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Home , Estrane, Fatty acid, Lathosterol oxidase

FattyFatty AcidAcid andand TriacylglycerolTriacylglycerol BiochemistryBiochemistry -- Omega-3Omega--33 FattyFatty AcidsAcids --

ThomasThomas Dayspring,Dayspring, MD,MD, FACPFACP ClinicalClinical AssistantAssistant ProfessorProfessor ofof MedicineMedicine UniversityUniversity ofof MedicineMedicine andand DentistryDentistry ofof NewNew Jersey,Jersey, NewNew JerseyJersey MedicalMedical SchoolSchool DiplomateDiplomate ofof thethe AmericanAmerican BoardBoard ofof ClinicalClinical LipidologyLipidology CertifiedCertified MenopauseMenopause Practitioner:Practitioner: NorthNorth AmericanAmerican MenopauseMenopause SocietySociety NorthNorth JerseyJersey InstituteInstitute ofof MenopausalMenopausal LipiLipidologydology Wayne,Wayne, NewNew JerseyJersey StStJoseph Joseph’s’s RegionalRegional MedicalMedical CenterCenter Paterson,Paterson, NJNJ LipidLipid ClassificationClassification

) are biologic substances that are generally hydrophobic or amphipathic in nature and in many cases soluble in organic solvents. ) Lipids are produced, transported and recognized by the concerted actions of numerous , binding proteins and receptors ) Lipids include: fatty , , sterols, , terpenes and others:

• Fatty acids • Sterol lipids • Glycerolipids • Prenol lipids • Glycerophospholipids • Saccharolipids • Polyketides • Sphingolipids • Polyketides

Fathy E et al. J Res 2005;46:839-861 PhospholipidsPhospholipids • Contain one or more fatty molecule and oneone phosphoricphosphoric acid radical and usually contain a nitrogenous

MajorMajor types:types: Polar End •: •Lecithins: phosphatidylcholine Non-Polar End •Cephalins: phospatidylethanolamine • Some formed from inositol • • 90% are formed in the , but all cells can make them • They are all lipid soluble and transported in and used throughout the body for structural purposes • Phospholipids are donors of phosphate radicals which are needed for different chemical reactions in tissues

Guyton & Hall Textbook of Medical Physiology Unit XII pp756-757 WB Saunders Philadelphia 2000 LipidLipid ClassificationClassification

) Lipid Nomenclature • Stereospecific numbering (sn) method is used in describing glycerolipids and glycerophospholipids • The group is typically acylated or alkylated at the sn-1 or sn-2 position • Core names are used for sterols (, and )

Fathy E et al. J Lipid Res 2005;46:839-861 LipidLipid ClassificationClassification

) Fatty acids (acyl) group: repeating series of methylene groups that impart hydrophobic character • First subclass is the straight chain saturated group with a terminal ) Glycerolpids are abundant as membrane constituents, metabolic fuels (acylglycerols) and signaling molecules ) Glycerophospholipids are ubiquitous and key components of the lipid bilayers of cells • Phospholipids may be divided by the nature of the polar head group at the sn-3 position of the glycerol backbone ) Sphingolipids are a complex family with a sphingoid base: , phosphosphingolipids & glycosphingolipids

Fathy E et al. J Lipid Res 2005;46:839-861 LipidLipid ClassificationClassification

) Sterol Lipids are important membrane lipids • and derivatives • Cholesteryl • Phyto, marine and fungal sterols

• (C18, C19, C21)

• (Vitamin D2 and D3) • Bile acids and derivatives • conjugates • Hopanoids

Fathy E et al. J Lipid Res 2005;46:839-861 LipidLipid ClassificationClassification

) Prenol lipids are synthesized from the five precursors isopentyl diphosphate and dimethylallyl diphosphate from the pathway. ) Saccharolipids (): Fatty acids are linked directly to a sugar backbone forming structures compatible with membrane bilayers. They exist as glycan or phosphorylated derivatives

Fathy E et al. J Lipid Res 2005;46:839-861 FattyFatty AcidAcid NomenclatureNomenclature

) Trivial and shorthand nomenclature have been in common usage. ) Gas liquid chromatography separates FA by chain length and saturation: nomenclature using this technology consists of two numbers separated by a colon. • The number before the colon gives the carbon number and the number after denotes the number of double bonds • Sometimes it is useful to number the double bonds from the methyl end: ie n-6 • Older literature uses ω instead of n • Δ refers to a double bond: Δ-9 is a double bond at #9 position • The isomeric configuration around a double bond can be cis/trans or as it is now called Z/E • In the cis form the two hydrogen substituents are on the same side of the molecule and in trans form they are on opposite sides. Trans FA are rare. • Older systems numbered carbon atoms with Greek letters: • α refers to C2, β to C3 and ending with ω at the last atom, furfurthestthest from the carboxyl chain

Gurr MI et al. Lipid 5th Ed 2002 Blackwell Science Malden, MA FattyFatty AcidAcid NomenclatureNomenclature

) Commonly occurring acids # Systematic Name Common Name

) 2 n-Ethanoic Acetic ) 3 n-propanoic Propionic ) 4 n-butanoic Butyric ) 6 n-hexanoic Caproic ) 10 n-decanoic Capric ) 12 n-dodecanoic Lauric ) 14 n-tetradecanoic Myristic ) 16 n-hexadecanoic Palmitic

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA FattyFatty AcidAcid NomenclatureNomenclature

) Commonly occurring acids # Carbons Systematic Name Common Name ) 18 n-octadecanoic Stearic ) 20 n-eicosanoic Arachidic ) 22 n-docosanoic Behenic ) 24 n-tetracosanoic Lignoceric ) 26 n-hexacosanoic Cerotic ) 28 n-octacosanoic Montanic

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA SaturatedSaturated FattyFatty AcidsAcids

) MajorMajor dietarydietary determinantdeterminant ofof LDLLDL--CC • For every 1% increase in calories from SF, serum LDL-C raises 2% or vice versa ) DELTADELTA andand beFITbeFIT studiesstudies havehave demonstrateddemonstrated benefitbenefit inin improvingimproving LDLLDL--CC ) ReducedReduced intakesintakes showshow nono compromisedcompromised growthgrowth oror developmentdevelopment issuesissues inin childrenchildren

NCEP ATP-III Circulation 2002;25:3260 UnsaturatedUnsaturated FattyFatty AcidsAcids

) MonoenoicMonoenoic (monounsaturated)(monounsaturated) • The more common have an even number of carbon atoms and a chain length of 16-22 carbons and a double bond with the cis configuration • Often the cis begins at the Δ9 position • The most common monoenoic acid is an 18 chain: • cis-9-octadecenoic acid or

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA UnsaturatedUnsaturated FattyFatty AcidsAcids

) PolyenoicPolyenoic (polyunsaturated)(polyunsaturated) • Unsaturated FA are very susceptible to oxidation; the more double binds the more the susceptibility

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA UnsaturatedUnsaturated FattyFatty AcidsAcids

) Commonly occurring acids

# Carbons Systematic Name Common Name ) Dienoic 18 Δ-6,9 octadecadienoic Linoleic ) Trienoic 18 Δ-6,9,12 octadecatrienoic γ-Linolenic 18 Δ-9,12,15 octadecatrienoic α-Linolenic ) Tetraenoic 20 Δ-5,8,11,14 eicosatetraenoic Arachidonic ) Pentaenoic 20 Δ-5,8,11,14,17 eicosapentaenoic ) Hexaenoic 22 Δ-4,7,10,13,16,19 docosahexaenic

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA SynthesisSynthesis ofof EPAEPA && DHADHA

H3C H3C COOH COOH

Δ15-desaturase α- (18:3n-3) (18:2n-6) (plants only)

Δ6-desaturase Δ6-desaturase (18:4n-3) γ-linoleic acid (18:3n-6) Elongase Elongase 20:4n-3) Di-homo-γ-linolenic acid Δ5-desaturase (20:3n-6) (20:5n-3) Δ5-desaturase Elongase (20:4n-6) (22:5n-3) Elongase Neither linoleic or linolenic acid can be synthesized de novo in (24:5n-3) Δ6-desaturase Conversion of α-linolenic acid into its (26:6n-3) longer chain derivatives is not very efficient in adult humans especially males β-oxidation (22:6n-3)

Calder P. Clinical Science 2004;107:1-11 DeDe NovoNovo FattyFatty AcidAcid SynthesisSynthesis

Carbohydrate, protein carbon sources

Small precursor Acetyl-CoA carboxylase Long chain FA synthase acetyl-CoA Other modifications Elongases Desaturases Unsaturated FA Modified FA

Elongases

Desaturases Very long chain FA (>18C)

Gurr MI et al. Lipid Biochemistry 5th Ed 2002 Blackwell Science Malden, MA TriglyceridesTriglycerides

) TriglyceridesTriglycerides areare waterwater--insolubleinsoluble O lipidslipids consistingconsisting ofof threethree fattyfatty acidsacids ║ H C-O-C-R linkedlinked toto oneone glycerolglycerol molecule.molecule. 2 1 O • They represent a concentrated source of ║ metabolic energy contributing 9 kcal/gm. HC-O-C-R2 O ) TGTG areare transportedtransported asas corecore ║ constituentsconstituents ofof allall lipoproteinslipoproteins,, butbut H2C-O-C-R3 thethe greatestgreatest concentrationconcentration isis inin TGTG-- richrich chylomicronschylomicrons andand VLDLVLDL particlesparticles

R = Fatty acid chain

Rafai, N et al. Handbook of Testing AACC Press Washington DC 2nd Ed 2000 SubstratesSubstrates forfor TriacylglycerolTriacylglycerol SynthesisSynthesis Plasma

NEFA Acyl-CoA Synthetase Glc-6-Pase Acyl-CoA CoA Multiple steps Glucose-6-P Hepatocyte Hepatocyte PEPCK CPT I Acyl-CoA Pyruvate kinase Acyl- CPT II PEP Malonyl-CoA Acyl-CoA Acyl-CoA Carboxylase Krebs Krebs Citrate Acetyl-CoA Pyruvate Cycle ATP Citrate Acetyl-CoA CO Pyruvate 2 HMG-CoA Synthase Citrate Bodies PEP = phosphoenolpyruvate Beta-oxidation PEPCK = PEP carboxylase CPT = Carnitine palmitoyl Mitochondria TriacylglycerolTriacylglycerol ()(Triglyceride) SynthesisSynthesis && DGATDGAT EnzymesEnzymes

Glycerol-3-phosphate Glycerol Phosphate FA CoA GPAT Pathway Pathway Lysophosphatidate FA CoA AGPAT Phosphatidate

Monoacylglycerol PPH-1 Pathway FA CoA Monoacylglycerol Diacylglycerol MGAT FA CoA DGAT 1&2 GPAT = glycerophosphate acyltransferase AGPAT = acylglycero-phosphate acyltransferase PPH-1 = phosphohydrolase-1 MGAT = AcylCoA:monoglycerol acyltransferase Triacylglycerol DGAT Diacylglycerol acyltransferase

Hubert C. Chen, Robert V. Farese, Jr ATVB 2005;25:482-486. HepaticHepatic TriacylglycerideTriacylglyceride SynthesisSynthesis

Mitochondria Glycerol DHAP FA FA Glycerol Dihydroxyacetone-P ACS ACS Acyl-CoA Synthetase FA-CoA FA-CoAFA-CoA FA-CoA G3P Glycerol-3-phosphate GPAT COCO2 GPAT Glycerol-P 2 acyltransferase LPA LPA KetonesKetones AGPAT AGPAT acylglycerol-P acyltransferase PA PA Phosphatidic Acid LPA PAP-1 PG Phosphatidylglycerol Phosphatidic acid Ether lipids phosphohydrolase PI Phosphtidylinositol CL Cardiolipin DAG DHAP-AT 1-acyl- DGAT acyltransferase DHAP diacylglycerol acyltransferase PC phosphatidylcholine FA-CoA TAG PE ACS DHAP PS Phosphatidylserine VLDL Lipid FA Droplet

Coleman R & Douglass L. Prog Lip Res 2004 43;134-176 RegulationRegulation ofof LipogenicLipogenic GenesGenes

Oxysterols gene SREBP-2 mRNA SREBP-2 precursor SREBP-2 protein protein sterol - - nuclear mature SREBP-2 form mature SREBP-2

CholesterolCholesterol SyntheticSynthetic GenesGenes

Coleman R & Douglass L. Prog Lip Res 2004 43;134-176 RegulationRegulation ofof LipogenicLipogenic && TGTG GenesGenes

+ SREBP-1c gene SREBP-2 + + LXR + - fasting mRNA SREBP-1c SREBP-2 PUFA precursor SREBP-2 protein SREBP-1c protein protein sterol - - + glucose nuclear mature SREBP-2 mature SREBP-1c form mature SREBP-2 mature SREBP-1c

Cholesterol Fatty Acid and Glycerolipid Synthetic Genes Synthetic Genes

Coleman R & Douglass L. Prog Lip Res 2004 43;134-176 NuclearNuclear ReceptorsReceptors

) Nuclear Receptors (NRs) exist in inactive forms as multi-protein complexes. ) Activation occurs when a ligand (fatty acid) binds to the NR and causes conformational changes • This alters the protein-protein interfaces of the molecule ) Heterodimerization with the Retinoid X Receptor occurs further changing the conformation NuclearNuclear ReceptorsReceptors

) At least 4 nuclear receptors (NRs) are affected by fatty acids and may regulate triglyceride . • (LXR) • Hepatocyte Nuclear Factor-4- (HNF-4-α) • Farnesol X Receptor (FXR) • Peroxisome Proliferator-Activated Receptors (PPARs) ) These often heterodimerize with Retinoid X Receptors (RXR)

xxxxxxxxx PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated SubfamilySubfamily ofof NuclearNuclear RReceptoreceptorss ((PPARsPPARs))

) PPARPPAR andand LXR subfamiliessubfamilies accountaccount forfor 55 ofof thethe 4848 nuclearnuclear receptorsreceptors thatthat havehave beenbeen identifiedidentified inin humanhuman andand mousemouse genomesgenomes

) TheyThey possesspossess aa conservedconserved DNADNA bindingbinding andand ligandligand bindingbinding domainsdomains • The DNA binding domain has two zinc C-terminal ligand finger motifs that mediate sequence- binding domain specific recognition of hormone-response elements in distinct target genes • The C-terminal ligand binding domain determines the specific binding properties of each receptor and mediates ligand-regulated interactions with effectors or repressors of transcription DNA Binding Domain

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 TranscriptionalTranscriptional ActivitiesActivities ofof PeroxisomePeroxisome ProliferatorProliferator--ActivatedActivated andand LiverLiver XX ReceptorsReceptors PPAR or LXR PPARs and LXRs possess the conserved DNA binding C-terminal ligand binding domains domain and the C-terminal C-terminal ligand binding domains ligand binding domain RXR characteristic of nuclear hormone receptors

PPARs and LXRs bind to DNA Binding Domains specific response elements in target genes as heterodimers with retinoid X receptors (RXRs) which are also members of the superfamily

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 LLiveriver XX RReceptoreceptorss ((LXRsLXRs))

LXR alpha

Tissue expression Ligands C-terminal ligand Liver, macrophages 24(S),25 epoxycholesterol, binding domain binding domain 22(S)-hydroxycholesterol, Biological Functions 24(S)-hydroxycholesterol Cholesterol absorption Disease Targets (intestine), cholesterol Atherosclerosis excretion (liver), DNA Binding cholesterol efflux Domain (peripheral cells), fatty acid (liver & peripheral cells)

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 LLiveriver XX RReceptoreceptorss ((LXRsLXRs))

LXR beta

Tissue expression Ligands C-terminal ligand Broadly expressed 24(S),25 epoxycholesterol, binding domain 22(S)-hydroxycholesterol, Biological Functions 24(S)-hydroxycholesterol Cholesterol absorption Disease Targets (intestine), cholesterol Atherosclerosis excretion (liver), DNA Binding cholesterol efflux Domain (peripheral cells), fatty acid biosynthesis (liver & peripheral cells)

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 LXRLXR ControlsControls SREBPSREBP--22 andand SREBPSREBP--1c1c

Citrate SREBP-2 ATP-citrate lyase Acetyl CoA synthetase SREBP-1c Acetyl CoA Acetoacetyl CoA Acetyl CoA carboxylase Malonyl CoA Acetoacetyl CoA HMG CoA synthase Malate NADPH Fatty acid synthase Long chain fatty acyl elongase HMG CoA Malic HMG CoA reductase NADPH Saturated fatty acids NADPH Mevalonate Stearoyl CoA desaturase Mevalonate kinase NADPH Phosphomevalonate kinase G6PD PGDH Mevalonate PP decarboxylase Monounsaturated GPP synthase NADPH fatty acids IPP Glucose-6-P 6-P-glucosonate FPP synthase Fatty Acyl CoA Squalene synthase Squalene GPAT Squalene expoxidase Lanosterol synthase NADPH CYP51 Monoglycerol 3-phosphate Lathosterol oxidase DHCR LDL receptor Triacylglycerols and Cholesterol phospholipids

Sterol Regulatory Element Binding Protein (SREBP)-2 regulates the genes involved with cholesterol synthesis while SREBP-1C stimulates the lipogenic enzyme genes LiverLiver XX ReceptorsReceptors

) LXR, which prevents excessive cellular cholesterol is activated by binding of ligands such as oxysterols ) LXR activation prevents excessive cellular cholesterol by • enhancing the expression of genes which stimulate synthesis (7-alpha-hydroxylase CYP7A) • Activates ABCA1 to promote cholesterol efflux into HDL • Activates ABCG5, G8 to increase cholesterol from hepatic cells into bile and from intestinal cells into the lumen: in effect the latter inhibits cholesterol absorption ) The net effect is reduced cellular cholesterol LXRLXR andand BileBile AcidAcid FormationFormation

Heterodimerization with Retinoid X Receptor Catalyzes formation of bile acids Mitochondria RXRRXR Mitochondria ↑ 7α

hydroxylase 27 OH-Cholesterol production

LXRLXR Liver X Receptor Upregulation

Increased oxysterols C24 acids

Increased Free Cholate, Chenodeoxycholate synthesis Hepatocyte Cholesterol (FC) LXRLXR && ApoAApoAApoA-I--II LipidationLipidation

Hepatocyte, enterocytes, or any other Heterodimerization with excess cholesterol with Retinoid X Receptor RXRRXR

Lipid–free apoA-I or

prebetaCE1 HDL LXRLXR Liver X Receptor Upregulation Pre-beta2 or Discoidal or Phospholipids Nascent HDL Free Cholesterol (FC) The FC is esterified by the ATP Binding Cassetteenzyme LCAT to cholesteryl (CE) creating larger Plasma Transporter A1 (ABCA1)ester (CE) creating larger alpha HDL particles Rye K-A & Barter PJ. ATVB 2003;24:1-8 Sahoo D et al. J Lip Res 2004;45:1122-1131 LXRsLXRs ActivateActivate ABCG5,ABCG5, ABCG8ABCG8

Increased hepatic cholesterol stores activates LXR ABCG5/G8 expression, which in turn increases expression of

hepatic and intestinal ABCG5/G8 Sterols RXR LXR Hepatocyte

LDL receptors

CE ↑Hepatic Sterol excretion of Cholesterol Micelle cholesterol & LDL stores campesterol, etc ↓Sterol Absorption Cholesterol & Campesterol, etc

VLDL NPC1L1 ABCG4 ABCG5/8 ABCB11

Bile Ductule Hepatic Sinusoid Gut Enterocyte lumen HepaticHepatic NuclearNuclear FactorFactor -- 44 --AlphaAlpha

) Hepatic Nuclear Factor - 4- alpha (HNF-4-α) binds to long chain fatty acyl-CoA with high affinity ) Binding of saturated FA stimulates whereas binding of polyunsaturated FA (PUFA) inhibits HNF-4-α ) HNF-4-α affects genes encoding proteins involved in both and including: • apoC-III, apoA-I, apoA-IV on lipoproteins • L-pyruvate kinase in carbohydrate metabolism • CYP 7A in bile acid synthesis ) Fibrates bind HNF-4-α and inhibit its transcriptional activity HepaticHepatic NuclearNuclear FactorFactor -- 44 --AlphaAlpha

Hepatocyte HNF-4-α

Long chain FA activation or PUFA inactivation of HNF-4-α

Potential Targets apoC-III, apoA-I, apoA-IV (LP) Endoplasmic L-pyruvate kinase (carbs) reticulum CYP 7A hydroxylase (BA)

Protein synthesis FarnesolFarnesol XX ReceptorReceptor (FXR)(FXR)

) Farnesol X Receptors are activated by bile acids and PUFA. They appear to protect cells from bile acid toxicity ) FXR controls bile acid synthesis by inhibiting expression of CYP 7A hydroxylase and other bile synthetic enzymes ) FXR controls expression of the bile acid export pump (BSEP) also termed ATP binding cassette transporter B11 (ABCB11) ) FXR expression has hypotriglyceridemic effects by: • Inducing PPAR-α expression • Modulating lipoprotein • Inhibition of Sterol regulatory binding element protein-1C (SREB-1c) which is mediated through the short heterodimer protein (SHP) negative effect on LXR FXRFXR andand BileBile AcidAcid FormationFormation

Heterodimerization with Retinoid X Receptor Catalyzes formation of bile acids Mitochondria RXRRXR Mitochondria ↑ Oxysterol 7α hydroxylase 27 OH-Cholesterol production

FXRFXR Peroxisomes Farnesol X Receptor Upregulation C24 acids

Cholate, Chenodeoxycholate synthesis Hepatocyte PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated RReceptoreceptorss (PPARs)(PPARs)

) Peroxisome Proliferator-Activated Receptors (PPARS) exist as three subtypes (alpha, beta or and gamma). They vary in their expression and biological function: • PPAR-α regulates genes in hepatocytes and vascular cells involved with multiple aspects of lipoprotein metabolism, fatty acid and vascular inflammation • PPAR-γ regulates genes in muscles and involved with glucose control and vascular inflammation • PPAR-Δ is widely expressed and its function is not thoroughly understood at present RoleRole ofof PPeroxisomeeroxisome PProliferatorroliferator-- AActivatedctivated RReceptoreceptorss αα ((PPARsPPARs))

PPARPPAR αα NucleusNucleus withwith genesgenes

EndoplasmicEndoplasmic reticulumreticulum PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated RReceptoreceptorss αα AgonismAgonism

The heterodimer NuclearNuclear ActivityActivity binds to response PPARPPAR αα elementsPPAR αin promoteragonist region of target genes Retinoid X Receptor PPARs and Retinoid X Receptor heterodimerize

Gene transcription Recruitment of transcriptional coactivators or repressors PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated RReceptoreceptorss αα AgonismAgonism

NuclearNuclear ActivityActivity

mRNA leaves the nucleus and enters the PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated SubfamilySubfamily ofof NuclearNuclear RReceptoreceptorss ((PPARsPPARs)) PPAR alpha

Tissue expression Ligands C-terminal ligand binding domain Liver, Heart, Kidney Adrenal PUFAs, 8(S)-HETE

Cell specific expression Disease Targets Endothelium, Macrophages, Hypertriglyceridemia Smooth Muscle cells DNA Binding Biological Functions Drugs Domain TG-rich lipoprotein synthesis Fibrates and metabolism, β-oxidation of FA, anti-inflammation HETE = hydroxyeicosatetraenoic acid

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated SubfamilySubfamily ofof NuclearNuclear RReceptoreceptorss ((PPARsPPARs))

PPAR gamma

Tissue expression Ligands C-terminal ligand binding domain , spleen, PUFAs, 15d-PGJ2, adrenal, colon 13-HETE, 9-HODE Cell specific expression Disease Targets Macrophages, T cells

DNA Binding Biological Functions Drugs Domain Fat cell development, TZDs Glucose homeostasis, HETE = hydroxyeicosoateraenoic acid 12,14 Anti-inflammation 15dPGJ2 15-deoxyΔ - J2 HODE = hydroxyoctadecadienoci acid

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 PPeroxisomeeroxisome PProliferatorroliferator--AActivatedctivated SubfamilySubfamily ofof NuclearNuclear RReceptoreceptorss ((PPARsPPARs))

PPAR delta

C-terminal ligand Tissue expression Ligands binding domain Many tissues PUFAs

Cell specific expression Disease Targets Many cell types Metabolic syndrome ? DNA Binding Biological Functions Drugs Domain Biological Functions Drugs Endothelial biology, - - - Energy utilization,

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 TranscriptionalTranscriptional ActivitiesActivities ofof PeroxisomePeroxisome ProliferatorProliferator--ActivatedActivated andand LiverLiver XX ReceptorsReceptors

Active Repression Ligand Dependent Ligand Dependent Transactivation Transrepression Fatty Acids HDACs PPARs Corepressor Coactivator Complexes Fatty Acids Complexes LXRs NCoR/SMRT PPARs Oxysterols PPARs LXRs RXRs LXRs RXRs Oxysterols TATA TATA TATA NF-κB PPRE/LXRE PPRE/LXRE Element In the absence of ligands, In the presence of ligands, PPAR/RXR PPARs and LXR agonists PPAR/RXR and LXR/RXR and LXR/RXR heterodimers activate can inhibit the activities heterodimers can bind to target transcription through the recruitment of of other signal- genes and actively repress diverse coactivator complexes that dependent transcription transcription through the include nucleosome remodeling activity, factors, such as nuclear recruitment of corepressor histone acetyltransferase and histone factor κB and activator complexes that contain NCoR, methyltransferase activities, and directly protein-1 (AP-1). This SMRT and histone deacetylases or indirectly recruit core transcriptional repression function (HDACs) machinery to the promotor contributes to their anti- inflammatory actions Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 TranscriptionalTranscriptional ActivitiesActivities ofof PeroxisomePeroxisome ProliferatorProliferator--ActivatedActivated andand LiverLiver XX ReceptorsReceptors

Active Repression Ligand Dependent Transactivation

HDACs Corepressor Coactivator Complexes Fatty Acids Complexes NCoR/SMRT PPARs PPARs LXRs RXRs LXRs RXRs Oxysterols

TATA TATA PPRE/LXRE PPRE/LXRE

In the absence of ligands, PPAR/RXR and In the presence of ligands, PPAR/RXR and LXR/RXR heterodimers can bind to target LXR/RXR heterodimers activate transcription genes and actively repress transcription through the recruitment of diverse coactivator through the recruitment of corepressor complexes that include nucleosome complexes that contain NCoR, SMRT and remodeling activity, histone acetyltransferase histone deacetylases (HDACs) and histone methyltransferase activities, and directly or indirectly recruit core transcriptional machinery to the promotor

Li, AC, & Glass CH. J Lipid Res 2004;45:2161-2173 NN--33 FAFA DirectDirectNEFANEFA NEFANEFA RegulationRegulation awayaway fromfrom TGTG ofof storagestorage GenesGenes toto oxidationoxidation Bile Acids SHP

LXR SREBP-1c FXR

Acetyl CoA Carboxylase Fatty Acid Synthase Spot 14 Acyl CoA NEFA & Unsaturated PPAR N3-FA Saturated

L-FABP HNF-4α

RXR Transport Apolipoproteins Oxidation Pyruvate kinase Fatty Acid Binding Protein Glucose-6-phosphalase Ketogenesis Transferin Bile Acids Adapted from Pegorier JP et al. J Nutr 2004;134:2444S-9S NN--33 FattyFatty AcidsAcids onon NuclearNuclear ReceptorsReceptors InvolvedInvolved withwith LipogenesisLipogenesis

Effects on Expected Changes Gene Regulation Triglycerides HDL LDL PPAR-alpha Increase ↓↓ ↑ ↓ LXR Decrease ↓↓ ↓ ↓ FXR Increase ↓↓ ↑ ↑ HNF-4-alpha Decrease ↓↓ ↓ _

Net Effects ↓↓↓↓ Neutral Neutral EffectsEffects ofof NN--33 FAFA onon ApoApo BB

Hepatocyte

Nucleus

Endoplasmic Reticulum + SFAs IDL

Lipids PERPP

apoB n-3 PUFA-ox

ERAD Large LDL Post ER Presecretory ER associated Proteolysis degradation X PERPP Plasma Large VLDL Peroxidation products of n-3 fatty acids increase PERPP which decreases the secretion of larger VLDL and subsequently lowers levels of its catabolic products (small LDL) This may explain how n-3 FA shift LDL size X Clinical trials reveal only a slight reduction in apoB Small LDL Adapted from Krauss RM. J Clin Invest 2004;113:1253-55 NN--33 FattyFatty AcidsAcids andand ChylomicronsChylomicrons

) n-3 FA decrease VLDL secretion which presents less competition for chylomicron ) n-3 FA supplementation does decrease chylomicron particle size thereby improving clearance

) n-3 FA increase pre-heparin activity in the fed state but had no effect on post-heparin lipase activity ) These combined effects support the use of n-3 FA as a valuable clinical tool for then treatment of hypertriglyceridemia

Park Y. et al. J Lipid res 2003;44:455-463 OmegaOmega--33--EthylEthyl EstersEsters andand LipidsLipids

60% Patients with TG > 500 mg/dL Placebo 45.0%

40% Omega-3 AEE 4g/day

20% p<0.0002 p<0.0001 9.1% 6.7% p<0.0001 0% 0% -3.6% -1.7% -0.9% -4.8% -9.7% -13.8% p<0.0001 -20% p<0.0059 p<0.0015

-40% -42.0% -45%

-60% TG HDL-C Non HDL-C TC VLDL-C LDL-C

Baseline Lipids 816 mg/dL 22 mg/dL 2711 mg/dL 296 mg/dL 175 mg/dL 89 mg/dL

Pownall JH. et al. Atherosclerosis 1999;143:285-297 OmegaOmega--33--EthylEthyl EstersEsters andand LipidsLipids

Placebo 20% Placebo Patients with TG > 200-499 mg/dL Omega-3 AEE 4g/day 11.3% 10% 8.4% 9.1% 1.7% 1.7% 0% 0% -1.2% -0.7% -0.6% -1.5% -0.6% -1.9% -1.0%

-10%

-20% -20.0% -24.3%

-30% TG HDL-C Non HDL-C TC VLDL-C LDL-C Apo B

Dara on file, Reliant Pharmaceuticals IntracellularIntracellular RegulationRegulation ofof ApoApo BB

Hepatocyte

Nucleus

Endoplasmic Reticulum + SFAs IDL

Lipids Lipid Droplet PERPP

apoB

ERAD Large LDL Post ER Presecretory ER associated Proteolysis degradation PERPP

Plasma The cotranslational binding of lipids to apoB in the ER by Large VLDL microsomal transfer protein is affected by the various fatty acids available for utilization Small ERAD and PERPP enhances apoB proteolysis and inhibits LDL secretion of apoB lipoproteins. Their inhibition would facilitate apoB particle secretion Saturated fatty acids protect smaller lipoproteins from PERPP Adapted from Krauss RM. J Clin Invest 2004;113:1253-55 leading to increased secretion of small VLDL and IDL