Cholesterol Transported in the Blood in Lipoproteins Because It Is Absolutely Insoluble

Cholesterol Overview

·  Cholesterol – transported in the blood in lipoproteins because it is absolutely insoluble in water

o  Serves as stabilizing component of cell membranes and as precursor of bile salts as well as steroids

·  Precursors of cholesterol converted to ubiquinone, dolichol, and (in the skin) cholecalciferol (active form of vitamin D)

·  As major component of blood lipoproteins, cholesterol can appear in its free, unesterified form in outer shell of macromolecules and as cholesterol esters in lipoprotein core

·  Precursor for cholesterol synthesis is acetyl coenzyme A (acetyl-CoA), which can be produced from glucose, fatty acids, or amino acids

o  2 acetyl-CoA molecules form acetoacetyl-CoA, which condenses with another acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA)

o  Reduction of HMG-CoA produces mevalonate (catalyzed by HMG-CoA reductase – rate-limiting step)

o  Mevalonate produces isoprene units that condense, eventually forming squalene

o  Cyclization of squalene produces the steroid ring system, and several subsequent reactions generate cholesterol

·  Adrenal cortex, gonads, liver, and intestine all produce cholesterol in significant quantities, but the process can be found in any cell in the body

·  Cholesterol is packaged in chylomicrons in intestine and VLDL in the liver and is transported in these forms along with triacylglycerol

o  As triacylglycerols of blood lipoproteins digested by lipoprotein lipase, chylomicrons are converted to chylomicron remnants, and VLDL is converted to intermediate-density lipoprotein (IDL) and subsequently LDL

o  Above products return to liver, where they bind to receptors and are taken up into vesicles which will then be digested by lysosomal enzymes

o  Cholesterol and other products of lysosomal digestion released into cellular pools

o  Liver uses recycled cholesterol and cholesterol synthesized from acetyl-CoA to produce VLDL and bile salts

o  Intracellular cholesterol obtained from blood lipoproteins decreases synthesis of cholesterol within cells, stimulates storage of cholesterol as cholesterol esters, and decreases synthesis of LDL receptors

·  HDL – contains triacylglycerols and cholesterol – exchanges proteins and lipids with other lipoproteins in blood

o  Transfers apolipoprotein E (apo E) and apo CII to chylomicrons and VLDL

o  After digestion of VLDL triacylglycerols, apo E and apo CII transferred back to HDL

o  HDL obtains cholesterol from other lipoproteins and from cell membranes and converts it to cholesterol esters by lecithin-cholesterol acyltransferase (LCAT) reaction

§  After the above, HDL either directly transports cholesterol and cholesterol esters to the liver or transfers cholesterol esters to other lipoproteins via the cholesterol ester transfer protein (CETP)

o  Lipoprotein particles carry the cholesterol and cholesterol esters to the liver, where endocytosis and lysosomal digestion occur

o  Reverse cholesterol transport (above return of cholesterol to liver) is major function of HDL

·  Elevated levels of cholesterol in blood associated with formation of atherosclerotic plaques

o  High levels of LDL are especially atherogenic

o  High levels of HDL are protective because HDL particles are involved in process of removing cholesterol from tissues and returning it to liver

·  Bile salts emulsify dietary triacylglycerols – digestive products absorbed by intestinal epithelial cells form bile salt micells (tiny microdroplets that contain bile salts at their water interface)

o  After contents of micells are absorbed, most bile salts travel to the ileum, where they are resorbed and recycled by liver

·  Fecal excretion of bile salts is major means by which body disposes of steroid nucleus of cholesterol

o  Because ring structure of cholesterol cannot be degraded in body, it is excreted mainly in bile as free cholesterol and bile salts

Intestinal Absorption of Cholesterol

·  Key regulatory point in sterol metabolism because it ultimately determines what percentage of biliary cholesterol produced by liver each day and what percentage of dietary cholesterol entering gut per day is eventually absorbed into the blood

o  In normal people, about 55% of intestinal pool enters blood through enterocytes each day

·  Unwanted or excessive cholesterol is removed and sterols from enterocyte are planted

o  ATP-binding cassette (ABC) protein family, specifically ABCG5 and ABCG8 couple ATP hydrolysis to transport of unwanted or excessive cholesterol and plant sterols (phytosterols) from enterocyte back into gut lumen

·  ABCA1 is required for reverse cholesterol transport and biogenesis of HDL

·  ABC protein expression increases amount of sterols present in gut lumen, with potential to increase elimination of sterols into feces

·  Phytosterolemia – rare autosomal recessive disease also called sitosterloemia – defect in function of either ABCG5 or ABCG8 in enterocytes, which leads to accumulation of cholesterol and phytosterols in these cells – eventually reaches bloodstream, markedly elevating level of cholesterol and phytosterol in blood, causing increased cardiovascular morbidity

·  Ezetimibe – structurally different from sterols – primary action in lowering serum cholesterol levels is to block cholesterol absorption through specific cholesterol absorption mechanism in brush border of enterocytes

o  Target is Niemann-Pick C1-like 1 (NPC1L1) protein, which transports cholesterol into cells via absorptive endocytotic mechanism (clathrin-dependent)

Cholesterol Synthesis

·  Cholesterol – alicyclic compound whose basic structure includes perhydrocyclopentanophenanthrene nucleus containing four fused rings (3 cyclohexanes and 1 cyclopentane)

·  About 1/3 of cholesterol exists in free (unesterified) form, and the other 2/3 exist as cholesterol esters in which a long-chain fatty acid (usually linoleic acid) is attached by ester linkage to OH at C3 (bottom left corner of above picture)

o  Percentage of free and esterified cholesterol can be measured using high-performance liquid chromatography (HPLC)

Cholesterol Synthesis Stage 1: Synthesis of Mevalonate from Acetyl-CoA

·  Committed, rate-limiting step in cholesterol formation

·  HMG-CoA synthase present in cytosol and distinct from mitochondrial HMG-CoA synthase that catalyzes HMG-CoA synthesis involved in production of ketone bodies

·  Committed step and major point of regulation is reduction of HMG-CoA to mevalonate (last step in picture to right)

o  HMG-CoA reductase embedded in membrane of ER and contains 8 membrane-spanning domains – amino-terminal domain faces cytoplasm and contains enzymatic activity

·  Rate of synthesis of HMG-CoA reductase mRNA controlled by a member of sterol-regulatory element-binding protein family (SREBPs)

o  Transcription factors belong to basic helix-loop-helix leucine zipper (bHLH-Zip) family of transcription factors that directly activate the expression of more than 30 genes dedicated to synthesis and uptake of cholesterol, fatty acids, triacylglycerols, and phospholipids as well as production of NADPH cofactors required to synthesize these molecules

o  SREBPs specifically enhance transcription of HMG-CoA reductase gene by binding to sterol-regulatory element (SRE) upstream of the gene – when bound, rate of transcription increases

o  SREBPs, after synthesis, are integral proteins of ER

§  SREBP is bound to SREBP cleavage-activating protein (SCAP) in ER membrane when cholesterol levels are high

§  When cholesterol levels drop, sterol leaves SCAP-binding site and SREBP-SCAP complex is transported to Golgi apparatus

§  In Golgi, 2 proteolytic cleaves occur (via site 1 and site 2 proteases [S1P and S2P]), which release N-terminal transcription factor domain from Golgi membrane

§  Once released, active amino terminal component travels to nucleus to bind to SREs

o  Soluble SREBPs are rapidly turned over and need to be continuously produced to stimulate reductase mRNA transcription effectively

o  When cytoplasmic sterol levels rise, sterols bind to SCAP and prevent translocation of complex to Golgi, leading to decrease in transcription of reductase gene and thus less reductase protein being produced

·  Rising levels of cholesterol and bile salts in cells that synthesize above molecules may cause change in oligomerization state of membrane domain of HMG-CoA reductase, rendering the enzyme more susceptible to proteolysis, decreasing its activity

o  Membrane domains of HMG-CoA reductase contain sterol-sensing regions similar to those in SCAP

·  Activity of reductase regulated by phosphorylation and dephosphorylation

o  Elevated glucagon levels increase phosphorylation of HMG-CoA reductase, inactivating it

o  Hyperinsulinemia increases activity of reductase by activating phosphatases, which dephosphorylate HMG-CoA reductase

o  Increased levels of intracellular sterols increase phosphorylation of HMG-CoA reductase (feedback suppression)

o  Thyroid hormone increases HMG-CoA activity

o  Glucocorticoids decrease HMG-CoA activity

o  AMP-activated protein kinase is the enzyme that actually phosphorylates HMG-CoA reductase

§  AMP-activated protein kinase is regulated by phosphorylation by LKB1

o  Cholesterol synthesis decreases when ATP levels low and increases when ATP levels high

Cholesterol Synthesis Stage 2: Conversion of Mevalonate to Two Activate Isoprenes

·  Purpose of phosphate transfers is to activate both C5 and hydroxyl group on C3 for further reactions

o  Phosphate group attached to C3 hydroxyl group of mevalonate in 3-phospho-5-phyrophosphomevalonate intermediate is removed along with carboxyl group on C1, producing the double bond in the second to last step (see pathway on previous page)

·  The bottom two products are the isoprenes – also used in synthesis of coenzyme Q and dolichol

Cholesterol Synthesis Stage 3: Condensation of 6 Activated 5-Carbon Isoprenes to Form the 30-Carbon Squalene

·  Head-to-tail condensation of isopentenyl pyrophosphate and dimethylallyl pyrophosphate

o  “head” is end of molecule to which pyrophosphate is linked

·  Geranyl pyrophosphate undergoes another head-to-tail condensation with isopentenyl pyrophosphate

·  2 molecules of farnesyl pyrophosphate undergo a head-to-head fusion, and both pyrophosphate groups are removed to form squalene

·  Geranyl pyrophosphate and farnesyl pyrophosphate are key components in cholesterol biosynthesis and both can form covalent bonds with proteins, particularly G proteins and certain proto-oncogene products involved in signal transduction

o  Hydrophobic groups anchor proteins in cell membrane

Cholesterol Synthesis Stage 4: Conversion of Squalene to the Four-Ring Steroid Nucleus

·  When squalene monooxygenase adds a single O from O2 to the squalene molecule, NADPH reduces the other O to H2O

·  The “many reactions” are a series of complex reactions, containing many steps, elucidated in the late 1950’s

Several Fates of Cholesterol

·  Placenta in pregnant women can also produce cholesterol

·  Fraction of hepatic cholesterol used for synthesis of hepatic membranes, but bulk is secreted from hepatocyte as

o  Cholesterol esters

o  Biliary cholesterol (cholesterol found in bile)

o  Bile acids

·  Cholesterol ester production in liver catalyzed by acyl-CoA-cholesterol acyl transferase (ACAT), which catalyzes the transfer of fatty acid from coenzyme A to hydroxyl group on C3 of cholesterol

·  Cholesterol esters more hydrophobic than free cholesterol

·  Liver packages some esterified cholesterol into hollow core of lipoproteins, primarily VLDL, which is secreted from hepatocyte into blood and transports cholesterol esters (triacylglycerols, phospholipids, apoproteins, etc.) to tissues that require greater amounts of cholesterol than they can synthesize de novo

·  Tissues use cholesterol for synthesis of membranes, formation of steroid hormones, and biosynthesis of vitamin D

o  Residual cholesterol esters not used stored in liver for later use

·  Hepatic cholesterol pool serves as source of cholesterol for synthesis of hydrophilic bile acids and their salts – very effective detergents because they contain both polar and nonpolar regions

o  Aid in digestion of intraluminal lipids by forming micelles with them, which increases surface area of lipids exposed to digestive action of intraluminal lipases

·  Free cholesterol also enters gut lumen via biliary tract, forming an intestinal pool, 55% of which is resorbed by enterocytes and enters bloodstream

·  Greater intake of dietary cholesterol suppresses rate of hepatic cholesterol synthesis (feedback repression)

Synthesis of Bile Salts

·  Bile salts synthesized by reactions that hydroxylate the steroid nucleus and cleave the side chain

·  First step is rate-limiting reaction (shown to right)

o  Activity of 7-α-hydroxylase decreased by an increase in bile salt concentration

·  pKa of bile acids is ~6

o  Contents of intestinal lumen usually around pH 6, so about 50% of molecules present are protonated, and 50% are ionized (forming bile salts)

ß second step of cholesterol conversion

·  Carboxyl group at end of side chain of bile salts activated by a reaction that requires ATP and CoA derivatives that can react with either glycine or taurine (which is derived from cysteine), forming amides (conjugated bile salts)

o  Glycine conjugated derivatives have a pKa of about 4

o  Taurine conjugates have pKa of about 2

Fate of Bile Salts

·  Bile salts serve as detergents that aid in digestion of dietary lipids

·  Intestinal bacteria deconjugate and dehydroxylate bile salts, removing the glycine and taurine residues and the hydroxyl group at position 7

·  Bile salts that lack a hydroxyl group at position 7 are secondary bile salts

·  Deconjugated and dehydroxylated bile salts are less soluble and thus less readily resorbed from intestinal lumen – they often get excreted

·  Greater than 95% of bile salts are resorbed in ileum and return to liver via enterohepatic circulation (portal vein)

·  Secondary bile salts may be reconjugated in liver, but they are not rehydroxylated

·  Because steroid nucleus cannot be degraded by body, excretion of bile salts serves as major route for removal of steroid nucleus, and thus cholesterol, from body

Transport of Cholesterol by Blood Lipoproteins

·  Cholesterol and cholesterol esters transported through bloodstream packaged as lipoproteins, where are composed of a core of hydrophobic lipids such as cholesterol esters and triacylglycerols surrounded by a shell of polar lipids (phospholipids), which allows hydration shell to form around the lipoprotein

o  Positive charge of nitrogen atom of phospholipid forms ionic bond with negatively charged hydroxyl ion of the environment

·  Shell of lipoprotein contains variety of apoproteins that increase water solubility of lipoprotein

·  Free cholesterol molecules dispersed throughout lipoprotein shell to stabilize it in a way that allows it to maintain its spherical shape

·  Lipoproteins are transported to tissues, where their components are either used in synthetic or oxidative processes or stored for later use

·  Apoproteins – “apo” describes protein within the shell of the particle in its lipid-free form

·  Apoproteins add to hydrophilicity as well as structural stability of the particle

o  Activate certain enzymes required for normal lipoprotein metabolism

o  Act as ligands on surface of lipoprotein that target specific receptors on peripheral tissues that require lipoprotein delivery for their innate cellular functions

·  Chylomicrons – largest of lipoproteins and least dense because of rich triacylglycerol content

o  Synthesized from dietary lipids (exogenous lipoprotein pathway) within epithelial cells of small intestine and secreted into lymphatic vessels draining gut