1. Cholesterol Metabolism
Dr Nancy Carmichael
Thursday 22nd November 2007

2. Objectives Roles of cholesterol in the body
Basic structure of cholesterol
Stages in the synthesis of cholesterol
Regulation of cholesterol synthesis
Cholesterol as a precursor for bile salts and steroid hormones
Cholesterol transport and the role of LDL
Diseases related to cholesterol metabolism and their treatment

3. Cholesterol Very important steroid
Has roles in modulating the fluidity of animal cell membranes and is the precursor of steroid hormones such as progesterone, testosterone, oestradiol, and cortisol
It can be consumed in the diet or synthesised de novo
Synthesis and utilization of cholesterol must be tightly regulated in order to prevent over-accumulation and abnormal deposition within the body
Accumulation of cholesterol can lead to atherosclerosis, a disease of the coronary arteries

4. Structure of Cholesterol
A,B,C,D rings in fused-ring system

5. Cholesterol in the Lipid Bilayer
Eukaryotic plasma membrane has large amounts of cholesterol – up to 1 molecule for every phospholipid molecule
Fills space between phospholipid molecules next to each other
Makes bilayer stiffer, less fluid, and also less permeable
Bacterial plasma membranes contain no cholesterol

6. Origin of Carbon Atoms in Cholesterol
All 27 carbon atoms in cholesterol come from acetate
Label acetate – feed to rats. Cholesterol synthesised by rats contained the label
Label acetate on either the methyl or carboxyl carbon
The results of isotope-labeling experiments reveal the source of carbon atoms in cholesterol synthesized from acetate labeled in its methyl group (blue) or carboxylate atom (pink)

7. Cholesterol Carbon Numbering
The numbering scheme for the carbon atoms in cholesterol and other steroids
2 angular methyl groups C19 attached to C10 and C18 attached to C13, these methyl groups are above plane of 4 rings

8. Synthesis of Cholesterol
Stage one is the synthesis of isopentenyl pyrophosphate, an activated isoprene unit that is the key building block of cholesterol.
Stage two is the condensation of six molecules of isopentenyl pyrophosphate to form squalene.
In stage three, squalene cyclises and is converted to cholesterol

10. Stage 1: Synthesis of Isopentenyl Pyrophosphate
First, 2 acetyl-CoAs form acetoacetyl-CoA
Then acetoacetyl-CoA and acetyl-CoA combine to make 3-hydroxy-3-methylglutaryl CoA (3-HMG CoA)
3-HMG CoA is then reduced to mevalonate in the cytosol
Mevalonate is converted to isopentenyl pyrophosphate
Cytosol
3HMG is membrane bound to endoplasmic reticulum

11. (i) 2 acetyl-CoAs form acetoacetyl-CoA

12. (ii) Formation of 3-HMG CoA

13. (iii) 3-HMG CoA to Mevalonate
3-HMG CoA has 2 fates:
Conversion to mevalonate for synthesis of cholesterol – cytosol
Cleaved to form acetyl CoA and acetoacetate - mitochondria
The synthesis of mevalonate is the committed step in cholesterol formation
The enzyme catalysing this irreversible step, (3-HMG-CoA reductase), is an important control site in cholesterol biosynthesis
Also important in regulation as bacteria often use a mevalonate independent pathway to synthesise cholesterol, which does not occur in animals so can be used as target for antibiotics

15. (iv) Mevalonate to Isopentenyl Pyrophosphate
Mevalonate is converted into 3-isopentenyl pyrophosphate in:
three consecutive reactions requiring ATP
a decarboxylation reaction

16. (iv) Mevalonate to Isopentenyl Pyrophosphate

17. (iv) Mevalonate to Isopentenyl Pyrophosphate

18. (iv) Mevalonate to Isopentenyl Pyrophosphate

19. (iv) Mevalonate to Isopentenyl Pyrophosphate

20. Stage 2: Isopentenyl Pyrophosphate to Squalene
Squalene is synthesised from isopentenyl pyrophosphate in reactions involving the following number of carbon atoms:
C5 → C10 → C15 → C30
isomerisation of isopentenyl pyrophosphate (C5) to dimethylallyl pyrophosphate (C5)
isopentenyl pyrophosphate and dimethylallyl pyrophosphate condense to form a geranyl pyrophosphate (C10) (enzyme; geranyl transferase)
Geranyl pyrophosphate then combines with isopentenyl pyrophosphate to form farnesyl pyrophosphate (C15) (enzyme: geranyl transferase)
two molecules of farnesyl pyrophosphate combine to form squalene (C30) (enzyme: squalene synthase)
Cytosol until stage iv, where process is continued in the endoplasmic reticulum

21. (i) Isomerisation of Isopentenyl Pyrophosphate to Dimethylallyl Pyrophosphate
Both 5 carbon
Position of the double bond changes

22. (ii) Formation of Geranyl Pyrophosphate
cytosol

23. (iii) Formation of Farnesyl Pyrophosphate
cytosol

24. (iv) Formation of Squalene
Membrane of the endoplasmic reticulum
tail to tail joining

25. Stage 3: Squalene forms Cholesterol
First stage, squalene to squalene epoxide, is a reduction reaction requiring O2
Squalene epoxide is cyclised to lanosterol by a cyclase enzyme
Migration of 2 methyl groups
movement of electrons through 4 double bonds
Lanosterol is converted to cholesterol by:
removal of 3 methyl groups
reduction of a double bond by NADPH
migration of another double bond
Requires O2 – thus cholesterol only evolved after earths atmosphere became aerobic
Cholesterol is absent from most prokaryotes, present in eukaryotes

26. (i) Squalene to Squalene Epoxide
Endoplasmic reticulum

27. (ii) Squalene Epoxide to Lanosterol
Migration of 2 methyl groups
Movement of electrons through 4 double bonds
Migration of 2 methyl groups
movement of electrons through 4 double bonds

28. (iii) Lanosterol to Cholesterol
HCOOH + 2CO2
Removal of 3 methyl groups
Reduction of one double bond by NADPH
Migration of another double bond
Endoplasmic reticulum

29. Summary of Cholesterol Biosynthesis

30. Regulation of Cholesterol Synthesis
Cholesterol can be obtained from the diet or it can be synthesised de novo
The liver is the major site of cholesterol synthesis in mammals, although the intestine also forms significant amounts
The rate of cholesterol formation by these organs is very responsive to the cellular level of cholesterol
This is called feedback regulation
Here, it is mediated mostly by changes in the amount and activity of 3-HMG CoA reductase
3-HMG-CoA reductase is controlled in many ways
3-HMG CoA to mevalonate was the committed step to cholesterol biosynthesis, this is the enzyme that carries that out
Cholesterol biosynthesis is comlpex energy expensive process, good to regulate cholesterol synthesis relative to dietary intake

31. HMG CoA Reductase Regulation
Rate of synthesis of 3-HMG CoA reductase mRNA
Rate of translation of 3-HMG CoA reductase mRNA into protein
Degradation of 3-HMG CoA reductase
Phosphorylation of 3-HMG CoA reductase

32. HMG CoA Reductase Regulation
DNA
mRNA
protein
degradation products P 1 2 3 4

33. 1.Synthesis of HMG CoA Reductase mRNA
The rate of synthesis of reductase mRNA is controlled by the sterol regulatory element binding protein (SREBP)
This transcription factor binds to a short DNA sequence called the sterol regulatory element (SRE) on the 5’ side of the reductase gene
In the presence of sterols, SRE inhibits mRNA production

34. 2. Translation of HMG CoA Reductase mRNA
The rate of translation of reductase mRNA is inhibited by nonsterol metabolites derived from mevalonate, as well as by dietary cholesterol.

35. 3. Degradation of HMG CoA Reductase
The enzyme is bipartite:
cytosolic domain carries out catalysis
membrane domain senses levels of derivatives of cholesterol and mevalonate
A high level of these products leads to rapid degradation of the enzyme

36. 4. Phosphorylation of HMG CoA Reductase
Phosphorylation decreases the activity of the reductase
Hormones regulate phosphorylation:
Glucagon stimulates phosphorylation (deactivation)
Insulin stimulates dephosphorylation (activation)

37. Negative Feedback of Cholesterol Synthesis
All four regulatory mechanisms are modulated by receptors that sense the presence of cholesterol in the blood
Negative feedback inhibition

38. Fates of Cholesterol Most cholesterol synthesis takes place in liver
Some of this is incorporated into membrane of liver cells
Most is exported in the forms of:
Bile acids and their salts
Cholesteryl esters
Cells use the cholesterol for membrane synthesis
They can also use it as a precursor for steroid hormone production and vitamin D production

39. Bile Acids and Salts
Bile acids and salts are derivatives of cholesterol
Make good detergents as they contain polar and non-polar regions
They are synthesised in the liver
They are the main constituent of bile
They emulsify dietary lipids, which increases their surface area to:
Promote hydrolysis by lipases
Facilitates their absorption by the intestine
Bile salts also aid in absorption of lipid soluble vitamins

40. Bile Salts as an Emulsifier
Action of bile salts in emulsifying fats in the intestine. Note polar and nonpolar regions on bile salt.

41. Excretion of Cholesterol in Bile
Synthesis of bile acids is one of the predominant mechanisms for the excretion of excess cholesterol
However, the excretion of cholesterol in the form of bile acids is insufficient to compensate for an excess dietary intake of cholesterol.

42. Synthesis of Bile Acids
Bile acids are synthesised from cholesterol via many reactions
The first reaction, cholesterol to 7a-hydroxycholesterol (enzyme: 7a-hydroxylase) is the rate limiting step in bile acid synthesis
Conversion of 7a-hydroxycholesterol to the bile acids requires several steps (not shown in diagram)

43. Synthesis of the 2 primary bile acids, cholic acid and chenodeoxycholic acid. The reaction catalyzed by the 7a-hydroxylase is the rate limiting step in bile acid synthesis. Conversion of 7a-hydroxycholesterol to the bile acids requires several steps not shown in detail in this image. Only the relevant co-factors needed for the synthesis steps are shown.

44. Bile Acids
The most abundant bile acids in human bile are chenodeoxycholic acid and cholic acid
These are the primary bile acids
In intestines, primary bile acids are converted to the secondary bile acids
deoxycholate (from cholate)
lithocholate (from chenodeoxycholate)

45. Synthesis of Bile Salts
In the liver the carboxyl group of primary and secondary bile acids is conjugated via an amide bond to either glycine or taurine
React with glycine to form glycocholate
React with taurine (a derivative of cysteine: H2N-CH2-CH2-SO3-) to form taurocholate
Glycocholate is the main bile salt
They are secreted into the intestine, where they aid in the emulsification of dietary lipids

46. Synthesis of Bile Salts
Glycocholiate (glycocholic acid)
Taurocholate (taurocholic acid)

47. Cholesterol used to Synthesise Steroids
Cholesterol is the precursor of the five major classes of steroid hormones:
progestagens
glucocorticoids
mineralocorticoids
androgens
oestrogens
These hormones are powerful signal molecules that regulate many processes in the body

48. Cholesterol and Steroid Hormones
Cholesterol (C27)
Pregnelonone (C21)
Progestagens (C21)
Glucocorticoids (C21)
Mineralocorticoids (C21)
Androgens (C19)
Oestrogens (C19)

49. Cholesteryl Esters
About 2/3 of cholesterol in blood is in form of an ester
Cholesteryl esters are formed in the liver via the action of acyl-CoA-cholesterol acyl transferase (ACAT)
Catalyses transfer of a fatty acid from coenzyme A to the hydroxyl group of cholesterol
This changes cholesterol into a more hydrophobic form
Can be stored in the liver or transported to other tissues which need cholesterol

51. Transport of Cholesterol in the Blood
Cholesterol and cholesteryl esters are virtually insoluble in water
They need to be transported from origin to other tissues for storage or use
They are transported in body fluids in the form of lipoprotein particles
These are complexes of an apolipoprotein combined with cholesterol, cholesteryl esters, phospholipids or triacylglycerol
Apolipoprotein is the protein in its lipid free form

52. Transport of Cholesterol in the Blood
Different combination of apolipoproteins and lipids produce particles of different density
Lipoproteins are classified according to increasing density
Chylomicrons
Chylomicron remnants
Very low density lipoproteins (VLDL)
Intermediate-density lipoproteins (IDL)
Low density lipoproteins (LDL)
High density lipoproteins (HLDL)
Can separate the lipoproteins by ultracentrifugation

53. Low Density Lipoprotein
Lipoprotein particle, spherical complex, hydrophobic lipids in core, hydrophilic amino acid side chains at the surface

54. Separate by ultracentrifugation. View on electron microscope
Separate by ultracentrifugation. View on electron microscope. Chylomicrons have diameter of nm, VLDL nm, LDL, nm, HDL 8-11 nm

55. Specific Function of Lipoproteins
Each class of lipoproteins has a specific function
This depends on its point of synthesis, lipid composition and apolipoprotein content
At least 9 apolipoproteins are found in lipoproteins of human plasma
The apolipoprotein has 2 roles:
Solubilise hydrophobic lipids
Contain targeting signals – target lipoprotein to a specific tissue or enzyme

56. Chylomicrons Largest, least dense, contain many triacylglycerols
Synthesised in endoplasmic reticulum of epithelial cells in small intestine, then enter blood stream
Carry fatty acids to where they are used or stored
The chylomicron remnants (without fats but still containing cholesterol) move to liver
apoE in the chylomicron remnants bind receptors in liver – taken up by endocytosis
In liver, they release cholesterol and are degraded in lysosomes

57. VLDL
If diet has more fatty acids than needed – converted to triacylglycerols in liver and packaged into VLDL
Excess carbohydrate can also be dealt with in the same way
VLDLs are transported in the blood from liver to muscle and adipose tissue
Here the fatty acids are relased from the triacylglycerols in VLDL
Adipocytes take up the fatty acids and resynthesise triacylglycerols to store
Most VLDL remnants (IDL) removed from circulation by hepatocytes: are uptaken by endocytosis (if apoE present)
Half the VLDL become LDL due to loss of lipids

59. Chylomicrons give plasma a milky appearance

60. LDL
Loss of triacylglycerols converts some VLDL to VLDL remnants (IDL) and then to LDL
LDLs are very rich in cholsterol and cholesteryl esters
They have apoB-100 as the major apolipoprotein
They carry cholesterol to extrahepatic tissue which has receptors to recognise apoB-100
The receptors mediate the uptake of cholesterol and cholesteryl esters

61. HDL These begin in liver and small intestine
Contains very little cholesterol and no cholesteryl esters
Very protein rich
Contain enzyme LCAT (lecithin-cholesterol acyl transferase)
LCAT catalyses formation of cholesteryl esters from lecithin (phosphatidylcholine) and cholesterol

62. Formation of Cholesteryl Esters from Lecithin
LCAT enzyme is present on the surface of HDL

63. HDL and LCAT
LCAT is on the surface of nascent (newly forming) HDL particles
It converts the cholesterol and lecithin of the chylomicron and VLDL remnants to cholesteryl esters
These form a core and makes HDL molecules from disc shaped to spherical
HDL particles are then mature
This cholesterol-rich lipoprotein then returns to the liver where it unloads some cholesterol which is converted to bile salts

64. HDL HDL may be taken up by the liver by receptor mediated endocytosis
Some is taken to other tissues
HDL binds to plasma membrane receptor proteins (SR-BI) in hepatic and steroidogenic tissues e.g. adrenal gland
These mediate the selective transfer of cholesterol and other lipids in HDL into the cell
The depleted HDL then dissociates to re-circulate in the blood stream and extract more lipids from chylomicron and VLDL remnants

65. Reverse Cholesterol Transport
Depleted HDL can also pick up cholesterol stored in extrahepatic tissues and carry it to the liver
This is called reverse cholesterol transport
In one reverse cholesterol transport pathway, interaction of nascent HDL with SR-BI receptors in cholesterol-rich cells triggers passive movement of cholesterol from the cell surface into HDL
HDL then carries it back to liver
HDL good cholesterol carrier

66. Reverse Cholesterol Transport
Another reverse cholesterol transport pathway – apoA-I in depleted HDL interacts with an active transporter in a cholesterol-rich cell
This is the ABC1 protein
The apoA-I and the HDL are taken up by endocytosis
They are then re-secreted with a load of cholesterol
It transports this back to the liver
ABC proteins – multidrug transporters, ABC transporters as have ATP binding cassettes

67. Receptor Mediated Endocytosis
LDL – apoB-100, recognised by LDL receptors
LDL receptors are present on cells which need to take up cholesterol
LDL binding to its receptor initiates endocytosis
This brings the LDL and its receptor iside the cell, within an endosome
This endosome fuses with a lysosome
The lysosome contains enzymes which hydrolyse the cholesteryl esters to cholsterol and free fatty acids
apoB-100 is degraded to amino acids

68. Endocytosis of LDL

69. Receptor Mediated Endocytosis
LDL also degraded to amino acids which are released into cytosol
LDL receptor is not degraded – it returns to the cell surface
apoB-100 present in VLDL, but it cannot bind to the LDL receptor
Conversion of VLDL to LDL exposes the binding domain of apoB-100
Binding domain not available to bind to LDL receptor

71. ACAT
Cholesterol which enters cell by receptor mediated endocytosis can be re-esterified by ACAT (acyl-CoA-cholesterol acyl-transferase)
Stored within cytosolic lipid droplets

72. Regulation of Blood Cholesterol
Accumulation of excess cholesterol prevented by reducing rate of cholesterol synthesis when sufficient cholesterol is available from blood LDL
Negative feedback inhibition
High intracellular cholesterol levels activate ACAT, increases esterification of cholesterol for storage
High cellular cholesterol reduces transcription of the gene which encodes LDL receptor
Reduces production of receptor and thus uptake of cholesterol from blood

73. regulation of cholesterol synthesis and levels of blood cholesterol
regulation of cholesterol synthesis and levels of blood cholesterol. Of course the best regulation is to control the amount of cholesterol in the diet

74. Unregulated Cholesterol Production
If more cholesterol is consumed in diet and synthesised than is needed for synthesis of membranes, bile salts and steroids:
accumulation of cholesterol in blood vessels
These are called atherosclerotic plaques
Obstruct blood flow leading to atherosclerosis

75. Atherosclerosis
Blocked vessels, occluded coronary arteries – heart attacks
Major cause of death in industrialised societies
Atherosclerosis linked to high blood cholesterol
Also to high levels of LDL-bound receptor
Negative correlation between HDL and arterial disease

76. Familial Hypercholesterolemia
Genetic disorder
High blood cholesterol
Develop atherosclerosis in childhood
Defective LDL receptor
Receptor mediated uptake of cholesterol by LDL does not occur
Cholesterol is not cleared from blood
Accumulated forming atherosclerotic plaques
Endogenous cholesterol synthesis continues despite excessive cholesterol in blood because extracellular cholesterol cannot enter cell to regulate intracellular synthesis

77. Treating Familial Hypercholesterolemia
Lovastatin and Compactin – derived from fungi
These, and other analogues resemble mevalonate
Competitive inhibitors of HMG-CoA reductase
Inhibit cholesterol synthesis
Lovastatin can lower blood cholesterol up to 30 %
Can combine drug with an edible resin which binds bile salts to prevent their re-absorption – makes drugs more effective

79. HDL Deficiency HDL levels low in familial HDL deficiency (FHA)
HDL almost undetectable in Tangier disease
Both result from a mutation in ABC protein
Cholesterol depleted HDL can’t take on cholesterol from cells that lack this protein
The cholesterol depleted HDL is rapidly removed from the blood and destroyed
Both diseases rare
Teach us about role of ABC1 protein
Low plasma HDL correlates with high incidence of coronary heart disease - ABC1 useful target for drugs to control HDL levels

80. Intermediates in Cholesterol Biosynthesis
Isopentenyl pyrophosphate has other roles apart from in cholesterol synthesis
Precursor for synthesis of many biomolecules
Vitamins A,E and K, natural rubber, essential oils and many others
These compounds are called isoprenoids

81. Isoprenoid Biosynthesis

82. Practice Questions
Which of the following is not an intermediate in the synthesis of lanosterol from acetyl-CoA?
Isopentenyl pyrophosphate
Malonyl-CoA
Mevalonate
Squalene
HMG-CoA
Cholesterol is synthesised from:
acetyl-CoA
choline
lipoic acid
malate
oxalate
1b 2a

83. Practice Questions A 30-carbon precursor of the steroid nucleus is:
farnesyl pyrophosphate.
geranyl pyrophosphate.
isopentenyl pyrophosphate.
lysolecithin.
squalene.
Which of the following is derived from a sterol?
Bile salts
Gangliosides
Geraniol
Phosphatidylglycerol
Prostaglandins
3e, 4a

84. Practice Questions
Which of these statements about cholesterol synthesis is true?
Cholesterol is the only known natural product whose biosynthesis involves isoprene units.
Only half of the carbon atoms of cholesterol are derived from acetate.
Squalene synthesis from farnesyl pyrophosphate results in the release of two moles of PPi for each mole of squalene formed.
The activated intermediates in the pathway are CDP-derivatives.
The condensation of two five-carbon units to yield geranyl pyrophosphate occurs in a “head-to-head” fashion. 5c

85. Practice Questions
Which of these statements about the regulation of cholesterol synthesis is not true?
Cholesterol acquired in the diet has essentially no effect on the synthesis of cholesterol in the liver.
Failure to regulate cholesterol synthesis predisposes humans to atherosclerosis.
High intracellular cholesterol stimulates formation of cholesterol esters.
Insulin stimulates HMG-CoA reductase.
Some metabolite or derivative of cholesterol inhibits HMG-CoA reductase. 6a

86. Practice Questions What are the functions of cholesterol in humans?
Which is the commited step in cholesterol synthesis? What enzyme catalyses this step?
How are the 2 activated isoprenes structurally related?
Which are the most dense lipoproteins? The least dense? How does this relate to their lipid:protein ratio?
What part of the LDL particle is recognised by the LDL receptor?
Based on the function of HDLs, why do you expect a negative correlation between HDL levels and arterial disease?