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Spring semester 2011

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• R.K.Murray, D.K.Granner, V.W.RodwellHarper‘s Illustrated Chemistry 27th, 28th Ed.

• Harvey R.A., Ferrier D.R : Biochemistry 4th ed.

• M.Lieberman, A.Marks: Basic medical Biochemistry(Clinical approach) 3rd edition

• Silbernagl, Stefan; Despopoulos, Agamemnon: Color atlas ofphysiology, 6th ed. Published Stuttgart: Thieme 2009.

• Lectures: – materials on IS MUNI

Recommended literature

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Lipids.Digestion and absorption, bloodplasma lipids, lipoproteins

2011 Department of Biochemistry FM MU(E.T.)

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Triacylglycerols (TG)-90%Phospholipids (PL)Cholesterolesters (CE)Glycolipids (GL)Lipophilic vitamins (LV)

Primary products:free FA2-monoacylglycerolslysophospholipidscholesterollipophilic vitamins

pancreatic lipases+ colipaseBile acids

Lingual andgastric lipase

Absorption into mucosalcells (enterocytes) inform of micelles (particles< 20 nm)

Digestion of lipidsWestern diet contains 40 % oflipids or more.

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Cleavage of lipids by enzymes in the smallintestine

• Pancreatic lipase

Triacylglycerol 2-monoacylglycerol + 2 FA

< 1/4 TGtriacylglycerol glycerol + FA

CH2

CH

O C

O

CH2

OC

O

O

C

O

Orlistat - drug designed to treat obesity, it inhibits lipases

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Orlistat

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• Cholesterol esterase:

Cholesterol esters cholesterol + FA

• Phospholipase A2

phospholipid lysophospholipid + MK

CH2

CH

CH2

O

O

O C

C

O

O

P

O

O

O CH2 CH2 NH2

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Emulsification of lipids

• is condition for effective digestion of hydrophobic lipids

• increase of effective surface oil-water, facilitation of contactwith enzymes

• is accomplished by action of detergents and mechanicalmixing due to peristalsis

Emulsificators in the small intestine

• salts of bile acid

• phospholipids

• salts of fatty acids

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Colipase

• protein secreted from pancreas

• binds the lipase at ratio 1:1

• anchors lipase to bile acids on the surface ofemulsified lipid droplets

• facilitates the action of lipase

bile acids

triacylglycerols

colipaselipase

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Absorption of lipids by mucosal cells(enterocytes)

Mixed micellesBrush bordermicrovilli

Mucosal cells

diameter < 20 nmPasive difusion of fatty acid and monoacylglycerols

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• long fatty acids and monoacylglycerol are resorbed bydiffusion

•short chain fatty acids (up to 10 C) do not enter micelles

• they are resorbed directly

• bile acids which remain in the intestine, are extensivelyabsorbed in ileum

• transport of cholesterol is mediated by NPC1L1 (Nieman-Pick C1 like 1) (see the lecture in Biochemistry I)

Absorption of lipids by mucosal cells– cont.

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Hormons affecting digestion of lipids

Secretin• intestinal “S-cells“ produce secretin into the blood after thestimulation by H+ entering the lumen• secretin stimulates release of secrets containing HCO3

- fromgallbladder and pancreas

Cholecystokinin (CCK)• intestinal “I-cells“ produce cholecystokinin into the blood after thestimulation of small peptides and lipids• CCK stimulates secretion of amylase, lipase a proteases fromexocrine cells of pancreas• potentiates effect of secretin on excretion of HCO3

-

• stimulates the secretion of bile from gallbladder

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Steatorrhoea (lipid malabsorption)

Loss of lipids by feces

(normaly is resorbed ~ 98% lipids of food)

Unsufficient supply of bile (damage ofliver, obstruction of bile duct

Posible causes

Disturbed function of pancreas

Disturbed function of intestinal mucose

Lipidmalabsorption

Unsufficient intake of lipophilicvitamins

lipids

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Resynthesis of lipids within the mucosal cells:

1. Activation of FA

FA + CoA + ATP Acyl-CoA + AMP + PPi

2. Resynthesis of triacylglycerols

Acyl-CoA + Monoacylglycerol Diacylglycerol + CoA

Acyl-CoA + Diacylglycerol Triacylglycerol + CoA

3. Resynthesis of phospholipids from lysophospholipids

4. Resynthesis of cholesterolesters

Processes are located in ER

FA with short chain and free glycerol do not také part in these processes andare transported directly into the portal vein

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TGchylomicron

PL

AA apoprotein B-48

(apoprotein A-I)

Portal veinLymphaticlacteals

FA with shortchain

glycerol

CHE

Transport of lipids from enterocytes

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Lipid Plasma concentration(mmol/l)

Triacylglycerol

Total cholesterol

Non-esterified cholesterol

Total phospholipids

Free fatty acids

0,9 - 2

3,8 - 5,8

1,3 - 1,9

1,8 - 5,8

0,4 - 0,8

Plasma lipids

Transport in form of lipoproteins

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Schematic structure of lipoproteins

Non-esterifiedcholesterol

Phospholipids

Apoproteins

Cholesterolesters

Triacylglycerols

Apoproteins

core superficial layer

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VLDL

LDL

Chylomicron CM

HDL

Plasma lipoproteins TG

Prot

CH

PL84%54%

45%

50%

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Lipoproteins characteristics

Class Diameter

(nm)

Half-life Main lipid

CM 100-1000 ~5-15 min TG

VLDL 30-90 ~2h TG

IDL 25-35 ~2h TG/CHE

LDL 20-75 ~2-4 d CHE

HDL 5-12 ~10 h PL/CHE

Separation of lipoproteins

• ultracentrifugation in gradient of salt

• elektrophoresis

(see seminars and practicals)

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Apoproteins

• protein component of lipoproteins

• function of AP: enzyme activators and inhibitorsinteraction with receptors

structural role

(transport)

• some of them are built in the lipoproteins, the other areperipheral and exchange between particles

• synthesis in rough ER, attachment to lipid micelles

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• they are formed in intestinal mucosal cells

• they carry TG, CH and lipophilic vitamins admitted in food

• main apoprotein is apo-B 48, minor is apo-A (the other cannotbe synthesized in intestinal cells), synthesis of apoB-48 limitsformation of CM

• they are relased by exocytosis into the lacteals (lymphaticvessels originating in the villi of the small intestine) – chylelymph

• they follow lymphatic veins and enter the blood in the thoracicduct

Chylomicrons (CM) – asembly and metabolism

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TGchylomicron

PL

AA apoprotein B-48

(apoprotein A-I)

Portal veinLymphaticvessels

SCFA glycerol

CHE

Assembly of chylomicrons

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Metabolism of chylomicrons in blood

• they enter the blood 1-2 hours after the meal –nascent chylomicrons

• modification of chylomicrons: in blood apo E andapo C-II are transfered from circulating HDL tochylomicrons

• in capilaries of most peripheral tissues are CMdegraded by lipoproteine lipase (LPL)

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LPL

ApoCII

ApoE

Chylomicron in blood (1st part)

Fatty acidsMucosalcells of theintestine

lymph

blood

Adiposetissue,muscles

glycerol

Ductus thoracicus

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• negatively charged enzyme on the surface of endotelial cells incapillaries (anchored by heparansulfate to the capillary walls)

• predominantly in adipose tissue and skeletal and cardiac muscle

• it is activated by apo-CII

• LPL can be released by heparin

• synthesis of isoenzyme in adipose tissue is stimulated by insulin

• deficit of LPL results in triacylglycerolemia

Lipoprotein lipase (LPL)

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Action of LPL

• lipoprotein is attached to the enzyme bonded on endothelialcells

• LPL catalyzes hydrolysis of TG contained in circulatinglipoproteins:

TG glycerol + 3 MK

CH2

CH

O C

O

CH2

OC

O

O

C

O

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Action of LPL on chylomicronsCM

• LPL hydrolyses TG to fatty acids andglycerol

• more than 90% of TG in CM isdegraded by LPL, the particle decrases insize and increases density remnant

CM-remnat

Is rapidly removedfrom circulation bythe liver játry(Apo-E receptor)

• free fatty acids aretaken up by tissues,small part returns backinto the plasma and istransported by serumalbumin

LPL

Apo CII

LPL

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liver

Fatty acids

LPL

ApoCII

ApoE

ApoCII

Glycerol

remnant

Chylomicron in blood (2nd part)

Adiposetissue,muscles

apoE, apoB/E,

apoE

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• LPL degrades about 90% TG in chylomicrons

• chylomicrons in blood are removed during aprox. 30min

The fate of fatty acids released by the action of LPL:

- oxidation in tissues (muscle, myocard) – yield of energy

• deposition in form of TG in adipous tissue

Removal of remnats from blood

•They bind to the receptors in liver and are taken up

• receptors recognize apo-E

• cholesterol taken in food is transported into the liver inthis way

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VLDL – asembly and metabolism

• they are formed in hepatocytes

• they are composed of 60% of TG that are synthesized inthe liver

• contains cholesterol

• content of apoproteins:apo-B 100, small amount of Apo-A andApo-C

• they are secreted into the blood as nascent particles

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Apo-B 100

• apoprotein in LDL and VLDL

•It is integral protein

• very long chain (4 536 AA)

• apo-B 48 and apo-B 100 have the same mRNA, but apo-B 48 has only 48% of chain leghth of apo-B 100)

• synthesis of apo-B 100 is inhibited by insulin, VLDL areformed in post-resorption phase

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What is the origin of TG in liver?

• fatty acids are synthesized in liver from acetyl-CoA

• acetyl CoA originates mainly from metabolism ofsaccharides (after the meal)

• free fatty acids can be also taken up from blood (duringstarvation)

• TG are synthesized from fatty acids

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IDL

LDL

VLDL

HDL

Apo-C,E

Apo C FA

G

Metabolism of VLDL-overview

Liver

LPA

HDL

CHE

Tissues

apoB/E

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Degradation of VLDL by lipoprotein lipase

Apo CIIfree fatty acids enter tissues

Taken up by liver (apo-Ereceptor)

IDL

LDL

particle becomesmaller in size

LPL

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Metabolism of VLDL in steps

•Nascent VLDL enter the blood

• apo E and apo CII are transfered from HDL

• triacylglycerols are degraded by lipoprotein lipase to fattyacid and glycerol (similarly like chylomicrons)

•VLDL changes to IDL

• IDL are either taken up by liver or are converted to LDL

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Hepatic lipase

• enzym on luminal wall of liver sinusoids

• it acts similarly like LPL

• it degrades TG in IDL, VLDL and HDL when they passthrough the liver

• it can be released by heparin

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Heart muscle and adipose LPL

KM of heart muscle LPL is aprox. 10x lower thanadipose LPL

Synthesis of adipose LPL is activated by insulin

What follows from it ?

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• IDL and LDL may be enriched by cholesterol esters fromHDL (role of cholesterolester transfer protein CETP)

• IDL are taken up by liver Apo-B/E receptors

• LDL are taken up by periferal tissues (1/3) and liver (2/3) bythe process of receptor mediated endocytoses (Apo-B/E)

• at physiological conditions 30-40% of new formed LDL iscatabolized during 24 h

Metabolism of IDL and LDL

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LDL receptor (apo B/Ereceptor)

Is regulated by intracelularcontent of cholesterol

Non specific (scavenger)receptors (SR-A)

membrane receptor with broadspecifity

Located on the surface ofmacrophages and Kupfer-cellsin liver

Are not down regulated

Uptake of modified andredundant LDL

LDL receptors

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Uptake of LDL by specific receptors (apoB/apoE)

•LDL receptor is negativelly charged membraneglycoprotein lokalized on the surface of clathrin coatedpits,

• it recognizes apo E and apo B 100

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• after binding, the complex LDL-receptor is internalized byendocytoses

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•vesicles containing LDL loses clatrin coat and fuses withlysosomes forming endosomes

•LDL disociates from its receptor, the receptor migrates to oneside of endosome, separates a recycles back to the membranemembrány

recyclation of areceptoru

endolysosom

Lysosome

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•pH in endolysosome falls, lipoprotein is degraded, cholesterol,amino acids, fatty acids, phospholipids releases

• cholesterol is esterified by ACAT (acylCoA-cholesterol-acyltransferase) and is stored in the cell

AK

MK PL

CH

ACAT

CHE

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The level of cholesterol in the cell is strictlyregulated

• down- regulation of cholesterol intake in form of LDL(increased level of cholesterol in the cell decreases the numberof receptors on the surface – expression of LDL-receptor geneis decreased)

• regulation of intracellular synthesis of cholesterol

regulation of transcription of HMG-CoA synthase geneby SREBP

Inhibition of HMG-CoA – reductase by cholesterol

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Role of sterol regulatory element-binding protein(SREBP) in regulation of intake and synyhesis ofcholesterol

• precursor of SREBP is an integral protein of ER membrane

• when sterol level in the cell is low, N-terminal peptide isreleased from precursor molecule and migrates to the nucleus,where it binds to sterol-regulatory element in promotor area ofgenes regulating cholesterol

• SREBP regulates synthesis of LDL receptors and HMGCoA

reductase

See also: http://www.biocarta.com/pathfiles/m_s1pPathway.asp

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Familiar hypercholesterolemia

(type II hyperlipidemia)

Defeciency in production ofLDL-receptors

High level of LDL in blood

Synthesis of cholesterol in thecell is not inhibited cellproduces excess of cholesterol

Increased risk of myocardialinfarction

Characteristic tendonxanthomas

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Uptake of lipoproteins by scavengerreceptor SRA

• SRA –receptors on surface of phagycytosing cells(macrophages in the cell wall, in lung alveoles andperitoneum, Kupfer cells)

• receptor does not have down-regulation

• it preferably take up modified LDL (oxidized, glycated)

• it can take up also undamaged LDL, if the capacity of down-regulated receptor is exceeded

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Formation of foam cells and plaque

•Makrophages filled by lipids become foam cells

• they canoothcumulate in subendotellial area of the cell wall

• growth factors and cytokines stimulate the migration ofsmooth muscle cells and their proliferation

• these processes can result in formation of atheroscleroticplaque

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Effect of hormones on LDL uptake in liver

Insulin and trijodthyronin increase uptake of LDL by theliver,

Glucocortikoids have opposite effect

(mechanism is not known)

Why non-controlled diabetes and hypothyroidism are riskfactors for development of atherosklerosis and are very oftenconnected with hypercholesterolemia?

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HDL and their role in metabolism of lipids

reverse transport of cholesterol (RTC)

- HDL take up cholesterol from peripheral tissues and

transports it to the liver

HDL exist in several modifications

They differ in size, shape, by content of lipids andapoproteins

• they have different functions

• main subfraction according to the density: HDL2, HDL3

• remodelation of HDL – changes of HDL in circulation asa consequence of exchange of lipids

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HDL – assembly and metabolism

• base of HDL structure are apo A I and apo A II, theycontain also apoC and apo E

• for transport of cholesterol from tissues are importantsmall particles so-called „lipid free“ a „lipid poor“ apoA

• thet are secreted by liver and enterocytes, can be alsoformed in circulation from larger HDL particles

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SR-B1 receptor

HDL-receptor with dual function in metabolism of HDL

• It binds HDL in liver and steroidogenic cells throughapo A-I and mediates transport of cholesterol inside thecells

• it mediates the transport of cholesterol from the cellsinto the HDL in periferal tissues

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How is cholesterol deposited in the cells?

•Cholesterol esters are present in form of lipid dropletsin cytoplasma

•Revers transport of cholesterol is started by theirhydrolysis by cholesterylester hydrolase

•Free cholesterol is transported to the cell membrane

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How is cholesterol taken up from tissues ?

Two mechanism are expected

difussion formation of new HDL

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SR-B1játra

CETP

LDL/VLDL

steroidogennítkáně

Lipid poorparticles

spherical HDL

Formation of new HDL

Periferaltissue

ABCA1

LCAT

Disk-shaped HDL

Tangier disease – extremlyrare genetic disease –functional ABCA1transporter is missing.

It results in intracellularaccumulation of lipids

* Gene expression for pro ABCA1 is regulated by the amount of cholesterolinside the cell

ABCA1 is an ATP-binding cassette

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Mechanism of cholesterol difusion into HDL

Periferaltissue

SRB-I

•HDL attaches to cell surfaceafter the interaction with SR-B1 receptor

•ABCG4 (ATP-binding casetteprotein G4) transportscholesterol into the HDL

•Gradient of cholesterolconcentration is mediated byLCAT on the HDL surface

ABCG4

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For further conversions of HDL areimportant:

Lecitin cholesterol acyltransferase (LCAT)

Cholesterol ester transfer protein (CETP)

LCAT

• transfers a fatty acid from lecitine (phosphatidylcholine)na cholesterol

• plasmatic enzyme, it acts on the surface of HDL,activated by apo A-I

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HO

• LCAT transfers acyl of fatty acid toOH group of cholesterol

• non-esterified cholesterol isconverted to esterified it is lesspolar and more voluminous – it issequestered into the core of HDL

CH2O CO

CH

CH2

O

O P

O

O

O

CO

CH2

CH2

N CH3

CH3

CH3+

Function of LCAT

-

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CETP

Cholesterolester –transfer-protein

It transfers esterified cholesterol from HDL to otherlipoproteins

CE

CE

VLDL

HDL

CETP

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• esterified cholesterol formed by the action of LCATaccumulates in the core of HDL

• particles become spheric

• spheric HDL interacts with the other lipoproteins

• uptake in liver is mediated by SR-B I receptor

Further metabolism of HDL

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Cholesterol uptake by SR-B1 receptor in the liver

•HDL binds to receptor on hepatocytes

• the complex is not endocytosed

• only cholesterol is transported into the cells, transportis mediated by ABCG transporter

•HDL dissociates from receptor and re-enters againcirculation

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Lipid poorparticles

Conversions of HDL - summarization

LCAT

SR-B1játra

Disk-shapedHDL

spherical HDL(HDL2 a HDL3)

CETP

LDL/VLDL

Remodelation by interaction with the otherlipoproteins

steroidogeniccells

lipid poor particles

SR-B1

ABCA1

SRB-I

Peripheraltissue

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Cholesterol balance

Food Biosynthesis

80-500 mg 800 – 1000 mg

Steroidal hormons,sebum, intestinalepitelium

200 mg

Bile acids

500 mg

Cholesterol(bile)

800 mg

Cholesterol pool

1000-1500 mg/day is secreted

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“Good” and “bad”cholesterol

HO HO

Cholesterol in HDL is considered to bethe good cholesterol, because it acceptsfree cholesterol from peripheral tissue

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Increased intake of cholesterol or defects of LDL receptors

Increased plasmatic LDL,

The long half-life of LDL possibility of oxidation

Damaged and redundant LDL are taken up by SRA receptors ofmakrophages, formation of foam cells

oxidized LDL are strongly aterogenic

LDL cholesterol – bad cholesterol

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High level of HDL-cholesterol

• HDL act as cholesterol scavenger, picking up excesscholesterol in blood and taking it back to the liver fordisposal. The higher HDL cholesterol level, the betterprognosis of coronary heart diseases risk.

HDL-cholesterol = good cholesterol

When the higher level of cholesterol in blood is found,the distribution between LDL and HDL fractions isdetermined – examination of HDL- and LDL-cholesterol is performed – see practicals

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Lipoprotein (a)

• Lp(a) has very similar structure as LDL

• it contains additional apoprotein molecule (a) [apo(a)], that iscovalently attached to apo B-100

• apo(a) has homologous structure with plazminogen

• large quantities Lp(a) in plasma are associated with the increasedrisk of coronary heart disease

• hypothesis: Lp(a) slows down the breakdown of blood clots thattrigger heart attack because it competes with plasminogen forbinding to fibrin