Fatty Acid synthesis and Cholesterol biosynthesis

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Lectures 45 and 46

Where does fatty acid biosynthesis occur?

brain, kidney, mammary glands, intestine, adipose tissue

Main site of synthesis

Liver

Problems associated with neonatal digestion

-Low pancreatic secretion of lipase
-Immature liver unable to produce bile

Overcome problems of neonatal digestion

- very active gastric lipase
- breast milk rich in MCFAs
- Breast milk also contains bile salt-stimulated lipase

Enzymes in De-novo Fatty acid synthesis

- ACCase (acetyl-CoA carboxylase)
- Fatty acid synthase (FAS)

End product of Fatty acid synthesis

Palmitate

Modifications of palmitate

Elongation
Desaturation
This takes place in the ER

**In mitochondria - Acyl oxidation, acetyl-CoA production, ketogenesis
**Cytosol - NADPH production, FA synthesis, isoprenoid synthesis

Where does fatty acid de novo synthesis take place?

Cytosol

Simplified version of fatty acid synthesis

1. Acetyl CoA» Palmitate (enz = ACCase and FAS)
2. Palmitate » Unsaturated fat (desaturase) »
LCFA, VLCFA (elongase)
Palmitate » LCFA, VLCFA (elongase)

How are acetyl CoA made in the mitochondria transported to the cytosol for FA synthesis?

Malate-citrate shuttle

Malate-citrate shuttle

1. Acetyl-CoA + OAA » Citrate (TCA) (Citrate synthase)
2. Citrate crosses mitochondrial membrane » cytosol
3. Citrate » acetyl CoA + OAA (ATP-Citrate lyase)

Citrate + CoASH + ATP » Acetyl-CoA + OAA + ADP + Pi

(ATP- Citrate lyase) Cytosolic reaction

Why can FAs not be converted to carbohydrate in animals?

*** Acetyl CoA cannot be converted to pyruvate/ OAA

What happens to th OAA released into the cytosol from the citrate lyase reaction?

1. OAA + NADH » Malate +NAD (malate dehydrogenase) --- Malate diffuses through membrane into mitochondria

Also in cytosol
2. Malate + NADP » Pyruvate (Malic enzyme)

CArboxylation of Acetyl-CoA by ACCase in cytosol

1. Acetyl-CoA » Malonyl-CoA ( ACCase)

**ACCase is the only enzyme involved in FA synthesis that is separate from the multifunctional enzyme FAS (Fatty acid synthase)

Components of ACCase

- Biotin carboxylase
- transcarboxylase
-Biotin-carboxyl carrier protein ( BCCP)

Human ACCase

- multifunctional protein
- exists in inactive form
-active when it forms a filamentous polymer

**Rate of FA synthesis dependent on the eqm of those 2 forms:

Protomer (inactive) »« Polymer (active)

ACCase reation

2 phases:

1. Carboxylation of biotin to form carboxybiotin
(Biotin carboxylase)

ATP + HCO₃⁻ + BCCP » CO₂---BCCP + ADP + Pi


2. Transcarboxylation of biotin
(Transcarboxylase)

CO₂---BCCP + acetyl-CoA » malonyl-CoA + BCCP

Overall ACCase reaction

ACetyl Co-A (2C) + ATP + CO₂ » Malonyl-CoA (3C) + ADP + Pi

FAS (Fatty acid synthase)

** located in cytoplasm
**Several sequential reactions

Simplified FAS reaction

1. ACetyl CoA (2C) »Malonyl CoA (3C) (ACCase)
2. Malonyl CoA (3C) » Palmitic Acid (C16:0) (FAS using ACP intermediates)
3. Palmitic acid » Palmitoyl-CoA (thioester bond formation) *** increases the solubility.

Characteristics of FAS enzyme

- phosphopantetheine binding domain (ACP - acyl carrier protein)
- 2 thiol grps must be loaded onto acyl groups before condensation
- acyl grp from acyl-CoA initially transferred to ACP
- acyl grp then transferred from ACP to ketoacyl-ACP synthase
- malonyl grp from malonyl-CoA transferred to thiol grp of ACP
***ACP arm is flexible - move substrate to active site.
*** FAS has several active sites - 2 FAS work in pairs.

Preliminary reactions of FA synthesis

1. Acetyl-CoA + ACP » Acetyl-ACP + HS-CoA
2. Malonyl-CoA + ACP » Malonyl-ACP + HS-CoA

Condensation reactions of FA synthesis

acetyl synthase (2C) + malonyl-ACP(3C) » acetoacetyl-ACP(4C) + synthase-SH + CO₂(1C)

* enzyme - β-ketoacyl-ACP synthase.

1st Reduction reaction of FA synthesis

acetoacetyl-ACP + NADPH + H⁺ «» βhydroxybutyryl-ACP + NADP⁺

**Enz- βketoacyl-ACP reductase

Dehydration reaction

βhydroxybutyryl-ACP »« crotonyl-ACP + H₂O

enz- βketoacyl-ACP dehydratase

2nd reduction reaction

crotonyl-ACP + NADPH + H⁺ »« butyryl-ACP (4C) + NADP⁺

enz- βenoyl-ACP reductase

Fate of butyryl-ACP

-enters 2nd round of FA synthesis similar to acetyl-ACP


*** Final product is PALMITOYL-ACP.
-palmitoyl-ACP » palmitic acid (thioesterase)

(All C's in palmitic acid are derived from malonyl-CoA except 2C's at methyl end (from original acetyl CoA molecule)

Effect of malonyl Co-A on CPT I

** inhibitory effect on CPT I
- prevents acetyl-CoA from β-oxidation

**Glucagon activates CPT I (liver)

Effect of insulin on FA synthesis

Insulin »» Acetyl CoA » Malonyl CoA » Pamitate

*Malonyl-CoA »»» inhibit CPT I»»»» no acetyl CoA goes into mitochondria»»» stays in cytosol for FA synthesis instead of oxidation

Elongation reactions of FA.

2 pathways:

1. ER (using malonyl-CoA)
2. Mitochondrial (uses acetyl-CoA)

Desaturation of FA reactions

-Aerobic
-2 H⁺ removed and H₂O produced
- NADPH used

**Acyl chain must be 16 or 18 C's before desaturation occurs.

Why can mammals not synthesize linoleic and α-linoleic acid?

- mammals cannot introduce Δ¹⁵ and Δ¹² double bonds.
-linoleate (C18:2) and α-linoleic (18:3) have this.

*** mammals can only synthesize up to Δ⁹ double bonds btwn the COOH and Δ⁹ of CH of chain.

**possible to desaturate oleate at Δ⁶ forming -
C18:2Δ⁶,⁹ FA.

Specific process : FA desaturation

Steroyl-CoA (C18:0) »»»oleoyl-CoA (C18:1 Δ⁹) + 2H₂O

*Aerobic desaturation
*microsomal enzymes
*Δ⁹ desaturation

Functions of essential FA's

- membrane structure
- specific enzyme-protein interaction in membranes
- synth eicosanoids
-syn arachidonic acid (C20:4)
-synth docosahexanoic acid (DHA C22:6)

PUFA

polyunsaturated FA
- not all are converted to eicosanoids bc of limiting activites of some enzymes (elongases and Δ⁵ and Δ⁶ desaturases)
- precursors for resolvins, docosatrienes, neuroprtectins (anti-inflammatory properties)
***** Humans rely on exogenous source of EPA and DHA in diet.

DHA

- key PUFA in brain tissue
-imp for brain and Nervous tissue development.

EPA and DHA

- not regarded as EFAs although they have to be taken in exogenously

What is linoleic acid used to make?

- C18: 2n-6 (linoleic acid) » C18:3n-6 (γ linoleic acid) » C20:3n-6 » C20:3n-6» C20:4n-6 (Arachidonic acid) » C22: 5n-6

What is α-linolenic acid used to make?

-C18:3n-3 (α-linolenic acid) » C18:4n-3 » C20: 4n-3 » C20: 5n-3 (Eicosapentanoic acid, EPA) » C22: 6n-3 (Docosahexanoic acid, DHA)

When there is an EFA deficiency what happens?

Stearic acid is used to make Mead acid, causes dermatosis

Regulation of FA synthesis

-ACCase is key regulatory enzyme.
** strict regulation
1. Short term Rapid Response
2. Long term response

Short term rapid response FA regulation

ACCase
-allosterically (stimulated) regulated by citrate
-inhibited by palmitoyl-CoA
-regulated by phosphorylation/dephosphorylation
(insulin, epinephrine, norepinephrine, glucagon)
-Activated by insulin in dephosphorylated form,
-Deactivated by glucagon and epinephrine in phosphorylated form.

Long term Response

- Increased expression of ACCase and FAS (at molecular level to maintain high carb diet)

TAG synthesis - Kennedy pathway

- takes place in the liver
- exported to peripheral tissue in blood
- Glycerol-3-Phosphate is immediate precursor for TAG synthesis.
** Glycerol-3-P derived directly from LPLase or indirectly from glycolysis.
- acyl-CoA transferred from cytosolic acyl-CoA to SER
-(TAG molecule is assembled here)

Formation of phosphatidic acid

-2 sequential acylations
1. Glycerol-3-P acyl transferase,
Glycerol-3-P + Acyl-CoA»» lysophoshatidic acid
2. Acylglycerol-3-P acyltransferase
Lysophosphatidic acid+ Acyl-CoA»» Phosphatidic acid

3. Phosphate removed by phosphatase
Phosphatidic acid»» Diacylglycerol + Pi
4. DAG acyl transferase
Diacylglycerol + Acyl-CoA»» Triacylglycerol

Specific to TAG biosynthesis

*** Acylation at the sn-3 position of DAG (DAG acyl transferase)

Important Sites of TAG synthesis

-SI (MAG pathway)
-Liver ( Kennedy pathway)
-Adipose tissue (Kennedy)
-Mammary gland (milk fat lactation) (Kennedy pathway)

** small amount of turnover in myocytes.
-TAG synthesis in enterocytes is primarily controlled by the rate of influx of exogenous dietery lipids.
-TAG synthesis in adipose tissue governed by the supply of glucose-derived glycerol-3-P.

MAG pathway in enterocytes

2 MAG + acyl-CoA »» DAG + Acyl-CoA »» TAG

** Enzyme = acyltransferase

Important points to note about FA synthesis

1. De novo FA synthesis from glucose (ACCase and FAS) --- not very active in adipocytes due to Western diet
2. TAG synthesis utilizing glycerol-3-phosphate
3. TAG mobilization by HSL
4. FA uptake from TAG-rich blood lipoproteins following LPLase activity.

Cholesterol facts

- most common steroid in animal cells
- absent from plant tissue and vegetable oils
- OH makes it amphipatic
- cholesteryl esters are hydrophobic
- vital in cell membranes
- precursor to steroid hormones
-cannot be degraded to CO₂
- liver converts it to bile acids

Plant sterols

- plants synthesize sterols: stigmasterol, campesterol, β-sistosterol
- not readily absorbed by human intestines
- inhibitory effect on cholesterol absorption
- use in cholesterol lowering drugs

Main site of cholesterol synthesis

Liver

Other sources of cholesterol

- dietary cholesterol
- Import of cholesterol from blood

Difference btwn testosterone and estrogen

- Testosterone has an =O grp while estrogen has an OH grp.

Cholesterol synthesis simplified

*** in cytosol
-------acetyl-CoA + acetoacetl-CoA » HMG-CoA

1. HMGCoA » Mevalonate (HMG-CoA reductase)
2. Mevalonate » C5 Isopentyl PPi
3. C5 Isopentyl PPi » C10 Geranyl PPi
4.C10 Geranyl PPi » C15 Farnesyl PPi
5. C15 Farnesyl PPi » Squalene
6Squalene » Lanosterol » Cholesterol

** Farnesyl is precursor for
- CoQ (Ubiquinone)
- Dolichol (glycoprotein synthesis)

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