Lipids and More

5/25/2018 Lipids and More

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Medical Biochemistry

99:163Lipids and moreDan Weeks, BSB 4-710

Email [email protected]

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Overview of the lectures Lectures 1 and 2- Brief review of lipids,

Phospholipids, lipid biomediators and cell signaling

Lecture 3- Sphingolipids and lipid storage disease Lecture 4- Cholesterol Metabolism and protein

prenylation

Lecture 5- Regulation of Cholesterol Metabolism Lecture 6- Lipoprotein structure and transport Lecture 7- Lipoprotein metabolism and

hyperlipidemias Lecture 8- Steroid Hormones Lecture 9- Lipids and nutrition, obesity and PPAR

nuclear receptors


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Where is this material coming from?Sources: 2006 Lecture presentations from Dr. ArthurSpector, professor emeritus in Medical Biochemistry.Textbook of Biochemistry with Clinical Correlations 6thed., Editor Thomas DevlinLehninger : Principles of Biochemistry, Nelson and Cox4th and 5th ed.Basic Medical Biochemistry, A Clinical Approach, 3rdedition, Lieberman and Marks.Lippincotts Illustrated Reviews: Biochemistry, editorsChampe, Harvey and Ferrier, 4th ed.The lipid libraryhttp://www.lipidlibrary.co.uk/Lipids/eicisop/index.htmPrimary literature as noted

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PHOSPHOLIPIDS, LIPIDBIOMEDIATORS, AND CELL

SIGNALING


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OBJECTIVES:

Know and recognize the main lipid classes.Know and understand how fatty acids are named.Know the essential fatty acids and what makes themessential.Understand the role and physical properties of

phospholipids in membranesKnow how phospholipids are synthesizedKnow the main phospholipases and the products formed ineach of these reactionsUnderstand the metabolism of phosphatidylinositol and thefunction of its inositol-phosphate derivatives in cell signaling

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OBJECTIVES:

*Understand the role of arachidonic acid in thesynthesis of the eicosanoid lipid biomediators.

*Know the Cycloxygenase reaction and thedifferences between COX-1 and COX-2.

*Understand how prostaglandins affect cellularfunction.

*Know how leukotrienes are produced.*Recognize the arachidonic acid products formed by thecytochrome P450 pathway.Know how isoprostanes are formed and their

significance as biomarkers.


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KEY WORDS Phospholipid, lipid biomediators,

and cell signaling

phosphatidylcholine (PC) phosphatidylinositol (PI, PIP, PIP2, PIP3)phosphatidic acid (PA) phosphatidylethanolamine (PE)cardiolipin phosphatidylserine (PS)alkyl ether phospholipids plasmalogenslysophospholipid platelet activating factor (PAF)phospholipase cytidylyl transferase1,2-diacylglycerol (DAG) inositol 1,4,5-trisphosphate (IP3)CDP-choline CDP-diacylglycerolprotein kinase C (PKC) PI 3-kinasePTEN prostaglandin (PG)cyclooxygenase (COX-1 and COX-2) arachidonic acideicosapentaenoic acid (EPA) eicosanoidlipoxygenase leukotriene (LT)cytochrome P450 isoprostane

hydroperoxyeicosatetraenoic acid (HPETE)

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What are lipids?Unique and diverse chemistrydue to structure: H2O insoluble; amphipathic

LIPIDS

I. Polar Heads, Non-polar tails/ Non-polara) fatty acids ! triacylglycerols, waxesc) sphingolipidsd) phosphoglycerolse) glycolipids

II. Fused-ring compounds: steroids: backbone cholesterol

What is their function?Diverse!!!

Storage of energy (fats/oils)Structural (membranes:phospholipids/sterols)Enzyme cofactors, pigments, hydrophobic anchors,chaperones for protein folding, hormones, intra-Cellular signaling molecules


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FATTY ACIDS Building blocks

Physical properties:Depend on C chainLength & amt. ofunsaturation

Free fatty acidscarried by albumin.

C=C: cis, C-9 & C-10,C-12 & C-15

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Essential Fatty Acids: Diet

Linoleate (-6) linolenate (-3): precursors of otherLipids (prostaglandins)

COMMON FATTY ACIDS


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Humans donthave the enzymes for these steps

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Nomenclature of FATTY ACIDS

Unfortunately, there are several naming schemes:CH3(CH2)14COOH CH3(CH2) 5CH=CH(CH2) 7COOHSimplest# of carbons:# of double bonds C16:0 C16:1

Common name: often derived from the first source forisolation and carries the acid designation- for examplePalmitic acid (Palmitate) IUPAC: Hexadecanoic acid(Hexa=6 deca=10)Location of double bonds:

Cis/trans -!x: counts from carboxyl group (X) andindicates cis or trans. cis!9 Hexadecanoic acid. All doublebonds are indicated if polyunsaturated.Omega-X (-X) counts from the methyl end.Palmitoleic acid(-7)


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Saturated vs. unsaturated Fatty Acid

Saturated Fatty acids come from animal fats andalso from tropical oils. One of the consequences ofeating saturated fatty acids is the the LDL-Cholesterol levels in your blood become elevated.The conclusion some years ago was that unsaturated fatis better for you.

But- unsaturated fatty acids have a shorter shelf life(they become rancid)- hydrogenation can make themmore stable but this process also generates trans-(incontrast to the more commonly occurring cis) double

bonds.

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Cis vs. trans Fatty Acid

Most of the fatty acids in your pre-french fry state

Are in the cis configuration.

http://content.nejm.org/cgi/content/short/354/15/1601

Review article in New England Journal of Med.:Volume 354:1601-1613 April 13, 2006


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Cis vs. trans Fatty Acid

http://www.elmhurst.edu/~chm/vchembook/558hydrogenation.html

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Trans Fatty Acid

The process of hydrogenation saturates some cis doublebonds, and turns others in to trans double bonds. The trans-configuration leads to a much straighter FA, and a highermelting temperature. They are look and behave more likesaturated FAs. The problem is that trans-fatty acids not onlyincrease LDLs, they decrease HDLs- (more on this later).What has trans-fatty acids?

Anything that says hydrogenated or partiallyHydrogenated vegetable oil.http://www.nationalacademies.org/headlines/20061212.html

New York City Bans Use of Trans Fats in Restaurants

Many cooking supplies, such as margarine, will have to be eliminated from dishes because of their high trans fat

content (Bruno Neves) By Lisa Pickoff-White

December 12 - The New York City Board of Health banned trans fat oils and shortenings from being used in an

estimated 24,000 restaurants. They are also requiring that restaurants with standard menu items, such as Starbucks

or McDonald's, prominently display the amount of calories in each item served, either on menus, menu boards, or

near cash registers. The trans fat and mandatory menu labeling law will be phased in, starting in July 2007, and

restaurants will have 18 months to come into complete compliance. Violations after October 2007 will result in a

fine.


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Adipose cells: Site of storage, synthesis, mobilization

into fuel for transport to tissue

Triacylglycerols: Fatty Acid + glycerol

C6H12O6+6O2

CH3(CH2)14CO2H+ 23O2

6CO2+ 6H2O

16CO2+16H2O

-15.9

-38.9Palmitic acid

Glucose

Energy

kJ mol-1

ester

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STRUCTURE OF LIPIDS


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Phospholipids:

Glycerophospholipids:Glycerol is thebackbone molecule, where does it come

from?

Adipose and most tissues:

Liver and Kidney:

Glycerol kinase reaction

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Phospholipids:

Stereo specific numbering (sn) of L-glycerol phosphate

sn 1 C OH#

sn2 HO C#

sn3 C O PO3H2

Fatty acids are added to positions sn1 and sn2.Usually sn1 has a saturated fatty acid and sn2 anunsaturated fatty acid.


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Phospholipids:

Exception (and an important one medically)

surfactant

Osn 1 C O-C-(CH2)14CH3

# Osn2 C -O-C-(CH2)14CH3

# Osn3 C O P-OCH2 CH2 NCH3

O

Dipalmitoyl phosphatidylcholine

=

=

=

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Pulmonary surfactant:

About 90% of the of pulmonary surfactant is lipid, largelyphosphatidylcholine with some phosphatidylglycerol,neutral lipid and cholesterol. Surfactant also hasproteins, including plasma proteins and theapolipoproteins SP-A, B, C and D.

Infant respiratory distress syndromeis common inpremature babies. Surfactant production begins at about20 weeks gestation, but is generally not adequate tosupport lung function until after 32 weeks or later.

For more information see Medline Plus:http://www.nlm.nih.gov/medlineplus/ency/article/001563.htm


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Clinical Correlations :

Acute respiratory distress syndrome

The lung of premature infants is not sufficiently developed to produce adequate amounts of surfactant, alipoprotein that contains a special form of phosphatidylcholine, called dipalmitoyl phosphatidylcholine. The lackof adequate amounts of surfactant causes poor gas exchange across the alveoli and leads to respiratorydistress.

Most phospholipids have a saturated fatty acid in the sn-1position and an unsaturated fatty acid in the sn-2position. An exception is phosphatidylcholine synthesized by the cells that line the alveoli (alveolar Type II cells)in the lung. O2passes in and CO2passes out through the alveolar membrane. The Type II cells synthesize aphosphatidylcholine that has palmitic acid (16:0) in both sn-positions, dipalmitoyl phosphatidylcholine.

The biochemical reason that the alveolar cells, as opposed to other cells, synthesize phosphatidylcholine with asaturated fatty acid in the sn-2 position is the specificity of the acyltransferase that acylates the sn-2 position.The sn-2 acyltransferase of most other cells is highly selective for polyunsaturated fatty acyl CoA. In contrast,the sn-2 acyltransferase of the alveolar Type II cells utilizes saturated fatty acyl CoA. Therefore, the sn-2position of the phosphatidylcholine in these cells becomes highly enriched in palmitic acid, a saturated fattyacid.

Dipalmitoyl phosphatidylcholine is the main lipid component of the lipoprotein material that is secreted by the

alveolar Type II cells, calledpulmonary surfactant. Surfactant coats the alveoli and thereby lowers the surfacetension. This facilitates the movement of O2and CO2across the alveolar membrane, which is necessary forefficient gas exchange in respiration.

Because the lung of many premature infants is not sufficiently developed to produce enough surfactant, theyoften have a serious respiratory problem and require intensive care and O2therapy to insure adequate tissueoxygenation.

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Pulmonary surfactant:

Most phospholipids have a saturated fatty acid in the sn-1position and an unsaturated fattyacid in the sn-2position. An exception is phosphatidylcholine synthesized by the cells thatline the alveoli (alveolar Type II cells) in the lung. O2passes in and CO2passes out throughthe alveolar membrane. The Type II cells synthesize a phosphatidylcholine that has palmiticacid (16:0) in both sn-positions, dipalmitoyl phosphatidylcholine.

The biochemical reason that the alveolar cells, as opposed to other cells, synthesizephosphatidylcholine with a saturated fatty acid in the sn-2 position is the specificity of theacyltransferase that acylates the sn-2 position. The sn-2 acyltransferase of most other cellsis highly selective for polyunsaturated fatty acyl CoA. In contrast, the sn-2 acyltransferase ofthe alveolar Type II cells utilizes saturated fatty acyl CoA. Therefore, the sn-2

position of thephosphatidylcholine in these cells becomes highly enriched in palmitic acid, a saturated fattyacid.

Dipalmitoyl phosphatidylcholine is the main lipid component of the lipoprotein material thatis secreted by the alveolar Type II cells, calledpulmonary surfactant. Surfactant coats thealveoli and thereby lowers the surface tension. This facilitates the movement of O2and CO2

across the alveolar membrane, which is necessary for efficient gas exchange in respiration.


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Phospholipids

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Phospholipids:

Cardiolipin

Lysophosphoglycerides

Lysophosphatidylcholine (LPC) L y sophosphatidic acid (LPA)


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Antiphospholipid Syndrome

Antibodies against protein-phospholipid complexes sometimes occur in humans.They include lupus anticoagulantand anticardiolipin antibodies. Both antibodiesare present in some patients, in others, only one is present.

The clinical syndrome is characterized by venous and arterial thrombosis,pulmonary embolism, thrombocytopenia, leg ulcers, migraine headaches, and infemales, spontaneous abortion. A high prevalence of valvular heart lesions occurs,characterized by thickening of the valve leaflets. There are two forms of theantiphospholipid syndrome. One form of the syndrome is primary; that is, there isno underlying disease. The other is secondary to the disease systemic lupuserythematosus. Most patients have the primary form.

This diagnosis should be considered in patients who have an unexplained

thromboembolic event. However, the production of the antiphospholipid antibodiescan be transient and decrease when thromboembolism occurs, making thediagnosis difficult.

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ETHER LINKAGES:


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ETHER LINKAGES:

Synthesis starts in the peroxisome- wherean acyl group in the 1 position is replaced with and alkyl. Thevinyl ether is generated at the membrane. Plasmalogens aremembrane lipids and serve as a specific substrate for some

lipases and a target for oxygen radicals as well as beingassociated with lipid rafts and synaptic vesicles.

Vs.

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ETHER LINKAGES:

Platelet-activating factor is not a membrane lipid- but ismade in response inflammatory stimuli, usinglysophosphotidylcholine and acetylCoA usinglysophosphotidylcholine transferase. Inactivation is via PAF

acetylhydrolase. It was the first lipid to be identified as asignaling molecule. Cells that respond to PAF have specificreceptors. PAF is a mediator of inflammation, vascularpermeability, platelet aggregation, bronchial constriction.Works via G-protein coupled reaction.

Vs.


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What does a membrane look like?

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Different membranes have unique compositions


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Fluid mosaic model for membrane

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The composition ofthe inner and outerleaflet need not bethe same.

Changes incomposition can beused to signal.


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Lipids (and proteins) in

membranes are notstatic

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Both diffusion of lipid and membrane proteins can beRestricted to a neighborhood.


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Scanning atomic force micrograph of membrane surface.

Lipid rafts.

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Liposomes and drug or gene delivery

Liposomes are vesicles formed in the laboratory from phospholipids. They can be used to transfer drugs or

genes into cells, and this has potential clinical utility.

When phospholipids are dispersed in an aqueous solution, they form multilamellar liposomes. Thephospholipids are in the form of a lipid bilayer. Multilamellar liposomes are relatively large, about 1 m indiameter. If the multilamellar liposomes are exposed to sonic irradiation, they form smaller vesiclescontaining a single lipid bilayer surrounding an internal aqueous core. This structure is called a unilamellarliposome. Think of a unilamellar liposome as a tennis ball, with the cover being a phospholipids bilayer anda center compartment filled with water. Almost all clinical applications use unilamellar liposomes.

The surface structure and physical properties of the liposome can be made to vary, depending on thecomposition of the lipid mixture used to make the liposomes. By varying the surface composition, theliposomes can be targeted to specific types of cells.

The aqueous core of the liposome will contain what is in the solution used to make the liposomes, such as achemotherapeutic drug or a plasmid. Liposomes fuse with cell membranes. When fusion occurs, thecontents of the aqueous compartment will spill into the cytosol. Thus, a drug or plasmid that ordinarily hasdifficulty in crossing the cell membrane can enter the cell. There are additional potential advantages of usingliposomes for drug delivery. If the liposome can be targeted specifically to the cancer cells, unwanted side

effects of the chemotherapeutic drug would be reduced. Also, by encapsulating the drug in a liposome, therate of inactivation and excretion should decrease, prolonging the chemotherapeutic effect of each dose andpossibly enabling the use of lower doses for treatment. Clinical trials are in progress to test the therapeuticeffectiveness of liposomes in drug delivery, particularly in cancer chemotherapy.


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Synthesis:Triacylglycerol,

Phospholipids

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2 Routes to Diverse Phospholipids

Formation of phosphodiester bond

1. Activation of OH with CDP

2. Activation of Head Group with CDP

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Phospholipid synthesisA. From activated DAG1) Phosphatidate + CTP to form activated intermediate:CDP-diacylglycerol + PPi

2) Reaction of hydroxyl group phosphodiester linkExamples: serine: phosphatidyl serine; inositol:

phosphatidyl inositol. Components vary; many species of

phospholipids.

B. Synthesis from activated alcohol

CTP + phosphorylethanolamine CDP-ethanolamine

CDP-ethanolamine + DAG phosphatidylethanolamine


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Salvage pathway for

lipid synthesis

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Synthesis of

phosphatidylcholine


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Phospholipases: These are enzymes that release thefatty acids or intermediates from phospholipids.Phospholipase A: A1 cleaves the sn-1 acyl chain, A2cleaves the sn-2 acyl chainPhospholipase B: can cleave either positionPhospholipase C: cleaves before the phosphategenerating DAG and a phosphorylated head groupPhospholipase D: cleaves after the phosphate leavingphosphatidic acid and the alcohol of the head group.

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What roles do phospholipases have?

Phospholipase A and B assist in remodeling phospholipidsor freeing a specific acyl chain (like arachidonic acid).


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What roles do phospholipases have?

Phospholipase C : Really a family of phosphodiesterases.Used in signal transduction involving the release of Inositoltriphosphate (IP3or inositol 1,4,5 trisphosphate) as asecond messenger.

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Note:PI- phosphatidyl inositol

PIP- inositol carries anadditional phosphate oftenat position 4PIP2--inositol has twophosphates at the 3,4 or 5position andPIP3--inositol has threephosphates (3,4,5)

IP3 released from sn3

position

50http://www.uni-tuebingen.de/dundee.ac.uk-PI3K/research.htm

PI3 kinase allows free PIP to serve a signaling role


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PI3 kinases and cancers:

Stein, R Endocrine-Related Cancer(2001) 8237!248

1. There is a family of PI3 kinase proteins. Many isoforms.2. Activation of PI3 kinases is important for cell growth andsurvival, intracellular trafficking and cellular motility.Some classes of PI3 kinase are strongly activated byinsulin.

3. Inappropriate activation of PI3 kinase or inhibition ofPTEN (phosphatase and tensin homolog) the enzymethat removes the activating phosphate can lead tocancer. PTEN is often called an anticancer or anti-oncogene.

Seminars in Cell & Developmental BiologyVolume 15, Issue 2, April 2004, Pages 171-176Signaling Downstream of Phosphoinositide 3-kinase and Ubiquitin -Like Proteins Meet the Family

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Inositol phosphates as cofactors:

Macbeth et al. (2005) showed that one enzyme responsible for RNAediting uses inositol hexakisphosphate (IP6) bound to the enzyme isrequired for activity.New studies by the York lab (Duke) indicate inositol phosphates arecritical to proper protein folding and stabilization.


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Inositol phosphates appear as regulators in:

Embryonic developmentRight left asymmetryRNA editingInsulin responseCalcium signalingMembrane dynamicsNuclear signalingChromatin dynamicsIon channel physiology

See: Tsui, M.M. and York, J.D. (2010) Advances in Enzyme Regulation

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What role do phospholipases have?

Phospholipase D : Generates phosphatidic acid. Oncethought to mainly contribute to head group exchange,newer studies implicate phosphatidic acid in the RASactivation pathway. This may be a breaking story as youcontinue to study medicine.

See>C. Zhao, G. Du, K. Skowronek, M. A. Frohman, D.Bar-Sagi, Phospholipase D2-generated phosphatidic acidcouples EGFR stimulation to Ras activation by Sos. Nat.Cell Biol. 9, 706-712 (2007).


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Lysophosphatidic acid generation and signalling

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Nature Reviews Cancer 3, 582-591 (August 2003)

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Eicosanoids:

1.All derived from arachidonic acid.2.Have Paracrine hormone function (act nearor at their point of generation).

3.Multiple effects of this family of compounds.For example:

Reproductive function:prostaglandinsFever and inflammation: prostaglandinsPain: prostaglandinsFormation of blood clots:thomboxanes

Regulation of Blood pressure: prostaglandins,epoxyeicosatrienoic acidGastric secretions: prostaglandinsSmooth Muscle contraction: prostaglandins,

leukotrienes, epoxyeicosatrienoic acid


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ARACHIDONIC ACID AND CELL SIGNALING

Intracellular mobilization of stored arachidonic acid

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Cycloxygenase reactions: Cox1 and Cox2

Cox 1 ( also called prostaglandin G/H synthase) activity isdetectable in most tissues, and is responsible for synthesisof PGE2 and PG 12 in the stomach, both have cytoprotectiveactivity. At the protein level two Cox1 isoforms have beenclearly recognized.

Cox2 (Prostaglandin-endoperoxide synthase) activity is notfound in most tissues but is induced in neutrophils,macrophages and mast cells as part of the inflammationresponse.

Note for the future: Things may get more complicated-recently alternative splicing analysis suggests at least 8different forms of Cox1 and 5 of Cox2 are generated.


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Cox reaction

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Prostaglandin Synthase(s) convert PGH2to other

compounds.


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2 COX isoenzymesCOX-1: synthesis thatregulate mucin secretionFound in gastric mucosa,platelets,vascularendothelium, kidney

COX-2: regulateinflammation, pain, feverInducible: activatedblood cells, smooth muscle,endothelial, epithelial,neurons

Prostaglandins:

mediators of inflammation

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Structures of prostaglandins:

From the lipid library


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Structures of prostaglandins:

From the lipid library

Each prostaglandin is named using the prefix 'PG' followed by a letter Ato K depending on the nature and position of the substituents on the ring.Thus PGA to PGE and PGJ have a keto group in various positions on thering, and are further distinguished by the presence or absence of doublebonds or hydroxyl groups in various positions in the ring. PGF has twohydroxyl groups while PGK has two keto substituents on the ring. PGGand PGH are bicyclic endoperoxides. An oxygen bridge between carbons6 and 9 distinguishes prostacyclin (PGI). Thromboxane A (TXA) containsan unstable bicyclic oxygenated ring structure, while thromboxane B(TXB) has a stable oxane ring. In addition, all prostaglandins have ahydroxyl group on carbon 15 and a trans-double bond at carbon 13 of thealkyl substituent (R2).

Further, a numerical subscript (1 to 3) is used to denote the total numberof double bonds in the alkyl substituents, and a Greek subscript ("or #)is used with prostaglandins of the PGF series to describe thestereochemistry of the hydroxyl group on carbon 9.

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Prostaglandins Action and function:1. Once made, PGs exit cell through a transporter that is amember of the ABC(ATP binding cassette) transporter family.2. Interact with specific G protein coupled receptors on cellsurfaces.

8 different receptorsEP1, EP2, EP3, EP4 - PGEDP PGDFP - PGFIP - PGITX - TXA

3. Response depends on prostaglandin and target cell.


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Main physiological functions of prostaglandins

Function Tissue EffectMuscle contraction Arteries Blood pressure

Bronchi AirwayGI tract MotilityUterus Contractility

Secretion Stomach HCl production

Permeability Capillaries Inflammation,swelling

Oocyte maturation Ovary ovulationElectrolytes Kidney tubule Na+,H2O retention

Blood Platelets Aggregation

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Cox-2 drugs


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COX inhibiting NSAIDs and the FDA

http://www.fda.gov/cder/drug/infopage/COX2/default.htm

Cox-1 is in almost all tissues, Cox-2 more selectivelyexpressed.The Cox-2 inhibitors were developed to be morespecific that existing drugs (like aspirin and ibuprofen)that hit both Cox enzymes. The result was an inhibitors likeVioxx that lowered the gastric side effects.

But- some studies show increased risk of heart attack

and stroke. The result is many of the original Cox-2 inhibitorsare no longer on the market.Note: over $20billion dollars spent annually in U.S.A. onNSAIDs

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Lipoxygenases:

Leukotrienes LT A4, LT B4(chemoattractant for neutrophils),LT C4(Bronchoconstriction) and LT D4involved in immunereactions and cellular permeability

HPETE-hydroperoxyeicosatetraenoic acid


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All those HPETEs are really intermediates, which pathway

predominates is often tissue specific. The designationleukotriene is conferred to products with three (that's the "tri" intriene) conjugated double bonds - though the the ones welooked at there are four total double bonds. Both 15- and 12-HPETES end up forming something that could be called aleukotriene - but I think more properly the product of 15-HPETEis called a lipoxin ( abbreviated LX A or B with a subscript) thathave four conjugated double bonds and three hydroxyls)- andhave a role in resolving inflammation-12-HPETE gives rise to Hepoxilins- pro inflammatory in skin-

anti inflamation in neutrophils- the Hepoxilins don't have threeconjugated double bonds- so would not be considered aleukotriene-

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Turning 5 HPETE into leukotrienes


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Cytochrome P-450:A family of enzymes (several hundred), usually found in theER. Reactions carried out by these enzymes includehydroxylation reactions important for detoxification ofbarbiturates and other compounds. Hydroxylation increasessolubility in water and allows excretion in the urine. In theproduction of eicosinoids or epoxyeicosatrienoic acids(EETs) they are called CYP epoxygenases. In addition to thereactions we are covering here Cytochrome P-450 is used infatty acyl-CoA desaturation, conversion of squalene to

cholesterol and steroid hormone synthesis.

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Acetaminophen and cytochrome P450 in the news:

One of the ways Acetaminophen (Tylenol) is metabolized inthe liver is via a P450 reaction, generating N-acetyl-p-benzo-quinone imine (NAPQI). NAPQI conjugates with glutathionerendering it an ineffective antioxidant leaving cells susceptibleto oxidative damage. Usually this is a minor pathway ofacetaminophen degradation, but under conditions wherelevels of cytochrome P450s are increased (think toxic loadsor TGIF) more of this toxic metabolite is made. The FDA hastaken steps to limit the addition of acetaminophen in mixedmedications like some over the counter formulations and

prescription drugs like vicoden or percoset ( a mix ofacetaminophen and a narcotic). Acetaminophen overdose isthought to be the most common cause for acute liver toxicityin America.


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The purpose of these diagrams is to show the structures of the arachidonic acid (AA) products made by thecytochrome P450 (CYP) pathway. The CYP enzymes are monooxygenases. They utilize O2but attach onlyone of the O atoms to the substrate. There are numerous genes that code for CYP-containing enzymes, and

they are divided into classes. AA is a substrate for two of the classes, the CYP epoxygenases which areClass 2 enzymes, and the CYP "-oxidases which are Class 4 enzymes.

CYP epoxygenases attach the O atom to AA in the form of an epoxide. The product is called anepoxyeicosatrienoic acid (EET). This can occur at each of the 4 double bonds of AA, and the double bond islost when the epoxide forms. The structures of the 4 different EETs synthesized by CYP epoxygenases areshown at the top of the diagram. Endothelial cells synthesize EETs, and one of their main functions is toactivate smooth muscle K+ channels and thereby relax the blood vessel. The vasorelaxation lowers bloodpressure.

EETs are metabolized by an epoxide hydrolase isozyme called soluble epoxide hydrolase. This reaction isillustrated for one of the EET regioisomers, 14,15-EET, in the middle part of the diagram. The enzymehydrates the epoxide group to form the corresponding diol, called a DHET. Thus, the epoxide hydrolaseconverts 14,15-EET to 14,15-DHET. Many of the functional effects of EETs are either reduced or totallyinactivated when they are converted to diols. Because many of the actions of EETs are thought to bebeneficial, drugs are being developed to selectively inhibit soluble epoxide hydrolase. The rationale is thatinhibition will increase the concentration and retention of the EETs, enhancing their beneficial actions.

CYP "-oxidases, the other CYP enzymes that utilize AA as a substrate, attach the O atom as an OH-groupto the methyl end of AA, carbon 20. The product formed is called 20-HETE, and the reaction is illustrated atthe bottom of the diagram. Vascular smooth muscle cells and kidney tubular epithelial cells synthesize 20-HETE, and it functions to constrict blood vessels and increase blood pressure.

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IsoprostanesLike the compounds we just covered isoprostanes arederived from arachidonic acid. Unlike the compounds wehave just covered isoprostanes are not enzymaticallycreated, rather they are the result of damage fromreactive oxygen species. They are generated in responseto cancer, cardiovascular and neurological disease andtoxin exposure. Urine analysis looking for isoprostanesreflects oxidative damage to lipids, and is also used tomonitor effectiveness of antioxidant treatment as acorrective measure.


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Reactions forming Isoprostanes:

ROS reactive oxygen species

H2O2, O2, OH

P er ox idative damage

More than 64 species

From: Pauling micronutrient center

ALA

EPA

DHA

EPO;LA


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Intervention in pathways that start with arachadonic acidEICOSAPENTAENOIC ACID (EPA) dietary fish oil ( 3-fatty acid) supplementation

E P A is the 3 analogue of arachidonic acid

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EPA competes with AA for incorporation into cell

phospholipids. Thus, it reduces the amount of AA availablefor release when cPLA2is activated. What is releasedinstead is a mixture of AA and EPA. Therefore, less AA isavailable for eicosanoid production by COX, LO and CYP.Furthermore, the released EPA competes with AA for theseenzymes. As a consequence, there is decreasedproduction of eicosanoids from AA, and substitution byeicosanoids produced from EPA. Some EPA eicosanoidsare less potent than the corresponding AA products. Inaddition, the EPA eicosanoids compete for access to thetargets and thereby interfere with the action of the AA

products. The overall effect is a decrease in the pro-inflammatory and pro-thrombotic actions of the AA-derivedeicosanoids.See also: http://www.americanheart.org/presenter.jhtml?identifier=4632


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Nutritional supplementation with fish oil (EPA, w3 fatty acids)

Vegetation that grows in cold water has a high content of the 18-carbon w3 fatty acid, a-linolenic acid(9,12,15-18:3). Fish that feed on this vegetation take in a relatively large amount of this w3 fatty acid

and elongate and desaturate it, forming EPA (20:5w3) and docosahexaenoic acid (DHA, 22:6w3).

These w3 fatty acids are enriched in the lipids of the fish, and the triglycerides in oils derived fromthese fish such as salmon oil, tuna oil, herring oil, and menhaden oil, are very rich in EPA and DHA.

Except for the retina and brain that normally have a high DHA content, w3 fatty acids usually accountfor only about 3% of the fatty acids in most tissues of humans who eat the ordinary Western diet.

However, humans whose diet contains several portions of salmon, tuna, herring or other fresh fish

(not farmed fish) each week have higher than ordinary amounts of EPA and DHA in their plasma and

tissue lipids.

Almost no attention was paid to w3 fatty acids until the late 1970s when two Danish physicians found

that the Greenland Eskimos, whose diet consists mostly of fish and seal products, had a very low

incidence of coronary heart disease. They traced this to the high EPA and DHA content of the diet

and showed that these w3 fatty acids prevented platelet aggregation and, hence, thrombosis.

The initial findings indicated that this was due to EPA, the w3 analogue of arachidonic acid. Like

arachidonic acid, EPA is a substrate for eicosanoid synthesis. In many cases, however, the

eicosanoids formed from EPA are less potent than the corresponding arachidonic acid products.

This is especially true for thromboxane, the cyclooxygenase product formed when platelets are

activated. TXA2 triggers platelet aggregation, causing blood clotting, but TXA3 (the thromboxane

synthesized from EPA) is formed in lesser amounts and is less potent. Furthermore, EPA competeswith arachidonic acid for incorporation into phospholipids and thereby reduces the arachidonic acid

content of the platelet phospholipids. When the platelets are activated, a mixture of EPA and

arachidonic acid is released, so there is less arachidonic acid for TXA2 synthesis. The released EPA

also competes with arachidonic acid for the COX active site, and a mixture of TXA2 and TXA3 areproduced. (continued)

86

Nutritional Supplementation continued: The result is less platelet aggregation, due to less production

of TXA2, competition between it and TXA3 for the platelet TX receptors, and the lesser potency of

TXA3. This affords protection against thrombosis, including coronary thrombosis.

There are other health benefits of w3 fatty acids. Elevated plasma triglycerides, a risk factor for

coronary heart disease, are reduced by increasing the dietary w3 fatty acid intake. w3 Fatty acid

supplements also reduce the inflammatory response in diseases like arthritis by decreasing the

synthesis of pro-inflammatory prostaglandins like PGE2 made from arachidonic acid. In addition, DHAhas beneficial effects for retinal and brain development and function.

Based on these findings, it is recommended that healthy humans should increase their dietary w3 fatty

acid intakes so a better balance between w3 and w6 fatty acids would be achieved in the tissues. The

ratio of w6 to w3 fatty acids in the diet of most Americans is between 10/1 and 20/1, and many expertsfeel that a ratio of 5/1 or even less would be more healthful.

In addition, people are taking w3 fatty acid supplements to prevent coronary thrombosis, to reduce

plasma triglycerides, and to reduce the pain and swelling of arthritis. These supplements are also

being tested for the prevention of cardiac arrhythmias and for the treatment of depression.

Some people are taking fish oil capsules enriched in EPA and DHA. Capsules containing the ethyl

esters of EPA and DHA also are now available. The dosage varies considerably, and humans havetaken as much as 15 grams/day without serious adverse effects. However, the usual dose today is

between 2 and 3 grams/day, and a clinical trial in Italy indicated surprisingly that a dose of only 1 gram/day was effective in reducing coronary events.


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87

SUMMARY

Phospholipids form the membrane lipid bilayer. Inaddition, some membrane phospholipids are substratesfor cell signaling pathways.

CDP-choline combines with the 1,2-diacylglycerolto form phosphatidylcholine (PC).

Phosphatidic acid and CTP are substrates forphosphatidylinositol (PI) synthesis. The inositol moiety ofPI can be phosphorylated, and the major products arePIP2and PIP3.

Phospholipases hydrolyze phospholipids. Thethree main forms for signaling use PLA, PLC and PLD.

88

PLA2hydrolyzes the sn-2fatty acid, which ordinarily ispolyunsaturated. There are secreted forms (sPLA

2) and

intracellular forms (cPLA2).

Generation of Lysophosphatidic acid serves a signalingfunction

PIP2is hydrolyzed by phospholipase C.Diacylglycerol, which activates PKC, is formed. IP3,which raises Ca2+in the cytosol by releasing it from theendoplasmic reticulum, also is formed.

PI 3-kinase produces PIP3, which stimulates cellproliferation. PTEN, a tumor suppressor gene, is the PIP3phosphatase. This produces apoptosis.

The arachidonic acid utilized for eicosanoidbiomediator synthesis is released from the sn-2 positionof membrane phospholipids by a cPLA2-mediatedhydrolysis.


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89

The main eicosanoids formed in humans arederived from arachidonic acid.

The cyclooxygenase pathway converts arachidonicacid to either prostaglandins or thromboxane.

There are two forms of cyclooxygenase. COX-1 isconstitutive, and COX-2 is induced by inflammatorystimuli. Some COX-inhibitor drugs like acetyl salicylicacid inhibit both forms, while celecoxib is selective forCOX-2 inhibitor.

Prostaglandins bind to membrane receptors thatare coupled to G-proteins. Adenylyl cyclase andphospholipase C are activated by prostaglandins binding

to their respective receptors.

90

Leukotrienes are arachidonic acid lipoxygenaseproducts that have pro-inflammatory actions.

Cytochrome P450 epoxygenases convertarachidonic acid to EETs which are vasodilators,whereas cytochrome P450 "-oxidases convertarachidonic acid to 20-HETE which functions to increaseblood pressure.

Isoprostane are prostaglandin-like compoundsformed by oxidation of arachidonic acid. They arebiomarkers of oxidative stress.

EPA, a fish oil ("3) fatty acid, is a competitor ofarachidonic acid and has anti-inflammatory and anti-thrombotic actions.


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Sphingolipids

Handout #2

2

OBJECTIVES:Understand the types of sphingolipids and thedi"erences in their structure -Know how ceramide is synthesized-Know how sphingomyelin and the glycosphingolipids

are synthesized-Understand the di"erence between a neutral

glycosphingolipid and a ganglioside-Understand the function of sphingolipids and their

role in membranes -Understand the enzymatic mechanism of

sphingolipid degradation

-Understand the metabolic basis of the sphingolipidstorage diseases

-Understand how sphingolipids are involved in cellregulation


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KEY WORDS- SphingolipidsCeramide

sphingomyelin Sphingomyelinase

cerebrosidesulfatide

neutral glycosphingolipid ganglioside

sialic acid (NANA) 3-phosphoadenosine 5-phosphosulfate (PAPS)

sphingolipid storage diseasesceramidase sphingosine 1-phosphate

4

STRUCTURE OF LIPIDS


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Biosynthesis of Sphingolipidsoccurs in every cell membrane, highest CNS!Palmitoyl-CoA + serine: starting point forsphingolipids!Requires fatty acid attachment: enter as CoA.!Uses NADPH/reducing power!C=C introduced by mixed function oxidases!Attachment of activated head group

6

Formation of Sphingosine Backbone

Pyridoxal Phosphate is used here


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Finishingtouches

Ceramides have sphingosineand a fatty acid.

FADFADH2

8

SPHINGOLIPIDS in plasma membrane,especially of CNS,Amino group always acylated; confer diversity

on lipid structure; source of second messengers incell, other functions yet to be confirmed.

CERAMIDE SUBGROUPS1. Sphingomyelinspolar head group2. Glycosphingolipidsone or moresugars attached: neutral charge3. Gangliosidesoligosaccharides,at least one sialic acid: negative charge


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CERAMIDE SUBGROUPS1. SphingomyelinsThese are phospholipids. The

polar head group is almost always choline

2. Neutral GlycosphingolipidsCerebroside- one sugar ( either glucose orgalactose) attached to the 1OH. For examplegalactocerebrosidewhich is the primaryglycosphingolipid in myelin.Ceramide oligosaccharides (Globosides) havemore than a single sugar attached- made usingglucocerebroside as a precursor.

3. Acidic Glycosphingolipids Gangliosidesoligosaccharides with at least one sialic acidSulfatides- have sulfated galactosyl residues

10

Synthesis occurs in the ER and golgi by sequentialaddition of monomers. Monomers come inactivated form- most often as UDP-sugars, or forsialic acid asCMP-NANA


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Reaction of -OH

Gangliosides:Activated sugars at leastone acidic (sialic acid);oligosaccharide chain

(or ethanolamine)

(globoside: 2 sugars)neutral

12

Additional sugars can be added with di"erent linkages:

Sugars:Glucose: GlcN-acetyl Glc: GlcNacGalactose: GalN-acetyl Gal: GalNac Fucose:Fuc


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Gangliosides and Sialic acid:Sialic acid is the common name for N-acetylneuraminic

acid or NANA.Notation for Gangliosides:G (for ganglioside)M, D, T, Q : for 1, 2, 3, or 4 NANA Number subscript refers to the orderof mobility on thin layer gelchromatogram.

Gangliosides can act as receptors for:Interferon and some growth factors,and bind to some viruses and bacterial

toxins.For example, Influenza virus and thebacterial toxins botulinum, tetanusand cholera bind to gangliosides.

14

Sulfatide: is the only class of ceramide that usesgalactocerebroside rather than glucocerebroside as thestarting material: sulfotransferase transfers the sulfateto the 3OH of the galactose.

PAPS-3phosphoadenosine5phosphosulfate: note thisis the obligate donor for allsulfation reactions.


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The composition of the inner and outer leaflet need notbe the same. Sphingolipids are mostly in the outer

leaflet.

16

Some blood groups are

determined by thecarbohydrate attachedto sphingoglycolipids.

The blood groupsubstancesare not only on bloodcells, but also on the plasmid membranes of many tissues.


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What di"erence doesIt make?

A and B individuals Are more resistant toCholera.AB even moreresistant.

O Individuals havea higher incidence ofpeptic ulcers.

A individuals havea higher incidence ofstomach cancer, heart disease, perniciousanemia.

18

Breakdown of the sphingolipids:Degradation of neutral glycosphingolipids:One sugar at a time, both the sugar and the link makea di"erence.


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19

Breakdown of the sphingolipids:Degradation of acidic glycosphingolipids

(gangliosides):

20

Breakdown of the sphingolipids:

Breakdown is a sequential process, each steprequires that the step before was completed- thisis the core cause of lipid storage disease.

Gaucher disease (glucocerebrosidase)Niemann-Pick disease (Type A and B:sphingomyelinase)

(Type C and D:NPC1 and 2proteins lacking)

Fabry disease (alpha-galactosidase-A)Farbers disease (ceramidase)GangliosidosesKrabb disease (galactosylceramidase)Metachromatic leukodystrophy (arylsufatase A)Sandho"s disease (hexaminidase)Tay-Sachs (hexaminidase A)Wolmans disease (acid lipase deficiency)


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21

22

Sphingolipidoseslack an enzyme involved in

sphingolipid catabolism

Lipid storage diseases:1) single sphingolipid accumulates; 2) ceramide portioncommon to storage lipids;3) rate of biosynthesis ofaccumulating lipid is normal;4) catabolic enzyme ismissing; 5) extent of enzyme deficiency in all tissues issame.Autosomal recessive, disease in homozygotes withdefect in both alleles. Diagnosis: enzyme assays,Morphological appearance in biopsy

http://www.jpathology.com/1(4).html


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23

No therapy except for

Gauchers and Fabry: treatedwith infusion ofglucocerebrosidase orgalactosidase A

24

SPHINGOMYELIN AND CELL REGULATION


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25

What can happen when sphingosine or ceramidelevels change?

Changes in ceramide levels seem to be anti-cancer/pro-apoptotic. Work done by Siskind and Colombinishow that increases in ceramide concentration in theouter membrane of the mitochondria leads toclustering and the formation of channels that allowthe release of pro-apoptotic proteins. TNF alpha,endotoxin, chemotherapeutic agents, interferon andreactive oxygen species have all been shown toincrease ceramide.

26

What can happen when sphingosine or ceramidelevels change?

Changes in Sphingosine 1-phosphate seems to beanti-apoptotic. Increases correlate with cellproliferation, motility and angiogenesis. Sphingosine1-phosphate binds to G-protein coupled receptors(GPCR) called EDG.EDG= Endothelium Di"erentiation GenesLysophosphatidic acid also binds to EDG receptors.


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Multiple Sph-induced mechanisms leading to apoptosis.

Suzuki E et al. PNAS 2004;101:14788-14793

2004 by National Academy of Sciences

Regulation and function of sphingosine kinase-1 (SK1).

Karliner J S Cardiovasc Res 2009;82:184-192Published on behalf of the European Society o f Cardiology. All rights reserved. The

Author 2008. For permissions please email: [email protected].


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29

New therapy for Multiple Sclerosis linked toSphingosine-1-phosphate.

The drug: Fingolimod-acts as an agonist on theSphingosine-1-P receptors. For T and B lymphocytesbinding of sphingosine-1-P to the S-1P-1 receptor isa critical signal for the immune cells to enter thecirculation. Fingolimod causes internalization of thesphingosine-1-P-1 receptor, and the cells stay in thesecondary lymphoid tissues.This reduces the immune induced damage to myelinsheaths of nervous tissue.

FDA approval: Sept. 2010-Novartis (Gilenya)

Oral Fingolimod (FTY720) for Relapsing Multiple Sclerosis

L.Kappos, J. Antel, G. Comi, X. Montalban, P. O'Connor, C. Polman, T. Haas, A. Korn, G. Karlsson, & E. Radue,

N Engl J Med 2006; 355:1124-1140

30

Summary:

There are two kinds of sphingolipids, sphingomyelin(a sphingophospholipid) and glycosphingolipids.

Sphingomyelin and the glycosphingolipids are synthesizedfrom ceramide.

Ceramide is synthesized in a series of reactions beginningwith the condensation of palmitoyl CoA and serine in areaction that requires pyridoxal phosphate.

Cerebrosides are formed when ceramide reacts with eitherUDP-glucose or UDP-galactose.

Sulfatides are formed from galactosylceramide in areaction that utilizes PAPS, an activated form of sulfate.


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Summary:Neutral glycosphingolipids and gangliosides are formedfrom glucosylceramide.

Gangliosides are glycosphingolipids that contain sialicacid (NANA).

Phospholipids are distributed asymmetrically across themembrane lipid bilayer. The choline containingphospholipids, sphingomyelin and phosphatidylcholine,are concentrated in the outer leaflet.

Glycosphingolipids also are present in the membrane

lipid bilayer. The carbohydrate chain project from thesurface into the surrounding aqueous environment.

Sphingolipids are degraded by lysosomal hydrolases.

32

Summary:Sphingolipid storage diseases, called sphingolipidoses

or lipidoses, are caused by genetic defects involving oneof the lysosomal sphingolipid hydrolases. The mostcommon forms (and the sphingolipids that accumulatein the tissues) are Niemann-Pick (sphingomyelin),Gauchers (glucocerebrosidase) and Tay-Sachs(ganglioside GM2).

One mechanism whereby certain cytokines regulate cellfunction is to activate a sphingomyelinase. The ceramidethat is formed can trigger apoptosis. However, theceramide also can be converted to sphingosine 1-phosphate.

Sphingosine 1-phosphate stimulates cell proliferationand angiogenesis by binding to the its receptors, whichare also called EDG receptors.


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33

Clinical Connections :

Niemann-Pick Disease Type AThe sphingolipid storage disease caused by sphingomyelinaccumulation in the tissues is

called Niemann-Pick Disease Type A.It is caused by a genetic defect in lysosomalsphingomyelinase,which has an acid pH optimum. Enlargement of the spleen and liver occurs because of intracellularsphingomyelin accumulation, and the presence of lipid-rich foam cells in the bone marrow are acharacteristic of the disease.

There are at two forms of acid sphingomyelinase, the lysosomal form and a secreted form.The lysosomal form degrades the excess sphingomyelin that accumulates intracellularly as a result ofeither endocytosis or phagocytosis. This is the enzyme that is defective in Niemann Pick disease TypeA. The acidic form that is secreted into the extracellular fluid and is not involved in Niemann Pickdisease Type A.

A second type of sphingomyelinase, called neutral sphingomyelinase, functions best atneutral pH.It hydrolyzes membrane sphingomyelin as part of a signal transduction mechanism thatcan trigger apoptosis, but it also is not involved in Niemann Pick disease Type A.

There are three forms of Niemann Pick disease Type A. Each has autosomal recessiveinheritance. The most serious form of Niemann Pick disease Type A occurs in infancy and is associatedwith very severe central nervous system damage. It is fatal by age 3 to 4 years. There is a less severeform that also a"ects the central nervous system, but these individuals can live into late adolescence.Finally, there is a mild form that does not involve the central nervous system, and these patients canlive a relatively normal life into adulthood. Each of these forms is caused by a di"erent mutation in the

lysosomal sphingomyelinase.A clinical diagnosis of Niemann-Pick Disease Type A should be confirmed by biochemical

analysis. Acid sphingomyelinase activity can be measured either in white blood cells or skin fibroblastscultured from the patient. The enzymatic assay is sensitive enough to detect heterozygotes, so carriersof the disease can be identified. Prenatal diagnosis also is possible by measuring acidsphingomyelinase activity in amniotic cells.

34

Gauchers Disease

Glucosylceramide, a cerebroside, accumulates in Gauchers Disease. It isthe most common inherited disorder of glycosphingolipid metabolism.The disease is due to a genetic defect in glucocerebrosidase, theenzyme that hydrolyzes the glucosylceramide. As a result of the enzymedeficiency, excessive amounts of glucosylceramide accumulate intissues throughout the body, especially in cells of thereticuloendothelial system. A characteristic of the disease in thepresence of large, lipid-filled cells called Gaucher cells in all parts of thereticuloendothelial system. This leads to enlargement of the spleen andhypersplenism, leading to anemia. The liver becomes enlarged due toaccumulation of glucosylceramide in the Kup"er cells, and erosion ofthe bones also occurs.

A suspected diagnosis of Gauchers Disease can be confirmedbiochemically by measuring the glucocerebrosidase activity in whiteblood cells isolated from the patients venous blood, or in fibroblastscultures obtained from a skin biopsy. The diagnosis also can be madeduring pregnancy by measuring glucocerebrosidase activity in cellscultured from the amniotic fluid.


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Fabrys Disease and Enzyme Replacement TherapyFabrys Disease is an X-linked genetic defect in the lysosomal !-galactosidase Aenzyme. This leads to anaccumulation of the neutral glycosphingolipid containing three carbohydrate residues, trihexosylceramide,also called globotriaosylceramide (Gal-Gal-Glc-Cer). The clinical onset usually occurs in childhood. There is

a progressive accumulation of trihexosylceramide in the endothelial lysosomes of the kidneys, heart, skinand brain. This leads to microvascular disease of the kidney, heart and brain, eventually causing death.

In the past the only treatment was management of pain and the complications of renal failure, heart diseaseand stroke. However, a multicenter placebo-controlled therapeutic trial in which the deficient enzyme wasreplaced with recombinant human !-galactosidase A was carried out. Some positive results were obtainedas reported in the New England Journal of Medicine (July 5, 2001).

Patients with Fabrys disease in this clinical trial were intravenously infused with 1 mg/kg of therecombinant !-galactosidase A or a placebo every two weeks for 20 weeks. Of the 29 patients who weregiven the recombinant enzyme, 20 had no kidney microvascular endothelial deposits of trihexosylceramideat the end of the treatment period, and decreased deposits in the skin and endocardium. A reduction in theplasma trihexosylceramide concentration also occurred. None of the placebo treated patients showed anyimprovement.

At the end of the 20-week period, everyone including those initially given the placebo was treated with therecombinant enzyme for 6 months. All but one of those who agreed to another kidney biopsy had clearanceof the trihexosylceramide deposits from the kidney microvascular endothelium. They also had less pain andimproved quality of life, indicating the success of the treatment.

Presumably, the !-galactosidase A was taken up by the microvascular endothelium, entered the lysosomes,and hydrolyzed the accumulated trihexosylceramide. The infused enzyme either also hydrolyzed thetrihexosylceramide in the plasma, or the reduction of the deposits in the tissues allowed the lysosomes totake up more of the circulating material, which was subsequently hydrolyzed by the recombinant !-galactosidase A.

These results are promising, but the need for repeated intravenous infusions is a cumbersome form oftherapy. It is likely that therapies eventually will be developed based on !-galactosidase A gene transferand, ultimately, on permanent transfection with the normal gene.

36

Tay-Sachs DiseaseThe ganglioside GM2(GalNAc-Gal[NANA]-Glc-Cer) accumulates in Tay-Sachsdisease, the most common of the genetic ganglioside storage diseases. It iscaused by a severe deficiency of hexosaminidase A, the lysosomal enzyme thatdegrades ganglioside GM2.

The main pathological changes occur in the neurons. The neuronal cytoplasm isdistended by the accumulation round structures consisting of tightly packedmembraneous material composed of GM2, and the nucleus is displaced. Thisleads to axonal degeneration and demyelination. Motor weakness usually occursby 3 to 6 months of age, and mental and motor deterioration progress rapidlyafter 1 year, feeding becomes a problem because of ine"ective swallowing,paralysis develops, and the child becomes deaf and blind. Head size increasesdue to cerebral gliosis, convulsions develop, and death usually occurs by age 3years.

There is no e"

ective treatment. The only e"

ective measure is to screen high-riskpopulations for detection of heterozygotes, or the identification of an a"ectedfetus in utero.


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Neutral Glycosphingolipids and Blood TypeThe blood type of a person is determined mainly by the kind of oligosaccharides on the surface ofthe erythrocytes. These carbohydrate chains are a part of membrane glycoproteins and neutralglycosphingolipids.

The main blood typing system is the ABO group. It consists of three structurally relatedoligosaccharide chains. The figure below shows the di"erence in the structure of the three chains,illustrating them as neutral glycosphingolipids (the chains are illustrated as being attached toceramide). However, the same chains also are attached to membrane glycoproteins.

The only di"erence between A and B is that the terminal Gal in acetylatedintype-A, but not in type-B. The enzyme that adds the terminal monosaccharide to the carbohydrate chain in a type-A personis GalNAc transferase. The enzyme that transfers the terminal monosaccharide in a type-B person isGal transferase. The di"erence in type-O is that the terminal Gal is not present because type-Oindividuals do not express either of these transferase enzymes.

Each of the three oligosaccharide chains contains fucose, abbreviated Fuc. Fucose is a hexose inwhich carbon-6, the carbon that is attached to the pyranose ring, is a CH 3-group rather than theCH2OH-group that is present in other monosaccharides.

The small structural di"erences in these oligosaccharide chains are of great importance in bloodtransfusion, and this major blood group typing system should always be matched when a blood

transfusion is given. Each person has either A, B, a mixture of A and B, or O chains on theirerythrocytes; in other words, humans are blood type A, B, AB, or O. The blood that is transfusedmust be the same type, or type O if the situation is urgent and there isnt time to type the patient. Ifnot, the incoming cells will be agglutinated, causing a life-threatening reaction to the transfusion.


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Cholesterol metabolism

and protein prenylation

Handout #3

2

Objectives Recognize the di#erent types of sterols Understand cholesterol nutrition and the concept

of feedback inhibition

Know the functions of the isoprenoid biosyntheticpathway

Know how cholesterol is synthesized Understand the role of HMGCoA reductase and

how the statin drugs a#ect this enzyme

Understand the mechanism that regulates SREBPand how cholesterol regulates this process

Understand how SREBP regulates cholesterolsynthesis

Understand the mechanism and function of proteinprenylation and the function of caveolae


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KEY WORDS - Cholesterol

metabolism

sitosterol ergosterol

SRE SREBP

SCAP Insig

HMG CoA reductase mevalonate

statin isopentenylpyrophosphate

farnesyl pyrophosphate geranylgeranylpyrophosphate

CAAX box farnesylation geranylgeranylation caveolae caveolin squalene

lanosterol7-dehydrocholesterol"7-reductase

4

STRUCTURE OF

CHOLESTEROL AND STEROLS

27 Carbons

4 rings; A, B and C are 6-membered, D is 5-membered

2 methyl groups A/B (C19) and C/D (C18)

One double bond in B-ring at C5; OH-group at C3

8 C saturated hydrocarbon chain attached to D-ring at C17


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Non-animal sterols

Ergosterol (yeast) Sitosterol(plants)

Sitosterolemia: this is a genetic deficiency that allows excessabsorption of plant sterols- cause is a mutation in an ABCG

transporter leading to defective sterol excretion. Plant sterols, are generally not well absorbed and have beenimplicated in reduced uptake of dietary cholesterol and reducedrecovery of bile acids- there are currently clinical trials testinge$cacy in improving lipid profiles. ClinicalTrials.gov Identifier:NCT00441480

Medical notes:

6

Sources of Cholesterol

Diet- cholesterol comes from foods derivedfrom animals

Synthesis- Cholesterol is not an essentialnutrient, there is plenty of synthetic capacityto take care of nutritional needs. Howevertypically about half of the cholesterol is fromthe diet.

The liver is the main site of synthesis butmany tissues can produce cholesterol. Thebrain seems to synthesize all of itscholesterol.


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Cholesterol production

regulated

Cholesterol serves as a feedbackinhibitor of the committed step in

cholesterol synthesis- the reaction

carried out by HMG-CoA reductase. As

dietary cholesterol is increased,

endogenous synthesis of cholesterol in

the liver is decreased.

8

CHOLESTEROL --Modulates membrane

fluidity, precursor of hormones

1. Condensation of 3 CH3COO-to form mevalonate #%

2. Synthesis of isopentenyl pyrophosphate, activated

isoprene unit (key building block).3. Condensation of 6 isopentenyl pyrophosphate to

form squalene.4. Squalene cyclizes, tetracyclic product converted

into cholesterol.

Liver:synthesis


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MEVALONATE SYNTHESIS: committed stepenzyme: HMG-CoA reductase

Stage 1

HMG CoA synthase-

HMG CoA reductase-

10

MEVALONATE SYNTHESIS: committed step

enzyme: HMG-CoA reductase

Stage 1

HMG CoA synthase-

HMG CoA reductase-


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Major intermediates in

Cholesterol synthesis

C2#C6#[C5] #C30#C27

Note: isoprene startingPoint for many natural

products (terpenes)Vitamins D, A, K, E

12

Stage 2:Squalene (C30

) from 6 x C5

condensation reactions:C5 C10 C15 C30

Geranyl - C10farnesyl - C15squalene - C30

using isopentenylpyrophosphate


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STAGE 3 CYCLIZATION

Conversion to cholesterol :removal of 3 methylgroups, reduction of C=Cby NADPH, migration ofother double bond.

14

Lanosterol to Cholesterol

6 genetic diseases mutations of 6 genes Sterol substrate of themutated enzymeaccumulatesSevere birth defects malformations,neurological defects,

mental retardation

D7 Reductase deficiency

Smith-Lemli-OpitzSyndrome


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Synthesized inliver; transportedby lipoproteins orstored in liver

16

Champe et al.


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Using the

Cholesterolpathway

Isopentenyl-tRNA

Sidechain of heme

N-linked glycoproteins

Co-enzyme Q

Modified from Wikimedia Commons

18

HMG-CoA reductase: mediator of regulation ofcholesterol biosynthesisIntervention is based on:

1) Inhibiting reabsorption of bile salts2) Blocking de novo synthesisCombine synthesis inhibitor (statins) with resin(cationic polymer) that prevents bile acidreabsorption


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19

Cholesterol level management by HMG-CoAinhibitors: statins: competitive inhibitors

Lipitor

Over $12 billion/ year on statinsin the U.S.A.

20

Regulation of Cholesterol Synthesis

HMG-CoA reductase: rate limiting stepRegulation by:1) Transcriptional control. Sterol regulatory element-binding protein (SREBP) that binds to a Sterolregulatory element (SRE) upstream of the HMG-CoAreductase gene.2) Protein modification ( phosphorylation) - ReductaseKinase and HMG-CoA reductase phosphatase. Active inunmodified state.3). Hormonal regulation of protein modification-insulin cascade leads to greater activity, glucagon lessactivity.4). Rate of translation of mRNA ( by Chl & mevalonate)somewhat controversial.5). Degradation of the reductase (senses %sterols)


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21

MEVALONATE SYNTHESIS: committed stepenzyme: HMG-CoA reductase

Stage 1

HMG CoA synthase-

HMG CoA reductase-

22

MEVALONATE SYNTHESIS: committed step

enzyme: HMG-CoA reductase

Stage 1

HMG CoA

synthase-Thereare two di#erentforms of thisenzyme- one inthe cytosol andone in themitochondria .They are encodedby di#erent genes

HMG CoA reductase-


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23

Mitochondrial shuttling

Lehninger Biochemistry 5th ed.

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Side e#

ects of Statins - muscle pain and weakness

1.SLCO1B1 variants and Statin-induced Myopathy- A Genomewide studyNEJM August 21, 2008.Background: Lowering low-density lipoprotein cholesterol with statintherapy results in substantial reductions in cardiovascular events, andlarger reductions in cholesterol may produce larger benefits. In rare cases,myopathy occurs in association with statin therapy, especially when thestatins are administered at higher doses and with certain othermedications.Conclusions: We have identified common variants in SLCO1B1 that arestrongly associated with an increased risk of statin-induced myopathy.Genotyping these variants may help to achieve the benefits of statin

therapy more safely and e#ectively.Note: SLCO1 encodes a transporting peptide that regulates statin uptake inthe liver. Muscle weakness side e#ect frequency increases with dose-article suggests that with normal doses incidence is 1/10,000. 2. The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statin-induced muscle toxicity.JCI December 2007. In patients with statininduced muscle pain atrogin is increased.


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Regulation of HMG-CoA reductase

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Linking transcription to cholesterol levels


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http://www4.utsouthwestern.edu/moleculargenetics/pages/brown/lab.html

There are at least 3 SREBPs-

Two made by SREBP1 ( a and c) and SREBP2SREBP1a and SREBP2 are involved in cholesterolmetabolismSREBP1c in fatty acid synthesis

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ER- endoplasmic reticulumInsig- ER retention protein- interact withSCAP preventing transit to golgi if

sterol levels are sufficient, whencholesterol levels fall in the

membrane then the SCAP-SREPB

complex can move to the gogiSCAP- SREBP cleavage activating protein

S1P - Site 1 protease that makes the f irstcut on SREBP

S2P- Site 2 protease that make the second

cut on SREBP

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Insig-1 and Insig-2

Insig-1 is binds to SCAP/SREBP1a and 2 complexes as longas there is adequate cholesterol in the ER membrane. Italso binds to the sterol sensing region of HMGCoAreductase if cholesterol levels are high enough, stimulatingthe ubiquitin mediated degradation of HMG CoA reductase.Insig-1 may also interact with NP-C.Protein and Patched, the receptor for Hedgehog. Insig-2associates with SCAP/SREBP1c has similar properties andparticular variants has been linked to obesity.


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Phosphorylation

and hormonalcontrol.

Figure and explanation come from: www.med.unibs.it/~marchesi/cholest.html

Hormones such as glucagon andepinephrine negatively a#ectcholesterol biosynthesis by increasingthe activity of the inhibitor ofphosphoprotein phosphatase-1, PPI-1.Conversely, insulin stimulates theremoval of phosphates and, thereby,activates HMG-CoA reductase activity.Additional regulation of HMG-CoAreductase occurs through an inhibition

of synthesis of the enzyme byelevation in intracellular cholesterollevels.

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Isoprenylation of proteins

The protein signal is a CAAX box- refers tothe amino acid sequence cys-aliphatic-aliphatic-X where the X amino acid specificethe prenyl group. The prenyl groupsattached this way are geranylgeranyl andfarnesyl. The cys is important because it isthe attachment site. Proteins that are

isoprenelated include the Rab family of Gproteins and the Ras oncogene.


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Nature Reviews Cancer 5, 930-942 (December 2005)

a | Rho, which has the carboxy-terminalcysteine-aliphatic-aliphatic-X (CAAX) motif,is combined with FPP at the cysteine residueby farnesyl transferase. b | The CAAXprotease, Ras-converting enzyme 1 (RCE1),then cleaves o#the AAX tripeptide from thecarboxy terminus of the protein at theendoplasmic reticulum membrane. c | Theisoprenylcysteine carboxyl methyltransferase(ICMT) enzyme then adds a carboxy methylgroup from S-adenosylmethionine (SAM) tothe prenylated cysteine residue. Rho, with itsnewly-attached hydrophobic anchor, thenmoves to the appropriate cellular membrane,remaining in the cytoplasmic compartment.

Isoprenylation of proteins

Isoprenylation of proteins is important in

compartmentalization. Often isoprenylatedproteins have a second Cys that ispalmitoylated. Giving three hydrophobic

reasons to associate with a membrane.

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Cholesterol in the membrane


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Membrane Lipids: Amphipathic, StructuralStructure determines packing, fluidity

PhospholipidsGlycolipidsCholesterol

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Signaling molecules are

disproportionately in lipid rafts


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The composition of lipid and protein in membranesis neither uniform nor random.

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The association ofcaveolin with areas ofthe membrane lead tothe formation of pitlike structures calledcaveoli.


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Summary

-Cholesterol is obtained from either the diet or bybiosynthesis from acetyl CoA.

-Foods containing animal fat are rich in cholesterol.

-Dietary cholesterol is absorbed well, but plant sterols are

poorly absorbed and very little plant sterol normally is

present in the plasma or tissues.

-Cholesterol is one of the products of the isoprenoid

biosynthetic pathway. This pathway also synthesizes

other important compounds necessary for glycoproteinsynthesis, tRNA function, mitochondrial electrontransport, and protein prenylation.

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Summary

-The isoprenoid pathway enzyme that is regulated bycholesterol availability is HMG CoA reductase. It is therate-limiting step in cholesterol synthesis and the site of

feedback inhibition, the process whereby cholesterol

synthesis is regulated by cholesterol availability.

-The statin drugs inhibit HMG CoA reductase.-Transcriptional regulation of the HMG CoA reductase

gene is mediated by SREBP, a protein bound to the

endoplasmic reticulum (ER) that is activated in response

to a decrease in the cell cholesterol content.

-Insig-1 is the cholesterol-sensing protein in the ER that

binds to SCAP when there is enough cholesterol in the

ER membrane. It keeps the SREBP-SCAP complex

bound to the ER.


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Summary

-When the ER cholesterol content decreases, Insig-1

releases SCAP and the SREBP-SCAP complextranslocates to the Golgi where SREBP is cleaved by two

proteases. This releases the transcription factor domain

of SREBP so it can enter the nucleus, bind to SRE in the

promoter of sterol responsive gene and thereby initiate

the expression of these genes.-Cholesterol synthesis proceeds from mevalonate

through farnesyl pyrophosphate, squalene, lanosterol,

and either 7-dehydrocholesterol or desmosterol to

cholesterol.

-Proteins can be prenylated, some with a farnesyl groupand others with a geranylgeranyl group. The

composition of the X in the CAAX box determines which

prenyl group is added. Prenylation targets these proteins

to specific domains in cell membranes.

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Summary

Membranes contain cholesterol. It is embedded inthe lipid bilayer between phospholipids. The OH-group is

oriented outward in the same region as the phospholipidhead groups, and the rings and hydrocarbon chain

project towards the center of the lipid bilayer and interact

with the phospholipid fatty acyl chains.

Most of the membrane cholesterol in a cell is

present in the plasma membrane. Caveolae are a specialized form of lipid rafts in

membranes that contain the protein caveolin. They are

rich in cholesterol and sphingolipids. Prenylated proteinsare targeted to caveolae and are components of signal

transduction complexes. Caveolae also regulate intracellular cholesterol

content, either redistributing cholesterol to the cell interior

or facilitating its excretion from the cell.


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CLINICAL CORRELATIONSHypercholesterolemia

This is a very common condition in which the plasma cholesterol concentration measured after anovernight period of fasting is elevated.

Plasma cholesterol is a component of plasma lipoproteins. Therefore, hypercholesterolemia is anelevation in lipoproteins that contain cholesterol. There are several forms of hypercholesterolemia,depending on which plasma lipoprotein is elevated. A gene defect that produces an elevation inlow density lipoproteins (LDL) is the most severe form. Hypercholesterolemia is a verycomplicated topic, and we will consider it in much more detail when we cover plasma lipoproteins.

Hypercholesterolemia is a serious risk factor for atherosclerosis, a disease of the arterial wall inwhich cholesterol accumulates and triggers an inflammatory reaction. Therefore,hypercholesterolemia requires treatment as soon as it is detected even though in can beasymptomatic for many years.

The cause of the hypercholesterolemia must be characterized before treatment is started in orderto know what type of treatment to prescribe. For example, one of the cholesterol-carryinglipoproteins is HDL (high density lipoproteins). HDL actually protects against atherosclerosis and,of course, cholesterol elevations due to HDL must not be treated.

The most serious cases of hypercholesterolemia are genetic in origin. They usually cause thehighest plasma cholesterol elevations and almost always require drug therapy. However, manycases in developed countries are diet induced. The diet in these cases contains too much saturated

fat and cholesterol, and the feedback inhibition of cholesterol biosynthesis is not enough toprevent the amount accumulating in the plasma from increasing. Other people who are eatingsimilar diets do not have hypercholesterolemia. Therefore, individuals who have diet-inducedhypercholesterolemia must have genetic di$erences from those of us who have similar diets butdo not develop cholesterol elevations. A number of candidate genes for diet-inducedhypercholesterolemia have been detected, but the cause has not yet been fully delineated. Theplasma cholesterol in the diet-induced form usually is only moderately elevated and often can beadequately controlled by dietary restriction, without the need for cholesterol-lowering drugs.

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SitosterolemiaOnly trace amounts of plant sterols normally are absorbed from the diet, and the

amounts in the plasma and tissues are extremely small. In a rare genetic disease calledsitosterolemia, large amounts of dietary plant sterols are absorbed and accumulate in the

plasma and tissues. Dr. William Connor discovered the disease in 1974 when he was a facultymember in Internal Medicine at the University of Iowa. Two sisters were referred to himbecause they had xanthomas. Xanthomas are cholesterol accumulations in the tendon sheaths,usually due to severe hypercholesterolemia. However, the sisters did not havehypercholesterolemia, at least according to the guidelines used in 1974.

Using gas-liquid chromatography, Dr. Connor discovered that the sisters had highlevels of plant sterols in their plasma and tissues. He correctly concluded that this was due to agenetic abnormality, and his further studies demonstrated that the mutation caused a muchhigher absorption of plant sterols from the diet.

Sitosterolemia is inherited as a recessive trait. We now know that the moleculardefect is caused by a mutation of an ABCG transporter, a membrane ATP cassette bindingprotein that utilizes the energy released by ATP hydrolysis to pump sterols out of theintestinal enterocytes so they are not absorbed and do not build up in the plasma. Normally,the ABCG transporters pump out almost all the plant sterols that are taken up from the diet bythe intestinal enterocytes, and only a very small amount remains in the body. Because the ABCGpumps are genetically defective in sitosterolemia, the absorbed plant sterols cannot beadequately excreted and accumulate in the plasma and tissues. There are many di$erent formsof plant sterols, and in addition to sitosterol, other plant sterols also accumulate. They include

campesterol, stigmasterol and avenosterol. Like sitosterol, these plant sterols di$er fromcholesterol only in the structure of the hydrocarbon chain attached to the steroid nucleus.

Sitosterolemia produces atherosclerosis and often leads to death from coronary heartdisease at middle age or even earlier. The atherosclerosis is due primarily to accumulation inthe arteries of cholesterol, not plant sterols, even though the plasma cholesterol is only slightlyto moderately elevated. The explanation is still uncertain. A likely possibility is that ABCGtransporters function to excrete excess cholesterol in addition to plant sterols. When thesetransporters are genetically defective, cholesterol cannot be adequately excreted from thetissues, and enough apparently accumulates in the arteries to produce atherosclerosis.


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CLINICAL CORRELATIONS

Smith-Lemli-Opitz syndromeThe Smith-Lemli-Opitz syndrome occurs in 1/20,000 live births in North AmericanCaucasians and has a carrier frequency of 2 %. Therefore, it is a fairly common genetic

disease. It causes severe developmental abnormalities, including microcephaly, pituitaryagenesis, midline facial anomalies, and defects in many internal organs. The disease alsoproduces neurological dysfunction and severe mental retardation. Interestingly, Dr. Opitzwas a faculty member in Neurology at the University of Iowa.

The cause of the Smith-Lemli-Opitz syndrome is a mutation in the sterol D7-reductasegene. The sterol D7-reductase operates in the isoprenoid synthesis pathway betweenlanosterol and cholesterol. Because of this genetic defect, cholesterol synthesis cannotoccur. Instead, there is an accumulation of two sterols that are substrates for this enzyme,7-dehydrocholesterol and cholest-5, 7, 24-trien-3b-ol .

A mother carrying a Smith-Lemli-Opitz syndrome fetus provides adequate amounts ofplasma cholesterol to the fetal circulation through the placenta, but this apparently doesnot compensate for the inability of the fetal tissues to synthesize cholesterol. It isuncertain whether the accumulation of 7-dehydrocholesterol and cholest-5, 7, 24-trien-3b-ol, or the failure to synthesize cholesterol, causes the Smith-Lemli-Opitzsyndrome abnormalities.

Attempts have been made to treat Smith-Lemli-Opitz syndrome patients by feeding themvery large amounts of cholesterol. The rationale for this approach is two-fold. One is toreplace the cholesterol that cannot be synthesized. The other is to down-regulate HMG CoAreductase through feedback inhibition and thereby reduce the production of 7-dehydrocholesterol and cholest-5, 7, 24-trien-3b-ol. While there are a few reportsindicating some improvement, the results have not been overly successful. This probably isdue to the fact that most of the severe abnormalities are imprinted in utero, and beginningtreatment after the child is born is far too late.


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Regulation of Cholesterol IIMetabolism

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Objectives

Know how cholesterol is esterified and thefunction of cholesterol esters

Know how bile acids are synthesized and theirfunction

Understand the term oxysterol Know the types of receptors that regulate

cholesterol metabolism

Understand the role of bile acids and neutralsterols in cholesterol excretion

Understand the mechanism of intracellularcholesterol transport

Understand the role of 24-hydroxycholesterolin brain cholesterol metabolism


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Key termscholesterol ester LCAT cholesterol ester hydrolase ACATlysosomal acid lipase bile acidsterol 7"-hydroxylase cholic acidsterol 27-hydroxylase chenodeoxycholic acid27-hydroxycholesterol24-hydroxycholesterol neutral sterolLDL receptor ABCA1 transporterNPC1 protein NCP2 protein

ABCG transporters

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What happens to cholesterol you eat?Hormonal signal made in duodenum: Cholecystokinin.

C= CholesterolsCE+ Cholesterol ester

Steps:1. Bile in small intestine acts as

an emulsifying agent. Bile isproduced in the liver andstored in the Gall Bladder.Cholecystokinin cause theGall bladder to contract.

2. Cholesterol esterase andother digestive enzymes are

made in the pancreasresponding tocholecystokinin. Conversion

of CE to free C. 3. Cholesterol is absorbed and

or has uptake facilitated ( by

NPCL1) into intestinalenterocytes ( jejunum andileum).

4. Some excretion by ABCG5and 8.

5. Reconversion into CE ( ACATreaction)


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5Lipid metabolic enzymes: emerging drug targets for the treatment of obesity Yuguang Shi & Paul Burn

Nature Reviews Drug Discovery 3, 695-710 (August 2004)

Digestion of lipid: Cholesterol esterscombine withtriacylglyceride,ApoB and MTP toform chylomicrons.

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a | Dietary lipid digestion begins in the stomach, where lipids are subjectedto partial digestion by gastric lipase and form large fat globules with

hydrophobic triacylglycerol (TAG) cores surrounded by polar molecules,including phospholipids (PLs), cholesterol (CL), fatty acids (FAs) and ionizedproteins. The digestive processes are completed in the intestinal lumen,where large emulsions of fat globules are mixed with bile salts (BS) andpancreatic juice containing lipid digestive enzymes to form an aqueoussuspension of small fatty droplets to maximize exposure to the pancreaticlipases for lipid hydrolysis. Monoacylglycerol (MAG), diacylglycerol (DAG)and free FAs that are released by lipid hydrolysis join BS, CL,lysophosphatidic acid (LPA) and fat-soluble vitamins to form mixed micellesthat provide a continuous source of digested dietary products forabsorption at the brush-border membranes of the enterocytes. b | FAs andMAG enter the enterocytes by passive di"usion and are facilitated bytransporters, such as intestinal FA-binding protein (IFABP), CD36 and FA-transport protein-4 (FATP4). They are then re-esterified sequentially inside

the endoplasmic reticulum by MAG acyltransferase (MGAT) anddiacylglycerol acyltransferase (DGAT) to form TAG. Phospholipids from thediet as well as bile mainly LPA are acylated by 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) to form phosphatidic acid (PA), which isalso converted into TAG. Dietary CL is acylated by acyl-CoA:cholesterolacyltransferase (ACAT) to cholesterol esters (CE). Facilitated by microsomaltriglyceride transfer protein (MTP), TAG joins CE and apolipoprotein B (ApoB)to form chylomicrons (CM) that enter circulation through the lymph.


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CHOLESTEROL ESTERS The intracellular storage and plasma transport form of

cholesterol

Cholesterol Ester Synthesis ACAT (acylCoA:cholesterol acyltransferase) intercellular

and ER

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CHOLESTEROL ESTERS The intracellular storage and plasma transport form

of cholesterol

Cholesterol Ester Synthesis LCAT (lecithin:cholesterol acyltransferase) for

plasma synthesis, liporoteins, HDL


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What is the role of cholesterol

esters?

Not generally found in membranes Makes cholesterol less polar and is the

preferred form for transport and storage.

Acyl components vary from tissue totissue.

Accu

Lipids and More

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