Cholesterol

Question 1

What are the saponifiable biological molecules?

Answer 1

FAs, Sphingolipids, glycerophospholipids, TGs

Question 2

Is cholesterol essential?

Answer 2

No, humans acquire it from diets and synthesize around 1000 mg/day

Question 3

How does cholesterol vary in membranes and what is its function?

Answer 3

1- Some membrane have a high ratio of cholesterol like RBC PM
2- Some membranes are devoid of cholesterol such as IMM
3- Some have moderate cholesterol levels
Function: maintain fluidity and permeability

Question 4

Cholesterol is the precursor for —?

Answer 4

Bile Acids (how .they’re excreted), Steroid hormones, and vitamin D

Question 5

How is it transported in the body and why?

Answer 5

It is hydrophobic so it is transported via Lipid Protein Complexes like CM, LDL, HDL, VLDL

Question 6

Dietary exogenous cholesterol is carried by —-. None of it is distributed to the —-, all goes to —-.

Answer 6

CM, tissues, liver

Question 7

Cholesterol exists in 2 forms:

Answer 7

free and esterified (60%).

Cholesterol esters are the storage forms.

Question 8

What happens to exogenous cholesterol in the liver?

Answer 8

The liver will combine it with endogenous cholesterol and secrete it in the form of VLDL, which can be metabolized inside the circulation into IDL, then LDL, which distributes cholesterol to extra-hepatic tissue. Any excess cholesterol will go back to the Liver.

Question 9

What type of apoprotein does LDL have?
What happens if there is a mutation in them?
What effect on the heart does this have?

Answer 9

LDL is characterized by apo B-100. Receptors of apo B-100 facilitate the entry of cholesterol into cells. Deficiency or mutations in these receptors impair their interaction with apo B-100. => cholesterol can’t enter the cells => cholesterol remains in circulation leading to Hypercholesterolemia.
Hypercholesterolemia is associated with increasing risk of heart problems: atherosclerosis, myocardial infarction

Question 10

LDL carries —- cholesterol mainly, while HDL carries —

Answer 10

free, cholesterol esters

Question 11

Cholesterol structure? Number of carbons, number of double bonds, number of rings, Functional groups? Conformation?

Answer 11

Cholesterol is C-27 molecule, made of 4 fused rings A, B, C and D, and a long hydrophobic saturated side chain. One (-OH) group is attached to C-3, in the beta orientation (above plane), and a double bond is present in ring B.
The cholesterol structure is not planar, each ring occurs has a chair conformation.

Question 12

What are steroids? What are sterols?

Answer 12

Steroids are lipid molecules (hydrophobic), with 4 fused carbon rings.
Many Steroids have at least one (-OH) group, not necessarily attached to the fused ring structure, so we refer to them as Sterols (cholesterol, cortisol, etc.)

Question 13

Cholesterol Biosynthesis Site in organs.

Site in cells?

Answer 13

Occurs in all nucleated cells but mainly active in liver, intestines, and kidneys. Other tissues get their need of cholesterol from the LDL and synthesize more if needed.
In cells, it occurs in involves cytosolic and microsomal fractions.

Question 14

What is a microsome?

Answer 14

It is a heterogenous vesicle made up of fragmented ER with ribosomes attached

Question 15

What is required for the synthesis of cholesterol?

Answer 15

18 Acetyl-coA, 36 ATP, and 16 NADPH

Question 16

Bloch Experiment

Answer 16

o Bloch demonstrated that when rats are injected with radioactive acetate, the label appeared in cholesterol. It also appeared in Squalene, a long (C30) hydrocarbon, that is polymer of isoprene, a (C5) branched molecule, whose structure is shown above.
- When labeled Squalene was injected into rats, cholesterol was also found to be labeled.
=> What we know from this, regarding cholesterol biosynthesis pathway is: acetate → X → isoprene → squalene → cholesterol. how a linear 2C molecule (acetate) gives rise to a branched molecule (isoprene)?

Question 17

How did scientists identify compound X in Bloch Experiment?

Answer 17

They did studies on Bacteria, and they found that bacteria growing on acetate were able to grow by substituting acetate with a 6-C acid growth factor: Mevalonic acid (MVA).
- Decarboxylation of MVA generates isoprene, which polymerizes to form squalene then cholesterol .
=> Cholesterol Biosynthesis Pathway is known as Mevalonate pathway, since MVA is an important intermediate in it.

Question 18

What is the first reaction in cholesterol biosynthesis? Where does it occur in the cell? Under what hormone effect?

Answer 18

o The 1st rxn in cholesterol biosynthesis is the Branching rxn, in which 3 acetyl CoA molecules condense to form: β-hydroxyl β-methyl glutaryl CoA (HMGCoA).
- This rxn occurs in the cytoplasm under insulin effect.

Question 19

The first step in the cholesterol biosynthesis is similar to the 1st step in —- which occurs in the — under the effect of —.

Answer 19

Ketogenesis, live mitochondrial matrix, glucagon

Question 20

What is the RDS in the cholesterol biosynthesis pathway? What enzyme is involved? What happens?

Answer 20

The 2nd step after the formation of the HMGCoA is then reduced at the thioester end to a 1⁰ alcohol, yielding Mevalonate. This rxn is catalyzed HMGCoA reductase enzyme which requires 2 NADPH. This rxn is IRREVERSIBLE + RDS => It is important in cholesterol homeostasis, where drugs are developed to target the reductase to treat hypercholesterolemia.

Question 21

What intermediate is common between ketone body synthesis and cholesterol synthesis pathway?

Answer 21

HMG-coA

Question 22

What inhibits the RDS? What activates it?

Answer 22

AMP and Glucagon inhibit, Insulin activates.

Question 23

By what transcription factor is the the HMG-coA synthesis triggered?

Answer 23

SREBP

Question 24

Drugs treating hypercholesterolemia will inhibit what enzyme and what are they called?

Answer 24

They inhibit HMG-coA reductase enzyme and they are called statins.

Question 25

Biosynthesis pathway, steps, enzymes

Answer 25

note on paper

Question 26

How are the enzymes changing squalene into cholesterol organized?

Answer 26

loose-multienzyme complexes not totally free

Question 27

What is the first non-phosphorylated compound in the pathway? What is the first steroid in the pathway?

Answer 27

Squalene, lanosterol

Question 28

All carbons of cholesterol were originally derived from?

Answer 28

Acetyl-coA

Question 29

Regulation of HMG-coA reductase enzyme is done by 3 ways. What are they?

Answer 29

Transcriptionally, allosterically, and covalently.

Question 30

Discuss the covalent modification of HMG-coA reductase.

Answer 30

When Glucagon / Insulin ratio ↑ => cAMP ↑:
• Reductase kinase kinase is activated. It phosphorylates Reductase kinase making it activate. => HMG-CoA reductase is phosphorylated (INACTIVE).
• Inhibitors of phosphatases are activated => Phosphatases are inhibited => HMG-CoA reductase remains phosphorylated (INACTIVE).
=> Cholesterol Biosynthesis is shut down.

• When Insulin / Glucagon ratio ↑ => Phosphatases are activated:
  • Reductase Kinase is dephosphorylated making INACTIVE.
  • HMG-CoA reductase is dephosphorylated (ACTIVE).
  => Cholesterol Biosynthesis is activated.

Question 31

Discuss the transcriptional control over the HMG-coA reductase.

Answer 31

HMGCoA reductase gene contains, in its Promoter region, a consensus sequence: Sterol Regulatory Elements (SRE) that is regulated by a TF called SRE Binding Protein (SREBP), the activity of which is triggered by cholesterol level:
As cholesterol ↓, SREBP dimerizes and enters the nucleus stimulating HMGCoA transcription, hence biosynthesis.
• As cholesterol ↑, SREBP remains trapped in the ER, hence not able to induce HMGCoA transcription.

• Note that dietary cholesterol exerts its effect only on the Liver HMG-CoA reductase. Neither the intestinal HMG-CoA reductase, nor that of other tissues are inhibited by dietary cholesterol.

Question 32

Allosteric Inhibition of Cholesterol.

Answer 32

• The HMGCoA reductase itself can be inhibited by: Cholesterol (final product of the pathway), oxygenated sterols (oxy sterols), Bile acids and Free FAs (FFAs).
• Mevalonolactone (Cyclic ester) was also reported to inhibit HMGCoA reductase by an unknown mechanism.
• Statins are a class of compounds that competitively inhibits HMGCoA reductase, since they are structurally similar to HMGCoA. (i.e. Lova/SimvA/Prava/Atorva/Fluva—statins)
• Statins binds to HMGCoA reductase => Intracellular Cholesterol ↓. => Body responds by increasing LDL receptors in the PM. => More LDLs are internalized. => Since LDL transports Cholesterol to extrahepatic tissues, Intracellular Cholesterol ↑ => Transcription of HMGCoA reductase ↓ and LDL receptors are down-regulated.

Question 33

See figure for LDL internalization process

Answer 33

LDL is the vehicle that transports cholesterol to periphery
1- Receptor mediated endocytosis …Apo B100
2- Intracellular increase in cholesterol will
3- Repress transcription and translation of HMGCoA reductase
4- Repress synthesis of LDL receptors: Down reguation
5- Activate ACAT … chol esters

Question 34

What is the fate of cholesterol? Exogenous? Endogenous? Storage form?

Answer 34

o Only part of exogenous cholesterol is absorbed and delivered to the Liver via CM.
- Unabsorbed Cholesterol is acted upon by intestinal bacteria, which convert it to Neutral Sterols: Coprostanol and Cholestanone (both are isomers), that are reduced and excreted as Cholestenol.

o Endogenous cholesterol ( CM cargo + de novo synthesized) is secreted as VLDL from he Liver, which gets metabolized in circulation to IDL and then LDL, which transports cholesterol to various cells & tissues.

• Inside the cell, cholesterol is stored as cholesterol esters, while 85% are converted into Bile Acids. => Most of the cholesterol pool is destined for excretion as Bile Acid, which is the major excretory product of cholesterol. Liver is the organ that is responsible for Bile Acid formation.
• Inside the cell, Cholesterol Esterification is catalyzed by ACAT. Cholesterol Esterification may also occur inside HDL (Clearing Lipoprotein; good lipoprotein) in the serum. HDL collects excess cholesterol from all tissues and organs, and re-transport it back to the Liver, which converts it into Bile Acids.
• HDL has:
  • apo D which recognizes cholesterol.
  • LCAT: Lecithin Acyl Cholesterol Transferase, activated by apo A.
  • apo E.
• Other fates of Endogenous Cholesterol include:
  • Conversion into various steroid hormones.
  • Conversion into vitamin D.

Question 35

What are the primary bile acids? the secondary? Why are they transformed from primary to secondary? How? What happens to the secondary bile acids?

Answer 35

Primary Bile Acids that are not reabsorbed by the enterohepatic system (recycling system in ileum absorbing cholesterol via active transport) are converted by intestinal bacteria into Secondary Bile Acids: Deoxycholic acids and Lithocholic acids.
How? Deconjugated then subjected to 7-alpha-dihydroxylation (loss of OH at C-7) catalyzed by bacterial enzymes, forming Deoxycholic acids and Lithocholic acids.
-Majority of Secondary Bile Acids are passively reabsorbed by the Liver.

Question 36

Pathway for formation of primary bile acids from cholesterol. Site of transformation?

Answer 36

site: liver
pathway:
•Mono-hydroxylation at C7 yielding Chenodeoxycholic acid, or di-hydroxylation at C7 & C12 yielding Cholic acid.
- This step is the differentiating step between the 2 Primary Bile Acids.
•Epimerization of the 3-β-hydroxyl group into an alpha hydroxyl group.
•Reduction of the double bond in ring B.
•Initial Omega oxidation followed by one β-oxidation of the side chain, resulting in cleavage of 3 carbons (propionyl-CoA). => C27 becomes C24: Cholyl-CoA and Chenodeoxycholyl-CoA (both are Primary Bile Acids )
-Each Primary Bile Acid is conjugated with taurine or glycine before it passes through the bile duct into the intestine.

Question 37

History of hypercholesterolemia drug.

Answer 37

One of the drugs thought to decrease cholesterol level was resins, which bind to Primary Bile Acids, preventing them from getting reabsorbed, hence will be excreted in feces.
- The idea was that by preventing reabsorption of Primary Bile Acids, we will be favoring more the conversion of cholesterol into bile, which decreases level of intracellular cholesterol. This is wrong because: Intracellular cholesterol level decreased. => RDS (HMG-CoA reductase) is stimulated, leading to more cholesterol. => Instead of getting large decrease, cholesterol level is maintained or even above the normal level.
CHOLESTYRAMINE MOST COMMON.

Question 38

Cholesterol is a precursor for —-

Answer 38

Cholesterol is the precursor of all the steroid hormones, mineralocorticoids, sex hormones (testosterone and estrogen), and the glucocorticoids (cortisone).

Question 39

What are 2 Vitamin D types and their names?

Answer 39

o Vit. D3: Cholecalciferol
o Vit. D2: Ergocalciferol
Cholecalciferol derived from Cholesterol is converted into active hormone by hydroxylation reactions in liver and kidney.