide - when blood glucose levels increase, protease
cleavage of peptide bonds releases fragments in the form of C-peptide [Proinsulin]
5. Carboxypeptidase E produces mature insulin - mature insulin is able to leave the beta
cells via exocytosis to be released into the bloodstream [Insulin]

Peptide Hormone Insulin

● It is secreted in response to increased blood glucose levels
● It binds to receptors in muscle, brain, liver, adipose tissue, and other fuel metabolizing
tissues
 ● In muscle, insulin facilitates glucose uptake
● In the liver, insulin promotes glycogen synthesis
● In adipocytes , insulin promotes glycerol synthesis and inhibits breakdown of fats
OBS. Adipose tissue and muscle are insulin dependent tissues, meaning that insulin binding
to these tissues causes the GLUT4 transporter expression on the cell surface which ensures
that the blood glucose gets into the cell. However that does not exclude the insulin ability to
bind to other types of tissues such as the liver and brain. This further stimulates other
metabolic processes.

Physiological Effects of C Peptide

C-peptide; the fragments formed in response to the release of proinsulin
● Acts through G protein coupled receptors (GPCRs)
● Mitigate effects of reduced insulin synthesis (such as diabetic nerve pain)
● C peptide replacement therapy - maintains health and proper function. Ex. diabetic
patients lacking insulin.
● Degrades more slowly, which makes it a more reliable biomarker in field studies. Ex.
C-peptide is a good biomarker for insulin availability in the human body.

Biogenic amines
Derived from amino acids (amino acid derived hormones), this includes catecholamines
● Act as neurotransmitters and hormones
● Examples: dopamine, noradrenaline, adrenaline, serotonin, histamine, GABA
● Decarboxylation (removal of carboxyl group) needed for activation from amino acids
● Neurotransmitters have a very short half life because we do not want to overstimulate
the nervous system, so they are cleaved immediately.
● Inactivation usually involves deamination and methylation
 ● Vast range of functions

Amino Acid Derived Hormones Dopamine and Catecholamines

Catecholamines - adrenalin, noradrenaline etc.
● Derived from amino acid Tyrosine
● Act as neurotransmitters in the brain and hormones released in the blood (e.g. from
adrenal glands)

Dopamine - cannot cross blood brain barrier

● Substantia nigra coordination of body movements; lack of dopamine causes
Parkinson's disease. The reason for the lack of dopamine could either be due to the
damaged brain cells being unable to form dopamine or the over activity of enzymes
degrading dopamine too fast.
- In this case, L-dopa is introduced as it is able to cross the blood-brain barrier
and treat Parkinson's disease (as opposed to its counterpart dopamine). Thus
the still healthy brain cells can pick it up and form dopamine in higher
concentration.
● Other brain regions - motivation, reward, reinforcement, sleep, mood, attention etc.
● Clinically used for shock treatment dilates - renal arteries and increase cardiac output ,
because inhibits norepinephrine by acting as a competitor for the same receptors
● Outside the central nervous system, dopamine functions primarily as a local paracrine
messenger.
➔ In the kidneys, it increases sodium excretion and urine output
➔ In the pancreas, it reduces insulin production
➔ In the digestive system, it reduces gastrointestinal motility and protects
intestinal mucosa
➔ In the immune system, it reduces the activity of lymphocytes
Adrenergic Receptors
Epinephrine Primarily Acts as Hormone while Norepinephrine as Neurotransmitter
Norepinephrine:
The effects of norepinephrine is active during our day to day life
● The most common neurotransmitter of the sympathetic nervous system , mostly acts
on α-receptors
● Sympathetic ganglion - cells increase heart rate
● Other brain regions - sleep and wakefulness, attention and feeding behavior

Epinephrine:

Degradation of Dopamine and Catecholamines by MAO and COMT

Degradation catalyzed by:
These enzymes can act in different order, but the end-product will always remain the same
(Metanephrines and Vanillylmandelic acid)
● Monoamine oxidase (MAO) [deamination]
● Catecholamine O methyltransferase (COMT) [methyl group added]
● Aldehyde dehydrogenase (AD) [breakdown of catecholamines]
Products:
● Metanephrines
● Vanillylmandelic acid can be measured in the urine as indices of the function of the
adrenal medulla
Particularly increased in patients who have the tumor of the adrenal medulla known as
pheochromocytoma causing hypertension because of the vasoconstrictor action of the
catecholamines it produces. By monitoring the levels of these given end-products, we can
understand the synthesis and degradation process levels in the patient's body.

OBS. These degradative enzymes are important as we do not want to overstimulate the cells,
as over stimulation can lead to lower levels of given catecholamines (epinephrine,
norapenine) and dopamine.

Amino Acid Derived Hormones Melatonin

Serotonin is produced as an intermediate during the synthesis of melatonin
● Derived from L-Tryptophan
● Act as neurotransmitter in the brain and as hormone
Melatonin
● Regulates sleep wake cycle (causes drowsiness and ↓body T). The darkness our eyes
detect signals to our brain that the night is approaching and the secretion of melatonin
is initiated in preparation for a nice sleep.
- Melatonin synthesis is disrupted when you surround yourself with bright lights
(Ex. watching TV) which makes it harder for you to fall asleep.
● The use of melatonin (naturally synthesized in the pineal gland and immune cells )), is
a potential treatment option to reduce the severity of COVID 19 symptoms due to its
known anti-inflammatory, immunomodulatory, and protective antioxidant
mechanisms. In summary, Micelles synthesize melatonin as a hormones and once
released into the blood it has an anti-inflammatory effect.

Amino Acid Derived Hormones Serotonin

(Intermediate in Synthesis of Melatonin)
● 10% of the total serotonin is produced inside the brain as a neurotransmitter. The
released serotonin in this case will have various behavioral effects; emotions,
sexuality, addiction, stress response, appetite as well central nervous system effects
(motor control, body temperature etc.)
● 90% of the total serotonin is produced in our gut. Serotonin is released from Colonic
enterochromaffin cells (ECs). These cells are stimulated by our gut bacteria, the
released serotonin in this case ensures gut motility.
● Regulate mood, memory processing, sleep, cognition
● Released from platelets and serves as vasoconstrictor, regulates blood clothing
● Alongside the catecholamines, serotonin can be metabolized by MAO and AD
OBS. Serotonin levels are reduced in case of depression.

Amino Acid Derived Hormones Histamine

Produced through decarboxylation of histidine.
Histamine; neurotransmitter and hormone.
● Produced in mast cells , basophils, eosinophils, neurons of
tuberomammillary nucleus (small region - regulates attention and arousal)
● Induce the contraction of smooth muscle tissues of the lungs, uterus, and stomach; the
dilation of blood vessels, which increases permeability and lowers blood pressure; the
stimulation of gastric acid secretion in the stomach; and the acceleration of heart rate
● Generate allergic response
● Mediate attention, arousal
● Metabolized by methyltransferase, MAO and DAO

Glutamate and its Derivative GABA

● Glutamate is the most important excitatory neurotransmitter in the CNS
 ● GABA is the major inhibitory neurotransmitter in the brain.
GABA is created from glutamate by a decarboxylation reaction. Glutamine was used in this
case as it was converted into glutamate in both the glutamatergic neuron and the GABAergic
neuron.

Glutamatergic neuron
Glutamine → Glutamate → Glutamate in vesicles → Synaptic cleft → Astrocyte
GABAergic neuron
Glutamine → Glutamate → GABA → GABA in vesicles → Synaptic cleft → Astrocyte

In both cases, the end product inside the astrocyte will be glutamine. The entering glutamate
will convert into glutamine through glutamine synthetase. The entering GABA will enter the
citric acid cycle and after a series of reactions it will leave the cycle as glutamate, which in
turn can be converted into glutamine through glutamine synthetase. The regeneration of
glutamine in astrocytes will allow for yet another cycle to repeat.

Importance of Balance in Neurotransmitters

Blance between serotoin and norpaeninhine: satsifaction with achiemvent, normal sexual
repsosne, no overeating.
Blance between serotoin and dopamine: satsifaction with achiemvent, normal sexual
repsosne, no overeating.
Blance between dopamine and norpaeninhine: newly seeking motivations

Eicosanoid Hormones Are Not Synthesized in Advance

● Produced in virtually every cell
● Short half life (minutes)
● Produced when needed from arachidonic acid (incorporated into the phospholipids of
membranes), once the phospholipase A2 cleaves it off then this acid can be turned
into eicosanoids.
- In response to stimuli (hormone, etc.), phospholipase A2 is activated and
attacks the C 2 fatty acid, releasing arachidonic acid
● Paracrine hormones (act nearby)
● Play a role in inflammation, smooth muscle contraction, platelet function

Eicosanoids Are Potent Short Range Hormones Made from Arachidonic acid
Eicosanoids are formed by either of these two enzymes below;
COX-1 catalyzes synthesis of prostaglandins that regulate gastric mucin secretion , controls
important blood clotting pathways synthesis of thromboxanes
COX-2 catalyzes synthesis of prostaglandins that mediate pain, inflammation, and fever
● Nonsteroidal anti-inflammatory drug [NSAIDs]; aspirin, ibuprofen, acetaminophen
inhibit COX
➔ Aspirin (acetylsalicylate) is irreversible inhibitor
➔ Ibuprofen and naproxen are competitive inhibitors

The mentioned medications are unable to distinguish whether they are inhibiting COX-1 or
COX-2, because both of these corresponding isozymes are really similar and the affinity of
the drugs to them are similar. So once we take a drug to reduce any of the factors ensured by
the various Eicosanoids (Ex. Pain, Inflammation, fever etc.), correspondly there is an
inhibition of the enzyme which ensures the blood coagulation.

This is important to keep in mind as patients with inner bleeding should be cautious of taking
these drugs as to not facilitate further bleeding with the inhibition of the coagulation process.
Transport Forms of Hormones (Steroid and Thyroid) with Low Water Solubility
Majority bound to protein carriers in the blood:
● Albumin - non-specific
● Thyroxine binding globulin (thyroid hormones)
● Sex hormone binding globulin (steroid hormones)
● Corticosteroid binding globulin (ex. cortisol)
Only the free fraction is biologically active - usually less than 10%

Free fraction - when hormone is not bound to the protein carrier. Such binding can be
considered as a type of storage for these hormones as they are not stored inside a cell.

Steroid Hormones Are Made from Cholesterol

Steroid hormones; longer half-life and longer effect
Bind to carrier proteins to travel through the bloodstream
Enter cell nucleus; bind to nuclear receptor to alter gene expression
● Some may also bind to a plasma receptor
● More than 50 corticosteroid hormones produced in adrenal cortex
Three groups:
● Sex hormones; secondary sexual charactericts - Tesetrone, Estrogen
● Corticosterone; regulation of ion flow will correspondly influence the blood pressure -
Aldosterone
● Glucocorticoid; affects protein and carbohydrate metabolism as well as the immune
response - Cortisol
Endocrine disruptors - xenoestrogens
Xenoestrogens do not need to resemble the steroid structure to take action.
3 methods of disruption:
1. Bind to nuclear receptors and stimulate hormone-like effects - Normally this should
not be the case, in case there is no neuronal signal sent to initiate this binding.
2. Block receptors, preventing stimulation by endogenous hormones. - No change
achieved in the body.
3. Interfere with normal metabolism of steroid hormones in the liver. - Elimination of
steroid hormones as well as synthesis could be influenced in this case.
Daily exposure of xenoestrogens could distrubt endocrine function (Ex. in terms of sexual
characteristics etc.)
Vitamin D Hormone
● It is obtained from food or from photolysis of 7 dehydrocholesterol in sun exposed
skin
● Calcitriol (1 ,25 dihydroxycalcitrol) is the active form
● It affects the transcription of genes that regulate [Ca 2+2+] and the balance between
Ca 2+ deposition and removal from bone
● Activates the synthesis of an intestinal Ca 2+ binding protein essential for uptake of
dietary Ca2+
Vitamin D plays an important role in the regulation of calcium as it stimulates calcium
absorption from intestines as well as stimulates calcium deposition and removal from inside
bones. Vitamin D is synthesized from cholesterol in the skin, UV light is needed for this
process. The active form of Vitamin D (calcitriol) is formed in the final step in the kidneys.
With the formation of calcitriol, the calcium homeostasis can be ensured.

Vitamin D Hormone as Immunomodulator

● Modulates the phagocytic activity of macrophages and natural killer cells
● Induces the microbicidal activity of phagocytes - elimination of microbes are a lot
more efficient in that way
● Suppresses differentiation and maturation of antigen presenting dendritic cells and B
lymphocytes; helps in diseases where they are over-exposed which leads to them
attacking their own cells.
● Inhibits proliferation of lymphocytes Th1 and Th17 cells
Vitamin D deficiency and COVID-19
● Positive association between vitamin D sufficiency and improved COVID 19 disease
outcomes
● Deficiency associated with poor COVID 19 disease outcomes
● Vitamin D deficient patients were more likely to die from COVID 19
● Vitamin D deficient people were more likely to develop severe COVID 19 disease
● Vitamin D deficient people were more likely to be COVID 19 infected
● Findings from these studies suggest that vitamin D may serve as a mitigating effect
for COVID 19 infection, severity, and mortality
● The current evidence supports the recommendations for people to eat foods rich in
vitamin D such as fish, red meat, liver, and egg yolks
● The evidence also supports the provision of vitamin D supplements to individuals
with COVID 19 disease and those at risk of COVID 19 infection in order to boost
their immunity and improve health outcomes

Retinoid Hormones
● They derive from vitamin A1 (retinol), which derive from beta-carotene primarily in
liver
● All cells have at least one form of retinoid receptor
● The hormone receptor complex regulates genes governing cell growth and
differentiation
● They are most active in cells experiencing rapid growth (lung epithelia, skin, immune
system, cornea, etc.). As they regulate the gene governing cell growth and cell
differentiation.
● More caution during pregnancy as excessive vitamin A can cause birth defects
OBS. All-trans-retinoic acid acts as a hormonal signal, more needed for cells experiencing
rapid growth.

Thyroid Hormones (Amino acid Tyrosine derivatives)

Tyrosine derivatives bind to nuclear receptors as they are more fat soluble than water
soluble.
● T3 (triiodothyronine) has three iodines at Tyr residues
● T4 (thyroxine) has four iodines
● Precursor thyroglobulin → T4, which is converted to T3
● The receptor hormone complex increases expression of enzymes that yield energy
❖ ↑ basal metabolic rate; achieved through up-regulation of uncoupling
proteins synthesis - are interrupting the ECT and allowing free hydrogen from
the intermembrane space → generation of heat instead of energy
❖ ↑ protein synthesis
❖ Regulate long bone growth and neural maturation
❖ Overexpression of thyroid hormones leads to excessive sweating as well as
weight loss.
❖ Thyroid hormones facilitate the action of catecholamines through
Permissiveness; Permissiveness is the situation in which a hormone cannot
exert its full effects without the presence of another hormone.
● In case of iodine deficiency; Goiter is an enlargement of the thyroid gland that can
occur in a futile attempt to produce thyroid hormone in the absence of iodine;
although generally not uncomfortable, a large goiter can affect your appearance and
may interfere with swallowing or breathing.
Nitric Oxide Interacts with an Intracellular Receptor
● NO is a free radical made from arginine and O2 by NO synthase found in many cell
types (neurons, macrophages, hepatocytes, endothelial cells and others)
● It acts near its point of release
● It enters the target cell and binds to the intracellular receptor for the activation of
guanylyl cyclase to increase cGMP
❖ leads to activation of cGMP dependent protein kinase
❖ leads to relaxation of contractile proteins in smooth muscle of blood vessels
→ the dilation of blood vessels lowers the blood pressure

General action mechanisms

Top Down versus Bottom Up Hormonal Signaling
Top down
Signals sent from the brain to the tissues
● Some signals originate in the brain, and the signal is sent out to the body
● Examples: oxytocin, vasopressin, cortisol
Bottom up
Signals sent from the tissues to the brain
● Some signals originate from elsewhere in the body and send messages to the brain
● Examples: epinephrine (adrenaline), insulin, leptin

The Hypothalamus Is the Coordination Center of the Endocrine System

● The hypothalamus is a small region of the forebrain in animals with skulls
● It receives and integrates nerve signals from the central nervous system
● Hypothalamus synthesizes either the small peptide hormones (oxytocin and
vasopressin) or several factors that regulate the function of the anterior pituitary. This
initiates the cascade of the signals that amplify the first received signal from CSN.
Pituitary Released Hormones Target Other Glands
Alongside the hypothalamus, the pituitary is important as the second major key in the
regulation of the whole endocrine system.
The posterior pituitary
Neurohypophysis - contains the end of axons from the hypothalamus
● releases short peptide hormones made in the hypothalamus (vasopressin, oxytocin)
The anterior pituitary
Adenohypophysis - is the endocrine organ that receives releasing factors from the
hypothalamus via blood vessels
● further on stimulates and produces long peptide hormones called tropins
● tropins activates second targets: adrenal cortex, thyroid, ovaries/testes

Hormones and Target Tissues

Central nervous system receives ther of these two signals from its environment:
● Endogenous signals
Volitionally guided mechanism and can be referred to as a top-down process, in
which change within an organism is enforced intracellularly
● Exogenous signals
More reflexive and can be described as bottom-up process, in which exterior stimuli
in the external environment will bring change intracellular
CSN receives signal → Sent to hypothalamus → Depending on what product the
hypothalamus creates, it is either sent to →
a. Short peptides → The posterior pituitary
b. Releasing factors → The anterior pituitary → Secrete hormones which
correspondingly affect these given secondary target tissues; renal cortex, thyroid,
ovaries/testes → hormones produced to affect these given ultimate targets; many
tissues, muscles, liver, reproductive organs.
 ❖ In some cases, the secreted hormones of the anterior pituitary will go straight
to the ultimate targets; liver, bone, mammary glands.
OBS. Central nervous system can directly affect the adrenal medulla for the immediate effect
in accordance to the stress response during a fight or flight situation

Hypothalamic Cortisol Cascade

● Fear, infection, hypoglycemia, and other factors send electric signal to the
hypothalamus
➔ ~nanograms of corticotropin releasing hormone
➔ anterior pituitary releases ug corticotropin
➔ adrenal gland releases mg cortisol
● The cortisol end product feeds back and can inhibit each of the previous steps of the
cortisol synthesis pathway. The inhibition prevents the overproduction of cortisol.

Immune system
● Important factor in a healthy body when dealing with an “attack”.
● Suppression of pro-inflammatory cytokines is valuable in case of COVID-19
treatment as they are able to reduce inflammation in lungs caused by an overactive
immune system.
● Cortisol is used in case of organ transplantation in order to reduce the action of the
immune system to lower the risk of rejection of the newly introduced organ.
● During long term stress; more cortisol is produced and results in a lower activity of
the immune system. This makes it easier for you to get sick with the likes of the cold
or the flu. Over a long term basis, the accumulation of stress over months or years will
suppress the immune system to the point where there is a higher risk of developing
pathologies when your body is not equipped to fight back mutations such as in the
case of cancer formation.
Bottom Up Signaling Requires Tissue Specific Responses to Fuel
A lot of hormones are involved in order for the hypothalamus to know our feeding state.
● When food intake and energy production are adequate, then the peptide hormones
released by the stomach, intestine, and adipose tissue will give a feedback regulation
on the hypothalamus to signal satiety and reduce feeding behavior
● Other tissue specific peptide hormones signal inadequate supplies of stored TAGs or
low blood glucose levels
● All of these signals impinge, directly or indirectly, on AMP activated protein kinase
(AMPK) in the hypothalamus, which integrates these signals and influences feeding
behavior and energy yielding metabolism in the tissues

The hormones mentioned below are released with the presence of food in the intestines:
GIP - gastric inhibitory polypeptide
GLP-1- glucagon-like peptide 1

GIP - inhibits alpha-cells of pancreas in order to prevent the release of glucagon. Glucagon
increases the breakdown of stored glucose to generate energy. In the absence of glucagon,
body understands that we already have enough energy with the intestines full.
GLP-1 - increases the activity of beta-cells of pancreas in order to release insulin for the
uptake of glucose. In this case, the hypothalamus interprets this signal as the body having
plenty of energy and we can downregulate the feeding behavior.
Adipose tissue - sufficient enough of fats/triacylglycerol stored in the adipose tissue. leptin as
a signaling molecule, will also reduce the feeding behavior.
while adiponectin will signal to the hypothalamus that there is reduced fat amount in adipose
so it can stimulate the feeding behavior.

Activity during fed state

This system and its components promote satiety and reduce feeding behavior.
GLP-1 → Insulin; Fasillate action of beta-cells, plenty of energy to down regulate.
GIP → Glucagon; Inhibition of alpha-cells to prevent the secretion of glucagon.. This is done
as we do not want to use up the stored glucose, so inhibition of glucagon is necessary in this
case.