Cholesterol Metabolism

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

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

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

7. Cholesterol Carbon Numbering

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

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

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)

21. (i) Isomerisation of Isopentenyl Pyrophosphate to Dimethylallyl Pyrophosphate

22. (ii) Formation of Geranyl Pyrophosphate

23. (iii) Formation of Farnesyl Pyrophosphate

24. (iv) Formation of Squalene

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

26. (i) Squalene to Squalene Epoxide

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

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

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

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

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

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)

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

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