D- Cholesterol Absorption, Synthesis, & Metabolism

1. D-Cholesterol Absorption, Synthesis, & Metabolism

2. Cholesterol Background • Atherosclerotic vascular disease • Stabilizes cell membrane • Precursor to bile salts and steroid hormones • Cholesterol precursors converted to ubiquinone, dolichol, & vitamine D

3. Cholesterol BackgroundSynthesis • Obtained through diet or synthesis • Synthesized in many cells, but mostly in the liver and intestine • Acetyl coenzyme A (acetyl CoA) is the precursor to cholesterol synthesis

4. Cholesterol Background(Transport) • Chylomicrons & VLDL transport cholesterol to other cells through the bloodstream • Chylomicrons package cholesterol in intestine, while VLDL package in liver • Triacylglycerols are also transported by Chylomicrons and VLDL • HDL – reverse cholesterol transport

5. Student Learning Outcomes • Describe the rate-limiting step in cholesterol synthesis and how the HMG-CoA reductase is regulated • Briefly describe the fates of cholesterol • Describe the VLDL to LDL pathway • The role of HDL • RCT, apoprotein & lipid exchange • Explain what occurs during receptor mediated endocytosis • Describe the aspects of Atherosclerosis

6. Cholesterol Synthesis • Perhydrocyclopentanophenanthrene structure consists of four fused rings • Cholesterol contains a hydroxyl group at C3, double bond between C5 & C6, eight-membered hydrocarbon chain at C17, & methyl groups at C10 & C13 Fig.2 Fig. 1 Perhydrocyclopentanophenanthrene Cholesterol

7. Cholesterol Synthesis Stage I: Acetyl CoA to Mevalonate A. B. C. Fig.3 Rate limiting step

8. Cholesterol Synthesis Stage I: Transcription Control • Feedback regulatory system • Rate of HMG-CoA reductase mRNA synthesis controlled by sterol regulatory element binding protein (SREBP) • Once in the Golgi, SERBP is cleaved twice by S1p & S2P to release the transcription factor Fig. 4A

9. Cholesterol Synthesis Stage I: Proteolytic Degradation of HMG-CoA Reductase • When sterol present, enzyme undergoes sterol accelerated ERAD (ER associated degradation) • HMG-CoA is ubiquitinated and extracted from membrane where it is then degraded by proteosomes Fig. 4B

10. Cholesterol Synthesis Stage I: Regulation by Covalent Modification Fig. 4C • Short-term regulation by phosphorylation & dephosphorylation • Adenosine monophosphate (AMP) activated kinase phosphorylates HMG-CoA • Glucagon, sterols, glucocorticoids & low ATP levels inactivate HMG-CoA • Insulin, thyroid hormone, high ATP levels activate enzyme

11. Cholesterol Synthesis Stage 2: Mevalonate to 2 Activated Isoprenes • Transfer 3 ATP to Mevalonate in order to activate C5 & OH-group of C3 • Phosphate group at C3 & Carboxyl group of C1 leave, which produces a double bound • This allows for two active isoprenes Fig.5

12. Cholesterol Synthesis Stage 3: Condensation of Isoprenes to for Squalene • 1) Head to tail attachment of isoprenes to form Geranyl pyrophosphate • 2) Head to tail condensation of Geranyl pyrophosphate and isopentenylpyrophosphate to form Farnesyl pyrophosphate • 3) Head to head fusion of two Farnesyl pyrophosphate to form squalene Fig.6

13. Cholesterol Synthesis Stage 4: Squalene to Four-Ring Steroid Nucleus • Squalene monooxygenase adds oxygen to form an epoxide • Unsaturated carbons (double bonds) are aligned to allow cyclization and formation of lanosterol • After many reaction get cholesterol Fig. 7

14. Fates of Cholesterol • Membranes • Cholesterol Ester • Biliary Cholesterol • Bile Acids

15. Cholesterol Esters • Acyl-CoA:cholesterol acyl transferase (ACAT) is an ER membrane protein • ACAT transfers fatty acid of CoA to C3 hydroxyl group of cholesterol • Excess cholesterol is stored as cholesterol esters in cytosolic lipid droplets Fig. 8

16. Bile Salts • Bile acids & salts are effective detergents • Synthesized in the liver • Stored & concentrated in the gallbladder • Discharged into gut and aides in absorption of intraluminal lipids, cholesteral, & fat soluble vitamines • Bile acid refers to the protonated form while bile salts refers to the ionized form • The pH of the intestine is 7 and the pKa of bile salts is 6, which means that 50% are protonated • These terms are sometimes used interchangeably

17. Synthesis of Bile Salts • Rate-limiting step performed by the 7α-hydroxylase (CYP7A1) and is regulated by bile salt concentration • End product: Cholic acid series & Chenocholic acid series • Bile salts can be conjugated & become better detergents Fig. 9 Fig. 10

18. Fate of Bile Salts Fig. 12

19. Cholesterol Transport by Blood Lipoproteins • Cholesterol, cholesterol esters, triacylglycerols, & phospholipids are insoluble and must travel via lipoproteins

20. VLDL to LDL • The TG, free & esterified cholesterol, FA, & apoB-100 are packaged into nascent VLDL • Nascent VLDL are secreted to bloodstream and acquire apoCII & apoE from HDL to form a mature VLDL • Hepatic triglyceride lipase (HTGL) hydrolyzes additional triglycerides to produce LDL • 40% of LDL transported to extrahepatic tissues • Excess LDL is taken up by macrophages Fig. 14

21. Reverse Cholesterol Transport (RCT) • HDL removes cholesterol from cells and returns it to the liver • ABC1 transport protein uses ATP hydrolysis to move cholesterol from inner leaflet to outer leaflet of membrane • HDL receives cholesterol and uses the LCAT enzyme to modify & trap the cholesterol Oram, JF & Vaughan, AM. (2000) ABCA1-mediated transport of cellular cholesterol & phospholipids to HDL apolipoproteins. Curr Opin Lipidol. June;11(3):253-60

22. Fate of HDL HDL binds SR-B1 receptor • HDL can bind to specific hepatic receptors, but primary HDL clearance occurs through uptake by scavenger receptor SR-B1 • Present on many cells • SR-B1 can be upregulated in cells that require more cholesterol • SR-B1 is not downregulated when cholesterol levels are high Transfers cholesterol & cholesterol ester to cell Depleted HDL dissociates & re-enters circulation

23. HDL Interactions with Other Particles • HDL transfers apoE & apoCII to Chylomicrons & VLDL • HDL either transfers cholesterol & cholesterol esters directly to liver or by means of CETP to VLDL (or other TG-rich lipoproteins) • In exchange, HDL receives triacylglyceroles • Prior to CETP mature HDL particles are HDL3, post CETP they become larger and are called HDL2 Fig. 16 Fig. 17

24. Receptor-Mediated Endocytosis of Lipoproteins • LDL receptor are located at coated pits, which also contain clathrin • Vesicles fuse with lysosome where cholesterol esters are hydrolyzed into cholesterol & re-esterified by ACAT • This avoids damaging effects of high concentrations of free cholesterol on membrane • Unlike cholesterol esters of LDL, these cholesterol esters are monosaturated Fig. 18

25. Feedback Regulation of Receptors • Regulation by SREBP or its cofactor • Low levels of cholesterol leads to up regulation of receptor genes • Increase amount of cholesterol in cells • High levels suppress expression of receptor genes • Reduces amount of cholesterol that enters cells

26. Lipoprotein Receptors • LDL receptor most well characterized & contains 6 different regions • LDL receptor-related proteins are structurally related but recognize more ligands • Macrophage scavenger receptor : SR-AI & SR-A2 • Take up oxidatively modified LDL • When engorged with lipids macrophages become foam cells

27. Anatomical & Biochemical Aspects of Atherosclerosis • Initial step is formation of fatty streak (foam cells) in subintimal space • Foam cells separate endothelial cells exposing them to blood, which leads to plaques & thrombin at these sites • When plaque content exposed to procoagulant elements in circulation, acute thrombus formation occurs • Further thrombus formation leads to complete occlusion of lumen & eventually AMI or CVA Fig 21. Layers of arterial wall

28. Key Concepts • HMG-CoA conversion to mevalonate is the rate limiting step of cholesterol synthesis • HMG-CoA reductase regulated by feedback, degradation, modification • Cholesterol fate: membranes, esters, biliary cholesterol, bile salts • Bile salts aide in absorption of lipids • Hydrolysis of VLDL leads to LDL, which transport TG & CE to peripheral cells & macrophages • HDL involved in RCT & apoprotein/lipid exchange • LDL enters cells via receptor-mediated endocytosis • Excess LDL taken up by macrophage leads to the formation of foam cells, which is the beginning of atherosclerosis