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Chapter 18: Metabolism and Movement of Lipids

Outline:1. Phospholipids and sphingolipids:

synthesis and intracellular movement2. Cholesterol: a multifunctional

membrane lipid3. Lipid movement into and out of cells4. Feedback regulation of cellular lipid

metabolism5. The cell biology of atherosclerosis,

heart attacks, and strokes

Cell use lipids for:1. Storing energy2. Building membranes3. Signaling within and between cells4. Sensing the environment5. Covalently modifying proteins6. Forming specialized permeability barriers (e.g., in skin)7. Protecting cells from highly reactive chemicals

Fatty acids – ATP(mitochodria), triglycerides, phospholipids, plasmalogens, sphingolipidsCholesterol – membrane components, precursors for steroid hormones and other biologically active lipids, fat-soluble vitamins (vit A - light detection, vit D - calcium metabolism, vit E - antioxidants, vit K – blood coagulation)Lipid biogenesis, lipid transport, cholesterol metabolism and atherosclerosis

Fig 18-1 Overview of synthesis of major membrane lipids and their movement into and out of cells

Proteolysis of an integral membrane protein precursor

Nuclear sterol regulatory element-bind proteins

18.1 Phospholipids and sphingolipids: synthesis and intracellular movement

Fatty acids are precursors for phospholipids, sphingolipids, and other membrane components:Fatty acid synthesis: – regulation of membrane synthesis

saturated fatty acids, unsaturated fatty acids, C14, C16, C18(stearate, oleate, linoleate, linolenate) C20(arachidonate)

C14, C16 – synthesized in cytosol from acetyl CoA by acetyl-CoA carboxylase & fatty acid synthetase

Palmitoyl CoA – elongated to 18-24 in ER or mitochondriaDe saturase – located in ER, introduce double bonds, kink -

unsaturated fatty acids – more fluidEssential polyunsaturated FAs – linoleic acid, linolenic acids (亞麻油酸)

Cell division need more membraneNew membrane not old membrane extension, but new synthesis

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Fig 18-2 Chemical structures of FAs and some of their derivatives

16C, saturated FA

20C, polyunsaturated FA

TG - 3 FFA (free fatty acid) + 1 glycerol

Fatty acid are precursor for phospholipids an other membrane components

Arachidonate, platelet activating factor….

TG - 3 FFA (free fatty acid) + 1 glycerol

Fatty acids are precursor for phospholipids and other membrane component

花生四烯酸

Unesterified FAs move within cells bound to small cytosolic proteins

Fig 18-2 Binding of a FA to the hydrophobic pocket of a fatty acid-binding protein (FABP)

FABP ( ): FA glucosein cardiac muscle

Fatty acid-binding proteins: FABP

Fatty acid

OFABP levels are high in active muscles using fatty acids for energyIn adipocytes + fatty acid stored as TG

- fatty acid use by other cellsIn liver 5% of all cytosolic proteins

Interacts non-covalently with hydorphobic amino acid

FABP expression is regulated for release or up-take FA

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Incorporation of FAs into membrane lipids takes place on organelle membranes

Fig 18-4 Phospholipid synthesis

Fatty acid didn’t directly pass membraneAcetyl CoA -------- saturated fatty acid

acetyl-CoA carboxylasefatty acid synthase

Annexin V ; binds to anionic phospholipidslong exposure of exoplasmic face of plasma memb.signal for scavenger cells to remove dying cells

Flippases move phospholipids from one membrane leaflet to the opposite leaflet

asymmetric distribution of phospholipids

senescence or apoptosis – disturb the asymmetric distribution

Phosphatidylserine (PS) and phosphatidylethanolamine: cytosolic leaflet

exposure of these anionic phospholipids on the exoplasmic face – signal

for scavenger cells to remove and destroy

Annexin V – a protein that specifically binds to PS phospholipids

fluorescently labeled annexin V– to detect apoptotic cells

flippase: ABC superfamily of small molecule pumps

Fig 18-5 In vitro fluorescence quenching assay can detect phospholipid flippase activity of ABCB4

Yeast sec mutant – at nonpermissive temp: secretory vesicle cannot fuse with plasma membrane – purify the secretory vesicles

(dithionite)

Phospholipid flippase activity of ABCB4

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18.2 Cholesterol: a multifunctional membrane lipid

50-90% cholesterol is present in plasma membrane and related endocyticvesicle membrane

Storage

release

massive

LCAT: lecithin:cholesterolacyl transferase; convert cholesterol to esterifiedcholesterol

Cholesterol is synthesized by enzymes in the cytosol and ER

(ER integral membrane protein:sterol-sensing domain -TMcatalytic activity-cytosol)

(Cytosol)

(Cytosol)

Rate limiting step

HMG-CoA: β-hydroxy-β-methylglutaryl CoA

Drugs used to inhibit cholesterol synthesis include competitive inhibitors of HMG-CoA Reductase.

Examples include various statin drugs such as lovastatin (Mevacor) and derivatives (e.g., Zocor), Lipitor, etc.

A portion of each statin is analogous in structure to mevalonate or to the postulated mevaldehydeintermediate.

Extensive clinical trials have shown that the statin drugs decrease blood cholesterol and diminish risk of cardiovascular disease.

Massive cholesterol → damage

Excess cholesterol → cholesteryl ester formed by acyl

cholesterol acyl transferase (ACAT) located in ER

membrane → stored →as cytosolic lipid droplets –

interfaced with coat protein (perilipins or perilipin-

related proteins) in mammal cell

Lipid droplets contain: fatty acid, cholesteryl ester

In plant, the related protein coat the surface of lipid

droplets called oil bodies.

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many bioactive molecules are made from cholesterol and its biosynthesic precursors

Cholesterol and phospholipids are transported between organelle by Golgi-independent mechanisms: three hypothesis

Classic secretory pathway disruption (chemical inhibitor, mutation) – do not prevent cholesterol or phodpholipidtransport between membranes although do disrupt transport of proteins and sphingolipids

lipid in the ER – can not move to mitochodrial membrane by classic secretory vesicle transport

Fig 18-8 Proposed mechanism of Golgi-independent transport of cholesterol and phospholipids between membranes

Vesicles transfer lipids, did not across Golgi

Cholesterol and phospholipids are transported between organelles by golgi-independent (non-classic secreted transport systems)-three pathway

membrane

Pathway I

Fig 18-8 Proposed mechanism of Golgi-independent transport of cholesterol and phospholipids between membranes

Membrane-embedded proteins Small lipid-transfer proteins

ER membrane extension for organelle

Pathway II Pathway III

StAR (steroidogenic acute regulatory) protein (key step of cholesterol transport for steroid hormone synthesis)Encoded in nuclear DNA, transfer of cholesterol from the rich outer mitochondiral m embrane to the poor inner membrane; First steps in steroid hormones synthesis-N-terminal targeting sequence to mitochondrial outer membrane-C-terminal START (StAR-related transfer) domain-> Has hydrophobic cholesterol binding pocket

-Key role in moving cholesterol into mitochondrion for steroid hormone synthesisrate-limiting step in steroid hormone synthesis

Ex) mutation in StAR geneCongenital adrenal hyperplasia

-a lethal disease-Drastic reduction in the systhesis of steroid hormone

-Phosphatidylcholine-transfer protein contains START protein

Intracellular cholesterol-transport system (StAR, NPC)

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Current working model of StAR mechanism of action via association of C´ with outer mitochondrial membrane

Cells with nonfunctional Niemann-Pick C1 (NPC1) proteinAccumulate cholesterol in late endosomal/lysosomal vesicles

NPC -integral memb. Protein-Rapidly moving endosome/lysosomal compartment-Contain mutiple membrane spanning segment

Sterol sensing domain

Mutation in NPC1-defect in intracellular cholesterol transport-accumulate excess cholesterol in the late endosomal/lysosomalcompartment

Defect in the regulation of cellular cholesterol metabolism

Rescued by Rab9 (small GTPase)-late endosomal vesicular transport

Intracellular cholesterol-transport system

18.3 Lipid movement into and out of cells

Fatty acid transport proteins (FATPs; cell surface protein CD36), concentration dependentAlbumin – lipid-binding groove, bilirubin (膽紅素) , others

Fatty acid binding protein

Two major pathways for importing and exporting cellular lipids

Poor water solubility, need binding protein

Lipids can be exported or imported in large well-defined lipoprotein complexes

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Cell-surface transports aid in moving fatty acids across the plasma membrane

Hydrophobic fatty acid → pass membrane , but rate limitingFatty acid transport protein (CD36) → help FA → pass membraneFA is hydrophobic did exit in water-environment → protein carrier → formed complex albumin

Cholesterol →Bile acids → bile salt → incorportatecholesterol

formed from cholesterol in the liverstored in the gall bladder in bile as bile salts (sodium and

potassium)utilized during digestion of fats and other lipid substances

(act as detergents)

Bile Salts• Breakdown products of cholesterol• Amphipathic molecules• Function to transport cholesterol in the digestive system

Bile salts are synthesized from cholesterol– Rate limiting Enzyme: 7-α-Hydroxylase– After that conjugated with glycin and taurin and secreted into the bile.

Mixed micelle formed by bile salts, triacylglycerols and pancreaticlipase

detergent character of bile salts is due to the hydrophobic-hydrophilic nature of the molecules

the presence of hydroxyl (or sulfate) and the terminal carboxyl group on the tail gives the molecule its hydrophilic face

the steroid ring with its puckered plane provides the hydrophobic face

Bile acids emulsify (solubilize) fats sopancreatic lipase enzymes can split fattyacids from triglycerides.

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ABC proteins mediate cellular export of phospholipids and cholesterol

Fig 18-11 Major transport proteins in the liver and intestine taking part in the enterohepatic circulation of biliary lipids

Binding protein

Na-linked symporter (NTCP)

IBAT: ileal bile acid transporter (IBAT)I-BABP: intenstinal bile acid-binding protein

95%

β-sitosterolemia :ABCG5 or ABCG8 mutation →cholesterol release X → cholesterol store

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Lipids can be exported or imported in large well defined lipoprotein complexes

Lipoprotein: packages from hundreds to thousands of lipid molecules into water-soluble, macromolecular carriers

Chylomicrons (乳糜微粒): contains highest proportion of lipoidVLDL: very low density lipoproteinLDL: low density lipoproteinHDL: high density lipoprotein

Only VLDL and Chyomicrons are fully formed by ER, via microsomal transfer protein. VLDL released from liver; Chyomicrons released from intestine

Lipid or cholesterol → formed lipoprotein → efficient transport

Lipid transport in the circulation

Lipids are insoluble in plasma. In order to be transported they are combined with specific proteins to form lipoproteins

Non polar lipids in core

(TAG and cholesterol esters)

Proteins (apoproteins)Cholesterol

HOHO

O

R

HOHO

O

R

HOHO

脂蛋白依照組成大約可分為四類：1. 乳糜微粒(chylomicron)：負責運送小腸細胞吸收的油脂，血中的濃度以飯後時最高，飯後的血漿呈混濁狀態就是乳糜微粒存在之故，其中的脂肪快速被組織利用與儲存，故濃度很快降低。組成份含三酸甘油酯82%，膽固醇9%，磷脂質7%與蛋白質2%。因為含脂肪量最多，所以密度最低。2. 極低密度脂蛋白(VLDL)：主要由肝臟合成，運送肝臟合成的脂肪供其他組織利用。組成份含三酸甘油酯52%，膽固醇22%，磷脂質18%與蛋白質8%。3. 低密度脂蛋白(LDL)：乳糜微粒與極低密度脂蛋白之代謝產物，經肝臟轉換而成，含膽固醇濃度最高，負責運送膽固醇供周邊組織利用，也是造成高血膽固醇的主要成分。組成份含三酸甘油酯9%，膽固醇47%，磷脂質23%與蛋白質21%。4. 高密度脂蛋白(HDL)：由肝臟與小腸所製造，在血液中可以回收血管壁堆積的膽固醇，死亡細胞釋出的膽固醇，並將膽固醇送回肝臟代謝，有助於保護心臟與血管。組成份含三酸甘油酯3%，膽固醇19%，磷脂質28%與蛋白質50%。

Lipoproteins differ in the ratio of protein to lipids, & in the particular apoproteins & lipids that they contain.

They are classified based on their density:

Chylomicron (largest; lowest in density due to high lipid/protein ratio; highest % weight triacylglycerols)

VLDL (very low density lipoprotein; 2nd highest in triacylglycerols as % of weight)

IDL (intermediate density lipoprotein)LDL (low density lipoprotein, highest in cholesteryl esters as % of weight)

HDL (high density lipoprotein; highest in density due to high protein/lipid ratio)

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Chylomicron &VLDL – TG; LDL &HDL – cholesteryl esters

Pre β-lipoprotein

β-lipoprotein α-lipoproteinLipoprotein Structure

LDL

What are lipoproteins?

• Lipoproteins are protein-lipid complexes.Hydrophobic

lipids (TG, CE,ester) in core;

Hydrophilic lipids (UC,PL) on surface 1. hydrolysis of TG and PL by lipases and esterification of cholesterol by an acyl transferase

2. Transfer of cholesteryl esters, TG, PL between lipoproteins by specific lipid-transfer proteins

3. Uptake by some particles of cholesterol and PL exported from cells4. Association and dissociation of some apolipoproteins from the surface

of the particles

Lipoproteins are made in the ER (with microsomal transfer protein activity), exported by the secretory pathway, and remodeled in the circulation

Chylomicron – intestinal epithelial cells; VLDL – liver cells

LDL, IDL, HDL – remodeled (modifications) for chlymicorn & VLDL

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Apolipoproteins

Apo AI (liver, small intestine)– Structural; activator of lecithin:cholesterol acyltransferase

(LCAT)

Apo AII (liver)– Structural; inhibitor of hepatic lipase; component of ligand for

HDL binding

Apo A-IV (small intestine)– Activator of LCAT; modulator of lipoprotein lipase (LPL)

Apo A-V (liver)– Direct functional role is unknown; regulates TG levels.

Apo B-100 (liver)– Structural; synthesis of VLDL; ligand for LDL-

receptorApo B-48 (small intestine)

– Structural; synthesis of chylomicrons; derived from apo B-100 mRNA following specific mRNA editing

Apo E (liver, macrophages, brain)– Ligand for apoE receptor; mobilization of cellular

cholesterol

Apo C-I (liver)– Activator of LCAT, inhibitor of hepatic TGRL

uptakeApo C-II (liver)

– Activator of LPL, inhibitor of hepatic TGRL uptakeApo C-III (liver)

– Inhibitor of LPL, inhibitor of hepatic TGRL uptake

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High density lipoprotiens HDL

HDL carries “used” cholesterol (as CE) back to the liver. Also donate some CE to circulating VLDL for redistribution to tissues.

HDL taken up by liver and degraded. The cholesterol is excreted as bile salts or repackaged in VLDL for distribution to tissues.

Cholesterol synthesis in the liver is regulated by the cholesterol arriving through HDL (and dietary cholesterol returned by chylomicrons remnants).

Cholesterol (CE) in HDL is referred to as “good cholesterol”

HDL may transfer some cholesterol esters to other lipoproteins. Some remain associated with HDL, which may be taken up by liver &

degraded. HDL thus transports cholesterol from tissues & other lipoproteins to

the liver, which can excrete excess cholesterol as bile acids.

Lipoproteins are made in the ER, exported by the secretorypathway, and remodeled in the circulation

Exogenous/chylomicron pathway (dietary fat)

Endogenous pathway (lipids synthesized by the liver)

HDL metabolism (apolipoprotein transfer, cholesteryl ester transfer, reverse cholesterol transport

Intermediate Density Lipoproteins (IDL)

Liver → VLDL →TG hydrolysis by lipoprotein lipase (extracellularenzyme) → loss some TG and lipoprotein →IDL→ LDL→ bind receptor →absorbed

(a) After having been secreted from the liver

apolipoprotein

Lipid uptake by Intestine →chyomicron→secreted → smaller chylomicron → taken up by hepatocytesreceptor-mediated endocytosis

(b) Dietary lipids are sbsorbed

殘餘物

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HDLs are generated extracellularlyLCAT: lecithin:cholesterol acyl transferase; convert cholesterol to esterified cholesterolCETP: cholesteryl ester-transfer protein

receptor

類固醇產生細胞

HDL is though to be formed after secretion of apolipoprotein A from cells

Contain apoA and very little lipid

, a acceptor for phospholipid and cholesterol from ABCA1

LCAT: in

plasma

lecithin:cholesterol acyl transferase

Cells use several protein-mediated mechanisms to import

lipoprotein lipids:

1. Local, partial extracellular hydrolysis of core TG followed

by transport protein-mediated uptake of the released fatty

acids: – lipoprotein lipase linked by glycosaminoglycan

(GAG) on endothelial cells, FATPs (Fatty acid transport

proteins; muscle cells, adipocytes)

2. Regulated expression of cell-surface lipoprotein receptors

that mediate the direct uptake of lipoprotein lipids:

receptor-mediated endocytosis & selective lipid uptake (

by SB-RI: scavenger receptor, class B, type I)

Analysis of familial hypercholesterolemia revealed the pathway for receptor mediated endocytosis of LDL particles

LDL + LDL-receptor → endocytosisFH: familial hypercholesterolemia, high LDL, LDL-receptor mutantFH: easy death

125I-LDL + apo-B-100 → FH cell → analysis

LDL + LDL-receptor → internalized → to lysosome → proteases →hydorlyze surface apolipoproteins and cholesteryl esterases hydrolyze their core cholesteryl ester → unesterified cholesterol for metab

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LDL receptor-mediated endocytosis

FS: HYPERCHOLESTEROLEMIA

Still bindingDid not metab

Pulse-chase experiment demonstrates precursor-product relations in cellular uptake of LDL

Binding → internalization → degradation

Cholesteryl esters in lipoproteins can be selectively taken up by the receptor SR-BI (scavenger receptor, class B type I)

SR-BI: 1. cluster on microvilli and in the cell surface lipid raft, not in coated pits

as does the LDL receptor 2. Mediate the transfer of lipid across the membrane, not endocytosis of

entire LDL particles

Can bind HDL, LDL, VLDL

Still unclear

Binds HDL, LDL, VLDL

18.4 Feedback regulation of cellular lipid metabolism

Cholesterol level rises:1. HMG-CoA reductase – suppressed2. LDL receptor – suppressed3. Acyl:cholesterol acyl transferase (ACAT) – increased

Cholesterol depepdent transcriptional regulation:• Sterol regulatory element (SRE)• SRE-binding proteins (SREBPs) in ER• SCAP (SREBP cleavage-activating protein), insig-1(2)

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SREBP: serol regulatory elements binding protein

1. DNA binding motif (N-terminal)2. Central membrane-anchoring domain3. C-terminal cytosolic regulatory SCAP

binding domain

SCAP:1. 8 transmembrane helices – sterol sensing

domain2. C-terminal cytosolic SREBP-binding

domain

• Sterol binding domain of SCAP binds tightly to insig-1(2), but only at high cellular cholesterol levels:- block the binding of SCAP to COPII vesicle coat proteins:-prevents incorporation of SCAP/SREBP complex into ER-to-Golgi transport vesicles

Target gene-HMG-CoA reductase-LDL receptor

Membrane bound proteases

C

N

ER-lumen

• In cholesterol loaded cells– Unprocessed SREBP resides in Endoplasmatic Reticulum (ER)– SREBP is tightly complexed with sterol sensor SREBP-cleavage

activating protein (SCAP)– SCAP is attached via its sterol-sensing domain (SSD) to Insulin-

induced gene (INSIG) retention protein– (INSIG-1 and -2)

N

C

SCAP SREBPINSIG

ER-lumen

N

C

• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in

conformation of SSD (sterol sensitive domaine)

C

N

SCAP SREBPINSIG

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ER-lumen

N

C

C

N

SCAP

• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in

conformation of SSD (sterol sensitive domaine)

SREBPINSIG

ER-lumen

N

C

C

N

SCAP SREBPINSIG

• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in

conformation of SSD (sterol sensitive domaine)– Which enables the dissociation of INSIG

Golgi-lumen

N

C

C

N

SCAP SREBP

• In cholesterol depleted cells– Conformation of SCAP is altered due alteration in

conformation of SSD (sterol sensitive domaine)– Which enables the dissociation of INSIG– SREBP is escorted to the Golgi apparatus

SREBP

Golgi-lumen

N

C

C

N

SCAP

• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially cleaved by

two proteases– Site-1 protease (S1P) and Site-2 protease (S2P),

respectively– S1P is cholesterol sensitive (inhibited by cholesterol and

SREBP is retained in Golgi if the content of cholesterol is high)

S1P

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SREBP

Golgi-lumen

C

C

N

SCAPS1P

N

S2P

• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially cleaved by

two proteases– Site-1 protease (S1P) and Site-2 protease (S2P),

respectively– S1P is cholesterol sensitive - low cholesterol content -->

cleavage by S1P, followed by S2P cleavage

SREBP

Golgi-lumen

C

C

N

SCAPS1P

S2P

N

• SREBP is cleaved in the Golgi apparatus– In Golgi apparatus SREBP is sequentially

cleaved by two membrane-bound proteases Site-1 protease (S1P) and Site-2 protease (S2P), respectively

– S1P is a serine protease– S2P is a zinc metalloproteinase

Target gene-HMG-CoA reductase-LDL receptor