16 - Biosynthesis of Cholesterol

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Biosynthesis of Cholesterol

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Origin of Carbon Atoms in Cholesterol

Tracer Experiment Evidence

The origin of carbon atoms in cholesterol can be deduced from tracer experiments using acetate labeled in:

• The methyl carbon (C-1 of acetate)
• The carboxyl carbon (C-2 of acetate)

The individual rings in the fused-ring system are designated A through D.

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Summary of Cholesterol Biosynthesis

Overall Pathway (Carbon Count)

$\text{Acetyl-CoA}\to \text{Mevalonate}\to \text{Isopentenyl diphosphate}\to \text{Squalene}\to \text{Cholesterol}$
$2\text{C}\to 6\text{C}\to 5\text{C}\stackrel{\times 6}{\to }30\text{C}\to 27\text{C}$

Info

Isoprene units in squalene are set off by red dashed lines in the original figure.

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Stage 1 — Formation of Mevalonate from Acetyl-CoA

Reactions

1. Thiolase condenses 2 molecules of acetyl-CoA → acetoacetyl-CoA (releases CoA-SH)
2. HMG-CoA synthase condenses acetoacetyl-CoA + acetyl-CoA → HMG-CoA (β-hydroxy-β-methylglutaryl-CoA) (releases CoA-SH)
3. HMG-CoA reductase reduces HMG-CoA → mevalonate (uses $2\text{NADPH}$, releases CoA-SH)

Rate-Limiting Step

The conversion of HMG-CoA to mevalonate by HMG-CoA reductase is the committed, rate-limiting step of cholesterol biosynthesis.

Cytosolic vs. Mitochondrial HMG-CoA

• In sterol biosynthesis, HMG-CoA is formed by a cytosolic HMG-CoA synthase.
• In ketone body synthesis, HMG-CoA is formed in the mitochondrial matrix.
• HMG-CoA lyase (for ketolysis) is present only in the mitochondrial matrix.

CC(=O)SCC(=O)SCC(=O)S

(Simplified; acetyl-CoA thioester moiety)

Origin of Mevalonate Carbons

C-1 and C-2 of mevalonate originate from acetyl-CoA.

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The rate-limiting enzyme of cholesterol biosynthesis is {1:HMG-CoA reductase}, which converts {2:HMG-CoA} to {3:mevalonate} using {4:2 NADPH}.

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HMG-CoA synthase involved in cholesterol biosynthesis is located in the {1:cytosol}, whereas the one involved in ketone body synthesis is in the {2:mitochondrial matrix}.

What are the three enzymes in the conversion of acetyl-CoA to mevalonate?

1. Thiolase (2 acetyl-CoA → acetoacetyl-CoA)
2. HMG-CoA synthase (acetoacetyl-CoA + acetyl-CoA → HMG-CoA)
3. HMG-CoA reductase (HMG-CoA → mevalonate; rate-limiting)

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Relationship with Ketone Body Synthesis

Shared Intermediate

HMG-CoA is a shared intermediate between cholesterol biosynthesis (cytosolic) and ketone body synthesis (mitochondrial). These pathways are spatially separated by subcellular compartmentation.

Ketone body formation from HMG-CoA (mitochondrial):

$\text{HMG-CoA}\stackrel{\text{HMG-CoA lyase}}{\to }\text{Acetoacetate}+\text{Acetyl-CoA}$

Acetoacetate is then converted to:

• Acetone (via acetoacetate decarboxylase)
• D-β-Hydroxybutyrate (via β-hydroxybutyrate dehydrogenase, using NADH)

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When acetyl-CoA accumulates (e.g., in {1:starvation} or {2:untreated diabetes}), thiolase condenses two acetyl-CoA molecules to form {3:acetoacetyl-CoA}, the parent compound of ketone bodies.

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Regulation of HMG-CoA Reductase

Hormonal Regulation

• Insulin → promotes dephosphorylation → activates HMG-CoA reductase
• Glucagon → promotes phosphorylation → inactivates HMG-CoA reductase
• Thyroxin → stimulates HMG-CoA reductase activity

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Insulin {1:activates} HMG-CoA reductase by promoting {2:dephosphorylation}, while glucagon {3:inactivates} it by promoting {4:phosphorylation}.

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Statins — Inhibitors of HMG-CoA Reductase

Mechanism

Statins are structural analogues of mevalonate that competitively inhibit HMG-CoA reductase, thus blocking cholesterol synthesis.

Statin	Generic Name	Trade Name
Compactin	$R_1=H$, $R_2=H$	—
Simvastatin	$R_1=C{H}_3$, $R_2=C{H}_3$	Zocor
Pravastatin	$R_1=H$, $R_2=OH$	Pravachol
Lovastatin	$R_1=H$, $R_2=C{H}_3$	Mevacor

What is the mechanism of action of statins?

Statins are structural analogues of mevalonate that competitively inhibit HMG-CoA reductase, thereby blocking the committed step of cholesterol biosynthesis.

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Stage 2 — Conversion of Mevalonate to Activated Isoprene Units

Reactions (3 ATP consumed)

$\text{Mevalonate}\stackrel{\text{mevalonate-6-phosphotransferase}}{\to }\text{5-Phosphomevalonate}$

$\text{5-Phosphomevalonate}\stackrel{\text{phosphomevalonate kinase}}{\to }\text{5-Pyrophosphomevalonate}$

$\text{5-Pyrophosphomevalonate}\stackrel{\text{pyrophosphomevalonate decarboxylase}}{\to }{\Delta }^3\text{-Isopentenyl pyrophosphate (IPP)}$

Decarboxylation Step

The C-3 phosphate and the adjacent carboxyl group are eliminated together, producing a double bond in the five-carbon product: isopentenyl pyrophosphate (IPP) (${\text{C}}_5$).

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Conversion of mevalonate to isopentenyl diphosphate requires {1:3} ATP molecules and produces {2:CO₂} and {3:inorganic phosphate} as byproducts in the final decarboxylation step.

Isomerization

${\Delta }^3\text{-Isopentenyl pyrophosphate}\rightleftharpoons \text{Dimethylallyl pyrophosphate (DMAPP)}$

Info

This isomerization yields a second activated isoprene unit: dimethylallyl pyrophosphate (DMAPP).

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The two activated isoprene units are {1:isopentenyl diphosphate (IPP)} and {2:dimethylallyl diphosphate (DMAPP)}, both containing {3:5} carbons.

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Stage 3 — Condensation of Six Isoprene Units to Form Squalene

Isoprenoids/Terpenes

Isoprenoids (terpenes) are hydrocarbon-containing biomolecules (and secondary metabolites) made up of multiples of the isoprene unit (${\text{C}}_5$).

Stepwise condensation (head-to-tail, then head-to-head)

Step	Reaction	Product	Carbons
1	DMAPP (${\text{C}}_5$) + IPP (${\text{C}}_5$) → head-to-tail	Geranyl pyrophosphate (GPP)	${\text{C}}_{10}$
2	GPP (${\text{C}}_{10}$) + IPP (${\text{C}}_5$) → head-to-tail	Farnesyl pyrophosphate (FPP)	${\text{C}}_{15}$
3	2× FPP (${\text{C}}_{15}$) → head-to-head	Squalene	${\text{C}}_{30}$

Head vs. Tail

The “head” is the end to which pyrophosphate is joined. Pyrophosphate groups are displaced in each condensation.

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Two molecules of {1:farnesyl pyrophosphate} join {2:head-to-head} with elimination of both pyrophosphate groups to form {3:squalene} ($C_{30}$).

What are the three intermediates in the synthesis of squalene from activated isoprene units?

1. Geranyl pyrophosphate (C₁₀) — head-to-tail of DMAPP + IPP
2. Farnesyl pyrophosphate (C₁₅) — head-to-tail of GPP + IPP
3. Squalene (C₃₀) — head-to-head of 2× FPP

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Overview of Isoprenoid Derivatives

Info

The isoprene unit (${\text{C}}_5$) is the building block of a wide range of biological molecules:

Derivative	Example
Bile acids	Taurocholic acid
Steroid hormones	Cortisol, testosterone
Vitamin D	Cholecalciferol
Electron carriers	Ubiquinone, plastoquinone
Plant hormones	Gibberellic acid
Fat-soluble vitamins	Vitamin A, K

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Stage 4 — Ring Closure: Squalene → Cholesterol

Squalene Epoxidation

$\text{Squalene}+O_2+\text{NADPH}\stackrel{\text{mixed-function oxidase (monooxygenase)}}{\to }\text{Squalene 2,3-epoxide}$

Epoxide

An epoxide is a cyclic ether forming a three-membered ring: two carbons and one oxygen.

Cyclization

$\text{Squalene 2,3-epoxide}\stackrel{\text{cyclase}}{\to }\text{Lanosterol}\to \cdots \to {\text{Cholesterol (C}}_{27}\text{)}$

Species Variation

• Animals → Cholesterol
• Plants → Stigmasterol
• Fungi → Ergosterol

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Ring closure of squalene begins with epoxidation by a {1:mixed-function oxidase (monooxygenase)}, requiring {2:NADPH} as cosubstrate, yielding {3:squalene 2,3-epoxide}, which is then cyclized to form the steroid nucleus.

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In animals, ring closure of squalene gives {1:cholesterol}; in plants, {2:stigmasterol}; in fungi, {3:ergosterol}.

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Cholesterol Export from the Liver

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Most cholesterol synthesis in vertebrates occurs in the liver. A small fraction is incorporated into hepatocyte membranes; most is exported in three forms:

1. Biliary cholesterol (free)
2. Bile acids (e.g., taurocholic acid)
3. Cholesteryl esters

Cholesteryl Ester Formation (ACAT)

$\text{Cholesterol}+\text{Fatty acyl-CoA}\stackrel{\text{ACAT}}{\to }\text{Cholesteryl ester}+\text{CoA}$

ACAT

Acyl-CoA–cholesterol acyl transferase (ACAT) transfers a fatty acid from CoA to the hydroxyl group of cholesterol, making it more hydrophobic.
Cholesteryl esters are then:

• Transported in lipoprotein particles to other tissues, or
• Stored in the liver

What is ACAT and what does it do?

ACAT (Acyl-CoA–cholesterol acyl transferase) transfers a fatty acid from coenzyme A to the hydroxyl group of cholesterol, forming a cholesteryl ester — a more hydrophobic storage/transport form of cholesterol.

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Steroid Hormone Synthesis from Cholesterol

Site of Production

Organs such as the adrenal gland and gonads use cholesterol as a precursor for steroid hormone production.

Side-Chain Cleavage (First Step)

$\text{Cholesterol}\stackrel{\text{mixed-function oxidase (Cyt P-450)}}{\to }\text{20,22-Dihydroxycholesterol}\stackrel{\text{desmolase}}{\to }\text{Pregnenolone}+\text{Isocaproaldehyde}$

Info

• Cytochrome P-450 acts as electron carrier in this mixed-function oxidase system
• Requires adrenodoxin and adrenodoxin reductase as electron-transferring proteins
• Located in mitochondria of the adrenal cortex
• Pregnenolone is the precursor of all steroid hormones

Major Steroid Hormones and Functions

Hormone	Class	Primary Function
Progesterone	Progestogen	Female reproductive cycle
Testosterone	Androgen	Male secondary sexual characteristics
Estradiol	Estrogen	Female secondary sexual characteristics; reproductive cycle
Cortisol	Glucocorticoid	Protein & carbohydrate metabolism; suppresses immune response, inflammation, allergy
Aldosterone	Mineralocorticoid	Renal reabsorption of ${\text{Na}}^{+}$, ${\text{Cl}}^{-}$, ${\text{HCO}}_3^{-}$
Prednisolone / Prednisone	Synthetic glucocorticoid	Anti-inflammatory

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Pregnenolone is formed from cholesterol by {1:cytochrome P-450}-dependent side-chain cleavage in the {2:mitochondria of the adrenal cortex}, and is the precursor of {3:all steroid hormones}.

Distinguish glucocorticoids from mineralocorticoids.

• Glucocorticoids (e.g., cortisol): regulate protein and carbohydrate metabolism; suppress immune response, inflammation, and allergic responses.
• Mineralocorticoids (e.g., aldosterone): regulate reabsorption of Na⁺, Cl⁻, HCO₃⁻ in the kidney.

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Vitamin D Synthesis from Cholesterol

Pathway

$\text{Cholesterol}\to \text{7-Dehydrocholesterol}\stackrel{\text{2 steps (UV, skin)}}{\to }{\text{Cholecalciferol (Vitamin D}}_3\text{)}\stackrel{\text{liver}}{\to }\stackrel{\text{kidney}}{\to }{\text{1,25-Dihydroxycholecalciferol (active Vit D}}_3\text{)}$

Step	Location	Reaction
1	Skin (UV light)	7-Dehydrocholesterol → Cholecalciferol (Vit D₃)
2	Liver	First hydroxylation (C-25)
3	Kidney	Second hydroxylation (C-1) → active $1,25(\text{OH}{)}_2{\text{D}}_3$

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Active vitamin D (1,25-dihydroxycholecalciferol) is produced from 7-dehydrocholesterol through: UV activation in the {1:skin}, then hydroxylation in the {2:liver}, then final hydroxylation in the {3:kidney}.

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Regulation of Cholesterol Biosynthesis

Enzyme-Level Regulation of HMG-CoA Reductase

Multiple Regulatory Mechanisms

1. Phosphorylation/dephosphorylation (short-term):
   • Insulin → dephosphorylation → active
   • Glucagon → phosphorylation → inactive
2. Proteolysis (long-term):
   • Glucagon stimulates proteolysis of HMG-CoA reductase
   • Unidentified cholesterol metabolites (X) also stimulate proteolysis
3. Feedback by cholesterol:
   • Intracellular cholesterol ↑ → suppresses HMG-CoA reductase activity
   • Excess cholesterol → esterified by ACAT → stored as cholesteryl esters
4. LDL receptor-mediated endocytosis:
   • Extracellular LDL-cholesterol taken up → raises intracellular cholesterol → inhibits synthesis

SREBP Pathway (Transcriptional Regulation)

Sterol Regulatory Element-Binding Proteins (SREBPs)

• SREBPs are embedded in the ER when first synthesized, in a complex with SCAP (SREBP cleavage-activating protein).
• When bound to SCAP, SREBPs are inactive.
• When sterol levels decline, the SREBP–SCAP complex migrates to the Golgi complex.
• SREBP is cleaved by two proteases in succession in the Golgi.
• The liberated amino-terminal domain migrates to the nucleus, where it activates transcription of sterol-regulated genes (e.g., HMG-CoA reductase gene).

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When sterol levels are low, the SREBP–SCAP complex migrates from the {1:ER} to the {2:Golgi}, where SREBP is cleaved by {3:two proteases}. The released N-terminal domain travels to the {4:nucleus} to activate transcription of sterol-regulated genes.

Summarise the four levels at which HMG-CoA reductase activity is regulated.

1. Phosphorylation: glucagon inactivates (phosphorylates); insulin activates (dephosphorylates)
2. Proteolysis: glucagon and cholesterol metabolites stimulate degradation
3. Feedback inhibition: intracellular cholesterol suppresses enzyme activity
4. Transcriptional regulation via SREBPs: low sterol → SCAP escorts SREBP to Golgi → cleavage → N-terminal domain activates HMG-CoA reductase gene

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Mnemonic

Mnemonic — Stages of Cholesterol Synthesis

“Acetyl Makes Isoprene; Squalene Converts”

• Acetyl-CoA → Mevalonate → Isopentenyl diphosphate → Squalene → Cholesterol

“Hungry Men Sell Pharmaceutical Drugs” — HMG-CoA reductase inhibitors:

• HMG-CoA reductase → blocked by statins
• Mevalonate — cannot be formed
• Statins: Simvastatin, Pravastatin, Lovastatin (all competitive analogues of mevalonate)

Mnemonic — Steroid Hormones from Pregnenolone

“Pregnant People Get Tall and Exotic”

• Pregnenolone → Progesterone → Glucocorticoids (cortisol) → Testosterone → Androgens → Estrogens

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TLDR

Cholesterol Biosynthesis — Summary

• All 27 carbons of cholesterol derive from acetyl-CoA (traced by isotope labeling).
• The pathway has 4 main stages:
  1. Acetyl-CoA → Mevalonate (3 enzymes; HMG-CoA reductase is rate-limiting; cytosolic)
  2. Mevalonate → Isopentenyl diphosphate (IPP, C₅) (3 ATP used; decarboxylation)
  3. IPP + DMAPP → Squalene (C₃₀) (head-to-tail condensations via GPP C₁₀ and FPP C₁₅; final head-to-head fusion)
  4. Squalene → Cholesterol (epoxidation by monooxygenase + NADPH; then cyclization)
• HMG-CoA is a shared intermediate with ketone body synthesis but they are compartmentally separated (cytosol vs. mitochondria).
• Statins competitively inhibit HMG-CoA reductase (simvastatin, pravastatin, lovastatin).
• Liver is the primary site of cholesterol synthesis; exports it as biliary cholesterol, bile acids, or cholesteryl esters (formed by ACAT).
• Cholesterol is the precursor of: steroid hormones (via pregnenolone), vitamin D (via 7-dehydrocholesterol), and bile acids.
• Steroid hormone classes: glucocorticoids (cortisol — metabolism/immunity), mineralocorticoids (aldosterone — renal ion reabsorption), androgens (testosterone), estrogens (estradiol).
• Vitamin D activation: skin (UV) → liver (C-25 hydroxylation) → kidney (C-1 hydroxylation) → active $1,25(\text{OH}{)}_2{\text{D}}_3$.
• Regulation of HMG-CoA reductase occurs at 4 levels:
  1. Covalent modification (insulin activates; glucagon inactivates)
  2. Proteolysis (glucagon + cholesterol metabolites stimulate)
  3. Intracellular cholesterol feedback
  4. Transcriptional regulation via SREBP (low sterol → ER→Golgi → protease cleavage → N-term enters nucleus → activates HMG-CoA reductase gene)