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1 lipoproteins

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  • 1. Spring semester 2011 1
  • 2. Recommended literature• R.K.Murray, D.K.Granner, V.W.Rodwell Harper‘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 of physiology, 6th ed. Published Stuttgart: Thieme 2009.• Lectures: – materials on IS MUNI 2
  • 3. Lipids.Digestion and absorption, bloodplasma lipids, lipoproteins  2011 Department of Biochemistry FM MU (E.T.) 3
  • 4. Digestion of lipids Western diet contains 40 % of lipids or more. Triacylglycerols (TG)-90% Phospholipids (PL) Cholesterolesters (CE) Lingual and Glycolipids (GL) gastric lipase Lipophilic vitamins (LV) pancreatic lipases Bile acids + colipase Primary products:Absorption into mucosal free FA 2-monoacylglycerolscells (enterocytes) in lysophospholipidsform of micelles (particles cholesterol< 20 nm) lipophilic vitamins 4
  • 5. Cleavage of lipids by enzymes in the small intestine• Pancreatic lipase Triacylglycerol  2-monoacylglycerol + 2 FA O O CH2 O C C O CH CH2 O C O< 1/4 TGtriacylglycerol  glycerol + FA 5Orlistat - drug designed to treat obesity, it inhibits lipases
  • 6. Orlistat 6
  • 7. • Phospholipase A2phospholipid  lysophospholipid + MK O O CH2 O C C O CH O CH2 O P O CH2 CH2 NH2 O• Cholesterol esterase:Cholesterol esters  cholesterol + FA 7
  • 8. 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 8
  • 9. 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 colipase lipase 9
  • 10. Absorption of lipids by mucosal cells (enterocytes) Brush border Mucosal cellsMixed micelles microvilli diameter < 20 nm 10 Pasive difusion of fatty acid and monoacylglycerols
  • 11. Absorption of lipids by mucosal cells– cont. • long fatty acids and monoacylglycerol are resorbed by diffusion •short chain fatty acids (up to 10 C) do not enter micelles • they are resorbed directly • bile acids which remain in the intestine, are extensively absorbed in ileum • transport of cholesterol is mediated by NPC1L1 (Nieman- Pick C1 like 1) (see the lecture in Biochemistry I) 11
  • 12. Hormons affecting digestion of lipidsSecretin• intestinal “S-cells“ produce secretin into the blood after thestimulation by H+ entering the lumen• secretin stimulates release of secrets containing HCO3- fromgallbladder and pancreasCholecystokinin (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 12
  • 13. Steatorrhoea (lipid malabsorption) Loss of lipids by feces (normaly is resorbed ~ 98% lipids of food) Posible causes lipids Unsufficient supply of bile (damage of liver, obstruction of bile duct Disturbed function of pancreas Disturbed function of intestinal mucoseLipid Unsufficient intake of lipophilicmalabsorption vitamins 13
  • 14. Resynthesis of lipids within the mucosal cells:1. Activation of FAFA + CoA + ATP  Acyl-CoA + AMP + PPi2. Resynthesis of triacylglycerolsAcyl-CoA + Monoacylglycerol  Diacylglycerol + CoAAcyl-CoA + Diacylglycerol  Triacylglycerol + CoA3. Resynthesis of phospholipids from lysophospholipids4. Resynthesis of cholesterolesters Processes are located in ERFA with short chain and free glycerol do not také part in these processes and 14are transported directly into the portal vein
  • 15. Transport of lipids from enterocytes AA    apoprotein B-48 (apoprotein A-I) CHE PL TG chylomicron FA with short chain glycerol Lymphatic Portal vein lacteals 15
  • 16. Plasma lipids Transport in form of lipoproteinsLipid Plasma concentration (mmol/l)Triacylglycerol 0,9 - 2Total cholesterol 3,8 - 5,8Non-esterified cholesterol 1,3 - 1,9Total phospholipids 1,8 - 5,8Free fatty acids 0,4 - 0,8 16
  • 17. Schematic structure of lipoproteins Apoproteins Phospholipids TriacylglycerolsApoproteins Cholesterol esters Non-esterified cholesterol core superficial layer 17
  • 18. Plasma lipoproteins TG 84% PL 54% CH ProtChylomicron CM VLDL 45% 50% LDL HDL 18
  • 19. Lipoproteins characteristics Class Diameter Half-life Main lipid (nm) 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/CHESeparation of lipoproteins• ultracentrifugation in gradient of salt• elektrophoresis(see seminars and practicals) 19
  • 20. Apoproteins• protein component of lipoproteins• function of AP: enzyme activators and inhibitors interaction 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 20
  • 21. Chylomicrons (CM) – asembly and metabolism• 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 21
  • 22. Assembly of chylomicrons AA    apoprotein B-48 (apoprotein A-I) CHE PL TG chylomicron SCFA glycerol Lymphatic Portal vein vessels 22
  • 23. 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) 23
  • 24. Chylomicron in blood (1st part) blood ApoCII Ductus thoracicus ApoE Adiposelymph tissue, muscles LPL Fatty acidsMucosalcells of theintestine glycerol 24
  • 25. Lipoprotein lipase (LPL)• 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 25
  • 26. Action of LPL• lipoprotein is attached to the enzyme bonded on endothelialcells• LPL catalyzes hydrolysis of TG contained in circulatinglipoproteins: TG glycerol + 3 MK O O CH2 O C C O CH CH2 O C O 26
  • 27. Action of LPL on chylomicrons CM • free fatty acids are Apo CII taken up by tissues, small part returns back into the plasma and is LPL LPL transported by serum albumin• LPL hydrolyses TG to fatty acids and CM-remnatglycerol Is rapidly removed• more than 90% of TG in CM is from circulation bydegraded by LPL, the particle decrases in the liver játrysize and increases density  remnant (Apo-E receptor) 27
  • 28. Chylomicron in blood (2nd part) ApoCII ApoE Adipose tissue, muscles LPL ApoCII Fatty acids liver Glycerol apoE apoE, apoB/E, remnant 28
  • 29. • LPL degrades about 90% TG in chylomicrons • chylomicrons in blood are removed during aprox. 30 min 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 tissueRemoval 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 in 29this way
  • 30. 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 30
  • 31. 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 31
  • 32. 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 32
  • 33. Metabolism of VLDL-overview VLDL Liver apoB/E Apo-C,E HDL Tissues LPA Apo C FA G IDL HDL LDL CHE 33
  • 34. Degradation of VLDL by lipoprotein lipase Apo CII free fatty acids enter tissues LPL IDLparticle becomesmaller in size LDL Taken up by liver (apo-E receptor) 34
  • 35. 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 35
  • 36. 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 36
  • 37. Heart muscle and adipose LPLKM of heart muscle LPL is aprox. 10x lower thanadipose LPLSynthesis of adipose LPL is activated by insulinWhat follows from it ? 37
  • 38. Metabolism of IDL and LDL• 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 38
  • 39. LDL receptorsLDL receptor (apo B/E Non specific (scavenger)receptor) receptors (SR-A)Is regulated by intracelular membrane receptor with broadcontent of cholesterol specifity Located on the surface of macrophages and Kupfer-cells in liver Are not down regulated Uptake of modified and redundant LDL 39
  • 40. Uptake of LDL by specific receptors (apoB/apoE) •LDL receptor is negativelly charged membrane glycoprotein lokalized on the surface of clathrin coated pits, • it recognizes apo E and apo B 100 40
  • 41. • after binding, the complex LDL-receptor is internalized byendocytoses 41
  • 42. •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 a receptoru Lysosome 42 endolysosom
  • 43. •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 ACAT CH CHE AK 43 MK PL
  • 44. 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 gene by SREBP Inhibition of HMG-CoA – reductase by cholesterol 44
  • 45. 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 HMGCoAreductaseSee also: http://www.biocarta.com/pathfiles/m_s1pPathway.asp 45
  • 46. Familiar hypercholesterolemia(type II hyperlipidemia)Defeciency in production ofLDL-receptorsHigh level of LDL in bloodSynthesis of cholesterol in thecell is not inhibited cellproduces excess of cholesterolIncreased risk of myocardial Characteristic tendoninfarction xanthomas 46
  • 47. 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 47
  • 48. 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 48
  • 49. Effect of hormones on LDL uptake in liverInsulin and trijodthyronin increase uptake of LDL by theliver,Glucocortikoids have opposite effect(mechanism is not known) Why non-controlled diabetes and hypothyroidism are risk factors for development of atherosklerosis and are very often connected with hypercholesterolemia? 49
  • 50. HDL and their role in metabolism of lipidsreverse transport of cholesterol (RTC)- HDL take up cholesterol from peripheral tissues andtransports it to the liver HDL exist in several modifications They differ in size, shape, by content of lipids and apoproteins • they have different functions • main subfraction according to the density: HDL2, HDL3 • remodelation of HDL – changes of HDL in circulation as 50 a consequence of exchange of lipids
  • 51. 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 51
  • 52. SR-B1 receptorHDL-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 52
  • 53. 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 53
  • 54. How is cholesterol taken up from tissues ? Two mechanism are expected difussion formation of new HDL 54
  • 55. Formation of new HDLLipid poor Periferalparticles tissue játra SR-B1 ABCA1 is an ATP- ABCA1 binding cassette steroidogenní tkáně Tangier disease – extremly rare genetic disease – LCAT CETP functional ABCA1 transporter is missing. It results in intracellular spherical HDL accumulation of lipidsDisk-shaped HDL LDL/VLDL * Gene expression for pro ABCA1 is regulated by the amount of cholesterol 55 inside the cell
  • 56. Mechanism of cholesterol difusion into HDL •HDL attaches to cell surface after the interaction with SR- Periferal B1 receptor tissue •ABCG4 (ATP-binding casette protein G4) transports SRB-I cholesterol into the HDL ABCG4 •Gradient of cholesterol concentration is mediated by LCAT on the HDL surface 56
  • 57. For further conversions of HDL are important: 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 57
  • 58. Function of LCAT CH2 O CO CO O CH O CH3 + CH2 O P O CH2 CH2 N CH3 O- CH3• LCAT transfers acyl of fatty acid toOH group of cholesterol• non-esterified cholesterol is HOconverted to esterified it is lesspolar and more voluminous – it issequestered into the core of HDL 58
  • 59. CETPCholesterolester –transfer-proteinIt transfers esterified cholesterol from HDL to otherlipoproteins CE VLDL CETP CE HDL 59
  • 60. Further metabolism of HDL• 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 60
  • 61. 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, transport is mediated by ABCG transporter •HDL dissociates from receptor and re-enters again circulation 61
  • 62. Conversions of HDL - summarizationLipid poorparticles Peripheral játra SR-B1 tissue SR-B1 ABCA1 steroidogenic cells SRB-I LCAT CETP Disk-shaped spherical HDL LDL/VLDL (HDL2 a HDL3) HDL Remodelation by interaction with the other 62 lipid poor particles lipoproteins
  • 63. Cholesterol balance Food Biosynthesis 80-500 mg 800 – 1000 mg Cholesterol pool Steroidal hormons,Cholesterol Bile acids sebum, intestinal(bile) epitelium800 mg 500 mg 200 mg 1000-1500 mg/day is secreted 63
  • 64. “Good” and “bad” cholesterolHO HO Cholesterol in HDL is considered to be the good cholesterol, because it accepts free cholesterol from peripheral tissue 64
  • 65. Increased intake of cholesterol or defects of LDL receptorsIncreased plasmatic LDL,The long half-life of LDL  possibility of oxidationDamaged and redundant LDL are taken up by SRA receptors ofmakrophages, formation of foam cells oxidized LDL are strongly aterogenic LDL cholesterol – bad cholesterol 65
  • 66. High level of HDL-cholesterol • HDL act as cholesterol scavenger, picking up excess cholesterol in blood and taking it back to the liver for disposal. The higher HDL cholesterol level, the better prognosis of coronary heart diseases risk. HDL-cholesterol = good cholesterolWhen 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 66
  • 67. 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 67