Cholesterol & lipoproteins

Introduction Lipidscholesterol triglycerides insoluble in plasma lipid is carried in lipoproteins

energy utilizationlipid depositionsteroid hormone productionand bile acid formation.

Cholesterol FunctionsMembrane componentPrecurser toBile acidsVitamin DSteroid hormones

Cholesterol makes phospholipid membranes more plasticHOcholesterol

Cholesterol associates with sphingolipids and certain proteins to form rafts in the plasma membraneLipids probably are more ordered in rafts than elsewhere in the membrane because sphingolipids usually have long, saturated fatty acid side chains

Cholesterol FunctionsMembrane componentPrecurser toBile acidsVitamin DSteroid hormones

Cholesterol serves as a precursor for steroid hormones & bile acids

Steroids are members of a large group of natural products with structures based on isopreneubiquinone-7 (35-carbon side chain)cholesterol(27 carbons)squalene(30 carbons)b-carotene(40 carbons)isoprene

Central Role of the Liver in Cholesterol Balance:Sources of hepatic cholesterolDietary cholesterolFrom chylomicron remnantsCholesterol from extra-hepatic tissuesReverse cholesterol transport via HDLChylomicron remnantsIDLDe novo synthesis

Central Role of the Liver in Cholesterol Balance:Fate of hepatic cholesterolVLDL -> LDLTransport to extra-hepatic tissuesDirect excretion into bileGallstones commonly are precipitates of cholesterolOccurs when bile becomes supersaturated with cholesterolObesity, biliary stasis, infectionsBile acid synthesis and excretion into bile

De novo Synthesis of CholesterolPrimary site: liver (~1g/d)Secondary sites: adrenal cortex, ovaries, testesOverall equation:

De novo Synthesis of Cholesterol:four stagesFormation of HMG CoA (cyto)

Conversion of HMG CoA to activated isoprenoids

De novo Synthesis of Cholesterol:four stagesFormation of HMG CoA (cyto)

Conversion of HMG CoA to activated isoprenoids

Mevalonate is synthesized from three molecules of acetylCoAvia b-hydroxy-b-methylglutarylCoA (HMG-CoA)2 NADPH + 2H+2 NADP+ + CoA-SHHMG-CoA reductasekey control point for cholesterol biosynthesis

The 5-carbon isoprenoid building block is synthesized from acetate by way of a 6-carbon intermediate, mevalonate

Formation of isopentenyl pyrophosphate from mevalonate consumes three molecules of ATPmevalonateATPATPATPisopentenyl pyrophosphate

De novo Synthesis of Cholesterol:four stagesCondensation of isoprenoids to squaleneSix isoprenoids condense to form 30-C molecue

Isopentenyl pyrophosphate tautomerizes to dimethylallyl pyrophosphate, which can form a relatively stable carbonium ionallyl carbonium ion

Isopentenyl pyrophosphate and dimethylallyl pyrophosphate combine to form geranyl pyrophosphateThis creates a new allyl-pyrophosphate derivative that can combine with a second molecule of isopentenyl pyrophosphate to form farnesyl pyrophosphate (15 carbons).dimethylallyl-pyrophosphategeranyl-pyrophosphate (10 carbons)

Two molecules of farnesylpyrophosphate condense head-to-head to form squalene

De novo Synthesis of Cholesterol:four stagesConversion of Squalene to Cholesterol

Formation of squalene epoxide requires O2 and NADPH

Cyclization of squalene epoxide zips up the sterol ringsProtonation opens the epoxide and generates a carbonium ion that reacts with the nearby C=C double bond, creating a new carbonium ion theres more

The cyclase reaction continuesI wont expect you to remember these details.

Experiments with radioactive tracers showed that rats synthesize cholesterol and squalene from acetate.The labeling patterns in squalene and cholesterol were similar, supporting the hypothesis that squalene is an intermediate in cholesterol biosynthesis.cholesterolacetateCH3CO2-squalene

De novo Synthesis of Cholesterol:What do you need to know?All carbons from acetyl-CoARequires NADPH, ATP, & O2StagesOne: forms HMG CoATwo: forms activated 5 carbon intermediates (isoprenoids)Three: six isoprenoids form squaleneFour: squalene + O2 form cholesterol

Regulation of Cholesterol SynthesisCellular cholesterol content exerts transcriptional controlHMG-CoA reductaseHalf life = 2 hoursLDL-receptor synthesisNutrigenomics:interactions between environment and individual genes and how these interactions affect clinical outcomes

Cholesterol prevents activation of transcription of the HMG-CoA reductase gene Cholesterol binds reversibly to a protein that holds the Sterol Regulatory Element Binding Protein (SREBP) in the ER membraneIn the absence of cholesterol, the proteins separate and SREBP is cleaved by proteasesA soluble fragment of SREBP diffuses to the nucleus, where it activates transcriptionto nucleus

Regulation of Cholesterol SynthesisCovalent Modification of HMG-CoA ReductaseInsulin induces protein phosphataseActivates HMG-CoA reductaseFeeding promotes cholesterol synthesisActivates reg. enzymeProvides substrate: acetyl CoAProvides NADPH

Regulation of Cholesterol SynthesisCovalent Modification of HMG-CoA ReductaseGlucagon stimulates adenyl cyclase producing cAMPcAMP activates protein kinase AInactivates HMG-CoA reductaseFasting inhibits cholesterol synthesis

Cholesterol biosynthesis is controlled primarily by HMG-CoA reductase b-Hydroxy-b-methyl-glutarylCoAmevalonatecholesterolHMG-CoA reductaseinsulinglucagonCholesterol or a cholesterol derivative inhibits synthesis and stimulates proteolysis of HMG-CoA reductase. enzymeproteolysisenzyme synthesisPhosphorylation inactivates HMG-CoA reductase; dephosphorylation activates it.Synthesis of the low-density lipoprotein (LDL) receptor, which mediates uptake of lipoproteins containing cholesterol, also is regulated. Cholesterol or a derivative inhibits synthesis of the receptor.

The lipoprotein ProteinEesterified and unesterified cholesterolTriglyceridesPhospholipids

Lipoproteins carry cholesterol between the liver and other tissuesThis is a model. The structures of lipoproteins are not known.

protein components apolipoproteins or apoproteins. cofactors for enzymes ligands for receptors

CLASSIFICATIONChylomicronsVery low density lipoproteinIntermediate density lipoproteinLow density lipoproteinHigh density lipoprotein

Chylomicrons very large particles carry dietary lipidThey are associated with a variety of apolipoproteins A-I A-II A-IV B-48 C-I C-II C-III E.

Very low density lipoproteincarries endogenous triglycerides to a lesser degree cholesterol The major apolipoproteins B-100C-I C-II C-III E

Intermediate density lipoprotein Carries cholesterol esters and triglycerides apolipoproteins B-100 C-III E.

Low density lipoprotein

cholesterol esters apolipoprotein B-100

High density lipoprotein

carries cholesterol esters associated with apolipoproteins A-IA-IIC-IC-IIC-IIIDE

ApolipoproteinsDefects in apolipoprotein metabolism lead to abnormalities in lipid handling

A-I Structural protein for HDL; activator of lecithin-cholesterol acyltransferase (LCAT)

A-II Structural protein for HDL; activator of hepatic lipase.

A-IV Activator of lipoprotein lipase (LPL) and LCAT.

B-100Structural protein for VLDL, IDL, LDL, and Lp(a); ligand for the LDL receptor; required for assembly and secretion of VLDL.

B-48 Contains 48 percent of B-100; required for assembly and secretion of chylomicrons; does not bind to LDL receptor.

C-I Activator of LCAT.

C-II Essential cofactor for LPL.

C-III Interferes with apo-E mediated clearance of triglyceride-enriched lipoproteins by cellular receptors inhibits triglyceride hydrolysis by lipoprotein lipase and hepatic lipase

D May be a cofactor for cholesteryl ester transfer protein (CETP).

E Ligand for hepatic chylomicron and VLDL remnant receptor leading to clearance of these lipoproteins from the circulation ligand for LDL receptor

Apo(a) Structural protein for Lp(a) inhibitor of plasminogen activation on Lp(a).

Partial structures of Apolipoprotein-E and the LDL receptor are knownReceptor-binding domain of Apo-Elipoprotein-binding portion of the LDL receptorApo-E, the only protein in most LDLs, has 3 common alleles: Apo-E3 is the most common form. Apo-E4 has been linked to Alzheimers disease & elevated risk of heart disease. Apo-E2 binds poorly to LDL receptors, & is associated with hyperlipidemia.

Cells take up low-density lipoproteins by receptor-mediated endocytosis

EXOGENOUS PATHWAY OF LIPID METABOLISM

ENDOGENOUS PATHWAY OF LIPID METABOLISMfour common sequence polymorphisms in the hepatic lipase gene promoter

Most trials of hypolipidemic therapy to prevent coronary heart disease (CHD) have evaluated patients with hypercholesterolemia due to elevations in LDL-cholesterol.

What is The role of HDL?

A growing body of evidence suggests that HDL cholesterol is an independent risk factor for CAD

The combination of high triglycerides and low HDL levels,

high serum HDL-cholesterol (>60 mg/dL is associated with a lower risk of CHD

One definition of dyslipidemia total cholesterol, LDL-cholesterol, triglyceride, apo-B, or Lp(a) concentrations above the ninetieth percentile HDL-cholesterol apo A-1 concentrations below the tenth percentile for the general population

HDL METABOLISM Hepatic and intestinal synthesis of small nascent HDL particles composed of phospholipid and apolipoproteins.

Procurement of surface components (phospholipids, cholesterol, and apolipoproteins) from triglyceride-depleted chylomicron and VLDL remnants.

HDL subfractions

HDL2 (density range, 1.063 to 1.125 g/mL) HDL3 (density range 1.125 to 1.21 g/mL)

The clinical importance of the different HDL subfractions is uncertain.

CLINICAL CLASSIFICATION OF DYSLIPIDEMIAS

Fredrickson phenotype

Cholesterol and Bile Acid/Salt MetabolismMajor excretory form of cholesterolSteroid ring is not degraded in humansOccurs in liverBile acid/salts involved in dietary lipid digestion as emulsifiers

Types of Bile Acids/Salts Primary bile acidsGood emulsifying agentsAll OH groups on same sidepKa = 6 (partially ionized)Conjugated bile saltsAmide bonds with glycine or taurineVery good emulsifierpKa lower than bile acids

Synthesis of Bile SaltsHydroxylationCytochrome P-450/mixed function oxidase systemSide chain cleavageConjugationSecondary bile acidsIntestinal bacterial modificationDeconjugationDehydroxylationDeoxycholic acid Lithocholic acid

Recycling of Bile AcidsEnterohepatic circulation98% recycling of bile acidsCholestyramine TreatmentResin binds bile acidsPrevents recyclingIncreased uptake of LDL-C for bile acid synthesis

High levels of cholesterol in the blood can result in atherosclerosis -- deposition of cholesterol and other materials in the inner walls of blood vesselsIndividuals with defective LDL receptors have exceptionally high plasma cholesterol (familial hypercholesterolemia). Because cholesterol does not enter their cells, HMGCoA reductase is not regulated properly and cholesterol biosynthesis remains switched on. If untreated, people with this condition tend to die of atherosclerosis at a young age.Major risk factors:high blood cholesterol (especially LDL cholesterol > 100 mg/dL)smokingdiabetes mellitusobesityphysical inactivityCoronary artery disease is the leading cause of death in industrialized countries.There usually are no symptoms until blood flow to the heart is seriously compromised.

Inhibitors of HMG-CoA reductase (statins) are used clinically to decrease cholesterol biosynthesisIn addition to decreasing LDL cholesterol, statins decrease the level of C-reactive protein in the blood. C-reactive protein is a marker of acute inflamation. Its role in atherosclerosis is unclear.Ridker et al., New Engl. J. Med. 325: 20 (2005)Nissen et al., New Engl. J. Med. 352: 29 (2005)

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIReducing intake of dietary saturated fat to < 7% of caloriesProposed mechanism:High saturated fat intake reduces activity of LDL-receptorsHigher unsaturated fat intake increases activity of LDL-receptorsSide effects: none

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIReduce intake of dietary cholesterol to less than 200mg/dayProposed mechanism:Reducing exogenous source of cholesterol reduces intracellular cholesterol pool and up-regulates LDL-receptorsSide effects: none

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIIncrease consumption of viscous soluble dietary fiber (10-25g/d)Proposed mechanisms:Impairs absorption of dietary cholesterolImpairs reabsorption of bile acidsBacterial fermentation of soluble fibers results in short chain fatty acids that may inhibit cholesterol synthesisSide effects: minimal (laxative)

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIConsume therapeutic doses of plant sterols and stanols (2g/d)Functional foodsBenecol, Take ControlProposed mechanismInhibit absorption of dietary cholesterolInhibit re-absorption of cholesterol in bileSide effects: none

Plant sterolsDifferent side chainsPlant stanolsNo double bond on B ring

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIHMG-CoA Reductase InhibitorsStatins18-55% reduction in LDL-CIncreases in HDL and decreases in TGProposed mechanism of actionInhibition of cholesterol synthesis reduces intracellular cholesterol pool and up-regulates LDL-receptorsSide effects: myopathy, increased serum hepatic enzymes

Structures of Common statindrugs

Statin drugs are structural analogs of HMG-CoA

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIBile acid sequestrantsReduces LDL by 15-30%Mechanism of actionBinds and prevents reabsorption of bile acidsIncreases hepatic synthesis of bile acids, reduces cholesterol pool, up-regulates LDL-receptorsSide effects: GI distress, constipation, decreased absorption of other drugs

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIPharmacological doses of niacin5-25% reduction in LDLIncreases HDL, decreases LDLProposed mechanismReduces VLDL synthesisDecreases lipolysis in adiposeIncreases LPL activityDecreases esterification of TG in liverSide effects: flushing, GI distress, hyperglycemia, hyperuricemia, hepatotoxicity

Nutritional and Pharmaceutical Means for Treating HypercholesterolemiaNCEP-ATP IIIFibric AcidsDecreases LDL by 5-20%Larger decreases in TG (20-50%), increases HDLMechanism of action: increases LPL activitySide effects: dyspepsia, myopathy, gallstones

The exogenous pathway starts with the intestinal absorption of dietary cholesterol and fatty acids Within the intestinal cell, free fatty acids combine with glycerol to form triglycerides, and cholesterol is esterified by acyl:cholesterol acyltransferase (ACAT) to form cholesterol esters. The important role of ACAT was established in an animal model of ACAT deficiency, which found complete resistance to diet-induced hypercholesterolemia due to lack of cholesterol ester synthesis and reduced capacity to absorb cholesterol [9]. Triglycerides and cholesterol are assembled intracellularly as chylomicrons. The main apolipoprotein is B-48, but C-II and E are acquired as the chylomicrons enter the circulation. Apo B-48 permits lipid binding to the chylomicron but not does not bind to the LDL receptor, thereby preventing premature clearance of chylomicrons from the circulation before they are acted upon by LPL.Apo C-II is a cofactor for LPL which makes the chylomicrons progressively smaller primarily by hydrolyzing the core triglycerides and releasing free fatty acids. The free fatty acids are then used as an energy source, converted to triglyceride, or stored in adipose tissue. The end-products of chylomicron metabolism are chylomicron remnants that are cleared from the circulation by hepatic chylomicron remnant receptors for which apo E is a high-affinity ligand. The chylomicron remnants contain a smaller core of lipids that is enveloped by excess surface components. These surface constituents are transferred from the chylomicron remnant for the formation of HDL.The endogenous pathway of lipid metabolism begins with the synthesis of VLDL by the liver . VLDL particles contain a core of triglycerides (60 percent by mass) and cholesterol esters (20 percent by mass). Microsomal triglyceride transfer protein is essential for the transfer of the bulk of triglycerides into the endoplasmic reticulum for VLDL assembly and for the secretion of apo B-100 from the liver. The surface apolipoproteins for VLDL are noted above. They include apo C-II which acts as a cofactor for LPL, apo C-III which inhibits this enzyme, and apo B-100 and E which serve as ligands for the apolipoprotein B/E (LDL) receptor .The triglyceride core of nascent VLDL particles is hydrolyzed by lipoprotein lipase. During lipolysis, the core of the VLDL particle is reduced, generating VLDL remnant particles (also called IDL) that are depleted of triglycerides via a process similar to the generation of chylomicron remnants. Some of the excess surface components in the remnant particle, including phospholipid, unesterified cholesterol, and apolipoproteins A, C and E, are transferred to HDL.VLDL remnants can either be cleared from the circulation by the apo B/E (LDL) or the remnant receptors or remodeled by hepatic lipase to form LDL particles. There are four common sequence polymorphisms in the hepatic lipase gene promoter; the most frequent is a C to T substitution [11]. The presence of a C allele is associated with higher hepatic lipase activity; smaller, denser, and more atherogenic LDL particles, and inversely with lower levels of HDL cholesterol even in the absence of increased LDL, is more common in patients with CRD than in the general population, and ignoring these lipid abnormalities in renal disease patients who are at increased risk for CAD may not be warranted.

The HDL2 density range is comprised of apo A-I HDL particles and the HDL3 density range is comprised of particles containing apo A-l and A-II. The apo A-I HDL particles are strongly associated with the cholesterol efflux-promoting effects of HDL, whereas the particles containing both apoproteins are less effective for mobilization of cholesterol from nonhepatic cells and appear to have other functionsJ. C. Cohen et al. New Engl. J. Med. 354: 1264 (2006) Sequence Variations in PCSK9, Low LDL, and Protection against Coronary Heart Disease. Lifelong reduction of LDL cholesterol is associated with reduced risk of heart disease.