Dr. Eman Shaat Professor of Medical Biochemistry and Molecular Biology Cardio Vascular System lipoprotein metabolism lecture 1 ( 43 slides)

Relatively water insoluble Introduction Lipid compounds: Relatively water insoluble Therefore, they are transported in plasma (aqueous) as Lipoproteins For triacylglycerol transport (TG-rich): Chylomicrons: lipids of dietary origin (exogenous) VLDL: lipids of endogenous (hepatic) synthesis For cholesterol transport (cholesterol-rich): LDL HDL

Function of lipoproteins Serve to transport lipids and lipid-soluble compounds between tissues and organs Substrates for energy metabolism (TG) Essential components for cells (PL, C) Precursors for hormones (C) Lipid soluble vitamins (D, E, K, A) Precursors for bile acids (C)

Classification of plasma lipoproteins Lipoproteins differ in the ratio of protein to lipids, & in the particular apoproteins & lipids that they contain. They are classified based on their density: Chylomicron (CM) (largest; lowest in density due to high lipid/protein ratio; highest % weight triacylglycerols) VLDL (very low density lipoprotein; 2nd highest in triacylglycerols as % of weight) IDL (intermediate density lipoprotein) LDL (low density lipoprotein, highest in cholesteryl esters as % of weight) HDL (high density lipoprotein; highest in density due to high protein/lipid ratio)

Plasma lipoproteins All lipids in plasma are transported in the form of lipoproteins. Lipoproteins solubilize lipids in plasma. The structure of lipoprotein is: Central core formed of non-polar lipids: triacylglycerols (TG; TAG) and cholesterol esters (CE). Outer layer contains polar lipids: phospholipids (PL). non-esterified cholesterol (C). proteins (apoproteins).

Hydrophobic lipids Amphiphilic lipids

Important enzymes and proteins involved in lipoprotein metabolism Enzymes (LPL, HL, LCAT, CETP). Apolipoproteins (A, B, C, D, E). Functions of apolipoproteins: Structural and transport function Enzymatic function Ligands for receptors

Types of apolipoproteins Apo Lp (a) E CI,II,III B100 B48 AI & AII In liver In liver & Intestine synthesized in the liver synthesized in the liver. synthesized in the intestine In Lp(a) in VLDL, HDL, chylomicron CII lipoprotein lipase In LDL & VLDL In chylom AI in HDL LCAT Peripheral Peripheral Transferable Integral Attached to B100; impairs fibrinolysis; atherogenic Helps in receptor recognition HDL chylomicron and VLDL Chylomic HDL + +

Lipoprotein classes TG TG, PL, C TG, cholesterol cholesterol Source Apo Proteins Protein/lipid % Main lipid component Diameter(nm) Function CM Intest. (A I,II,III,IV) (B48) (C I,II,III) (E) 1/99 % TG 90-1000 Dietary: FFA  Adipose/muscle CE  Liver via remnants CM remnants 6/94 TG, PL, C 45-150 VLDL liver (B100) (CI,II,III) 7/93 30-90 Synthesized: FFA adipose/muscle CE  LDL IDL 11/89 TG, cholesterol 25-35 LDL Blood (VLDL) B100 21/79 cholesterol 20-25 CE to liver (70%) and peripheral cells (30%) HDL 1,2,3 Liver, intes., VLDL, CM (A1,II,IV) (D in HDL2,3) PL, cholesterol HDL1: 32/68 HDL2: 33/67 HDL3: 57/43 preβHDL: 70/30 HDL1: 20-25 (only apo-E) HDL2: 10-20 HDL3: 5-10 preβHDL:<5 supplies apo CII, E to chylomicrons and VLDL; mediates reverse cholesterol transport Albumin/FFA Adipose tissue Free fatty acids 99/1

A A apo-E receptor

Metabolism of CM I- Synthesis of nascent chylomicrons Nascent chylomicrons are formed in the intestinal mucosa and pass to the chyle and then to the blood stream through the thoracic duct. Triglycerides are the predominant lipid in chylomicrons and form 90% of the total lipid present in them. ( Exogenous ; dietary triglycerides ) Its protein content is about 1%. Apo-A and apo-B48 are component of the nascent cylomicrons. II- conversion of nascent chylomicrons to mature chylomicrons After entering the blood stream, the chylomicron particles transfer apo-A to HDL and acquire apo-C from HDL.

Metabolism of CM III- Degradation of chylomicrons Apo-CII activates lipoprotein lipase (LPL= clearing factor) LPL located in the capillaries of adipose and other peripheral tissues as cardiac and skeletal muscles and lactating mammary gland. LPL Hydrolyze triglyceride (TG) in the core of CM and VLDL to free fatty acids and glycerol. Insulin enhances the synthesis of this enzyme and heparin increases its activity. Lipoprotein lipase (LPL)

Metabolism of CM III- Degradation of chylomicrons 2. Triglycerides in chylomicrons are hydrolyzed into glycerol and FFA. The fatty acids are released and oxidized in muscles or stored as triglycerides in adipose tissue. They are transported bound to plasma albumin. Glycerol is taken by the liver cells mainly (due to high activity of glycerol kinase). IV- Formation of chylomicron remnants 1. As triglycerides are removed from the hydrophobic core of chylomicrons, the surface area of cylomicrons decreases. 2. The more hydrophilic surface components (apo-C, unesterified cholesterol and phospholipids) are transferred to HDL. The remaining particles are termed chylomicron remnants.

Metabolism of CM V- Fate of chylomicron remnants They are triglycerides poor particles and consist mainly of cholesterol esters, apo-B48, apo-E. They interact with receptors on liver cells and are taken by endocytosis, where they are catabolized within the hepatic lysosomes and the products (amino acids, fatty acids, cholesterol) are released into the cytosol and reutilized by the hepatic cells. remnants are taken up by liver cells due to apoE recognition sites.

70% HDL 30% apo-B/E receptor

Metabolism of VLDLs Assembled and secreted by liver directly into blood as nascent form. Carry lipids (hepatic origin; endogenous TG) from liver to peripheral tissues. Mature VLDLs: contain Apo B-100 plus Apo C-II and Apo E (both from HDL). ApoC-II is required for activation of lipoprotein lipase. Lipoprotein lipase is required to degrade TG into glycerol and fatty acids. As TG is degraded, VLDLs become Smaller in size More dense Apo C back to HDL

CE HDL Metabolism of VLDLs VLDL TG Exchange of TG with cholesterol ester (HDL) by cholesterol ester transfer protein (CETP) End products: IDL (returns Apo E to HDL) and become LDL Most core lipid in LDL is cholesterol ester. ApoB100 is only apolipoprotein in the surface. VLDL HDL TG CE Choleteryl Ester Transfer Protein (CETP)

Summary of CM, VLDL, LDL LDL VLDL Chylomicron Synthesized in small intestine Transport dietary lipids 98% lipid, large sized, lowest density Apo B-48: Receptor binding Apo C-II: Lipoprotein lipase activator Apo E: Remnant receptor binding VLDL Synthesized in liver. Transport endogenous TG. 90% lipid, 10% protein. Apo B-100 Receptor binding Apo C-II LPL activator Apo E Remnant receptor binding LDL Synthesized from IDL. Cholesterol transport. 78% lipid, 58% cholesterol & CE. Apo B-100 Receptor binding

LDL receptor Also named as apoB-100/apoE receptors. because of its ability to recognize particles containing both apos B and E (apo E>> apo B-100) LDL receptors exist in the liver and in most peripheral tissues The complexes of LDL and receptor are taken into the cells by endocytosis, where LDL is degraded but the receptors are recycled LDL cholesterol levels are positively related to risk of cardiovascular disease Therefore, cholesterol in LDL has been called “bad cholesterol”

Fates of cholesterol intracellular. Non-hepatic cells obtain cholesterol from the plasma LDL rather than by synthesizing it. LDL uptake occurs by receptor mediated mechanism. Receptor-LDL complex fuses with lysosomes. cholesterol esters are hydrolyzed by lysosomal acid lipase to free cholesterol that is either. Used unesterified for biosynthesis of cell membrane. Reesterified for storage inside the cell by ACAT. Synthesis of steroids in specified tissues

Fates of cholesterol intracellular. Apo-B100 Apo-B100/E receptor C, CE> TG LDL TG Membrane C C HL ACAT FA+Glycerol CE Storage Steroids Liver

Hepatic Lipase (HL) Bound to the surface of liver cells. Hydrolyzes TG → FFA + glycerol Unlike LPL, HL does not react readily with CM or VLDL but is concerned with TG hydrolysis in VLDL remnants and HDL metabolism

Coordinate control of cholesterol uptake and synthesis Increased uptake of LDL-cholesterol results in: inhibition of HMG-CoA reductase (reduced cholesterol synthesis). stimulation of acyl CoA:cholesterol acyl transferase (ACAT) increased cholesterol storage decreased synthesis of LDL-receptors “down-regulation” decreased LDL uptake

High density lipoprotein (HDL)

Functions of HDL Transfers proteins to other lipoproteins Picks up lipids from other lipoproteins Picks up cholesterol from cell membranes Converts cholesterol to cholesterol esters via the LCAT reaction HDL can transfer cholesterol ester into the liver: directly or through other lipoproteins “reverse cholesterol transport) High blood levels of HDL ("good" cholesterol) correlate with low incidence of atherosclerosis.

Functions of HDL Function: Transports C from peripheral tissues to liver (reverse C transport). Only HDL can pick up cholesterol from peripheral tissues Only the liver can degrade cholesterol HDL acts as a circulating store for apo-C & E required in the metabolism of CM & VLDL.

HDL: structure These are the smallest and the most dense lipoprotein particles. They contain large amount of proteins (50-60%) and very little TG, while the predominant lipid is phospholipid. Synthesized de novo in the liver and small intestine, primarily as protein-rich disc-shaped particles. Newly formed HDL are nearly devoid of C & CE. Apoproteins : apoA, apoC, apoD and apoE. Apo C- & E- are synthesized in liver and transferred from liver HDL to intestinal HDL when the latter enter the plasma. apoA-I is synthesized by liver & intestine, which is secreted in a lipid-poor form and then immediately recruits additional PLs and free cholesterol (C) via the ABCA1 pathway, forming nascent HDL.

HDL: synthesis Nascent HDL consists of discoid PL bilayers containing apo E & free cholesterol (C). On entering the circulation: apo-A, the major HDL apoprotein is acquired from CM apo-C is transferred to CM and VLDL. LCAT and its activator apo-A1, bind to the discoidal particles, and the surface PL & free C are converted to CE & lysolecithin. The non-polar CE move into the hydrophobic interior of the bilayer, whereas lysolecithin is transferred to plasma albumin. Thus, a spherical HDL is formed.

HDL metabolism Liver C + FA C C CE CE C C CM VLDL Bile acids Apo-A CM VLDL Liver Apo-C Apo-E Bile acids LCAT Nascent HDL C + FA C Apo-A C CE Peripheral tissue CE C LCAT C Other lipoproteins HDL receptor Mature HDL

HDL metabolism Apo-AI activates LCAT (lecithin – cholesterol acyl transferase) which is present on the HDL surface. Free cholesterol, present on the HDL surface is esterified by LCAT. Formation of cholesterol esters in lipoproteins

CETP

Class B scavenger receptor B1 (SR-B1) In liver and in steroidogenic tissues: it binds HDL via apo-A1, then CE is selectively delivered to the cells (the particle itself, including apo-A1 is not taken up). In the tissues on the other hand: SR-B1 mediates the acceptance of cholesterol effluxed from the cells by HDL, which then transports it to the liver for excretion.

2nd mechanism for reverse C transport ATP-binding cassette transporters It involves the ATP-binding cassette transporters A1 & G1 (ABCA1 & ABCG1). They are transporter proteins that couple hydrolysis of ATP to the binding of a substrate, enabling it to be transported across the membrane. ABCG1: transports C from cells to HDL. ABCA1: preferentially promotes efflux to poorly lipidated particles such as preβ-HDL, which are then converted to discoidal HDL then to HDL3 and finally to HDL2.

HDL cycle: interchange of HDL2 & HDL3 HDL3 generated from discoidal HDL by the action of LCAT, accepts cholesterol from the tissues via SR-B1 and C is then esterified by LCAT, increasing the size of particles to form the less dense HDL2. HDL2 is formed due to the action of cholesterol ester transfer protein (CETP). Exchanges CE with CM & VLDL remnants; gets TG in return. By transferring CE out of HDL, product inhibition of CE on LCAT is eliminated allowing more cholesterol removal from peripheral tissues. When C efflux followed by esterification by LCAT is effective, HDL2 will be high. HDL3 is then reformed, either after selective delivery of CE to the liver via the SR-B1 or by hydrolysis of HDL2 PL & TG by hepatic lipase (its activity is increased by androgens and decreased by estrogens → higher conc. of plasma HDL2 in women) and endothelial lipase. Free apo-A1 is released by these processes and forms Preβ-HDL after associating with a minimum amount of PL & C. Surplus apo-A1 is destroyed by kidney.

HDL metabolism

HDL: types Pre-β-HDL (<5nm): Hydrolyzed CM & VLDL donate PL & apo-A1. This induces cholesterol efflux leading to discoidal HDL. Discoidal HDL (nascent HDL): newly secreted HDL from intestine with additional C & PL. Contains Apo-A1. Does not contain apo C or E; synthesized in liver & transferred to HDL. HDL3 (5-10nm):formed due to the action of LCAT. HDL2 (10-20nm): formed due to the action of cholesterol ester transfer protein (CETP). CE transferred to CM & VLDL remnants is degraded to bile acids. HDLc (HDL1) (20-25nm): is found in blood of diet-induced hypercholesterolic animals. It is rich in C, and its sole apolipoprotein is apo-E.

Liver C C C TG CE Intest CM Apo-A1 SR-B1 ABCA1 HDL R PL & C preβHDL Peripheral tissue C Discoid HDL ABCG1 C LCAT + A1 SR-B1 HDL3 TG VLDL IDL LDL LCAT CETP HL EL HDL2 (↓↓ CE) (↑↑TG) CE

Reverse Cholesterol Transport (RCT) The process whereby excess cholesterol in peripheral cells, especially foam cells, is returned to the liver for degradation and excretion.

HDL summary HDL ACAT LCAT Cholesterol ester formation Site Intracellular Extracellular in HDL (in blood) Acyl donor Acyl Co-A Lecithin Function Storage of cholesterol Transport of cholesterol from tissues to HDL then to the liver HDL Synthesized in liver and intestine. Reservoir of apoproteins. Reverse cholesterol transport. Liver is the only organ capable of metabolizing and excreting cholesterol. 52% protein, 48% lipid, 35% C & CE Apo A: Activates LCAT Apo C: Activates LPL Apo E: Remnant receptor binding Cholesterol ester formation

Lipoprotein (a) [Lp(a)] Lp(a) When present, is attached to apo-B100 by a disulfide bond. in some normal population, there is no detectable level of Lp(a) in serum. High blood level is linked to susceptibility for heart attack at a younger age. Lp(a) is similar in structure to plasminogen. So, it interferes with plasminogen activation and impairs fibrinolysis. This leads to thrombosis & MI.

Separation of Lipoproteins Why do we have different types of lipoproteins? They differ in lipid and protein composition and therefore. So, they differ in Size and density Electrophoretic mobility 1- Ultracentrifugation: Lipoproteins are separated according to their density. The higher the protein content the higher the density of the particles. The main fractions separated are: CM, VLDL, LDL, HDL & Free fatty acids-albumin complex.

Separation of Plasma lipoproteins 2- Electrophoresis: - It depends on the charge and molecular weights of various particles. - Plasma lipoproteins are separated according to their mobility in electric field into: 1.  lipoproteins. ( HDL) 2. Pre -lipoproteins. (VLDL) 3.  lipoproteins. (LDL) 4. Chylomicrons. (immobilized fraction) [Free fatty acids-albumin complex is not considered typical lipoprotein fraction]

- + Thank you & good Luck Dr. Eman Shaat Chlomicrons VLDL LDL HDL Albumin+FFA Ultracentrifugation fractions Electrophoresis fractions Non-mobile lipoproteins B-Lipoproteins Pre-B Lipoproteins α-Lipoproteins + Thank you & good Luck Dr. Eman Shaat