Cholesterol Absorption, Synthesis, & Metabolism I Chapter 34 Nov. 4th 2011

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

Cholesterol Background Synthesis 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

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

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

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

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

Cholesterol Synthesis Stage I: Transcription Control Fig. 4A 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

Cholesterol Synthesis Stage I: Proteolytic Degradation of HMG-CoA Reductase Fig. 4B 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

Cholesterol Synthesis Stage I: Regulation by Covalent Modification 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 Fig. 4C

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

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

Cholesterol Synthesis Stage 4: Squalene to Four-Ring Steroid Nucleus Fig. 7 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

Fates of Cholesterol Membranes Cholesterol Ester Biliary Cholesterol Bile Acids

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

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

Synthesis of Bile Salts Fig. 9 Fig. 10 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

Fate of Bile Salts Fig. 12

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

VLDL to LDL Fig. 14 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

Reverse Cholesterol Transport (RCT) Oram, JF & Vaughan, AM. (2000) ABCA1-mediated transport of cellular cholesterol & phospholipids to HDL apolipoproteins. Curr Opin Lipidol. June;11(3):253-60 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

Fate of HDL HDL binds SR-B1 receptor Transfers cholesterol & cholesterol ester to cell Depleted HDL dissociates & re-enters circulation 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

HDL Interactions with Other Particles Fig. 16 Fig. 17 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

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

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

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

Anatomical & Biochemical Aspects of Atherosclerosis Fig 21. Layers of arterial wall 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

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