1. 1) OBESITY & high plasma triglycerides Adipose cells, adipocytokines. White fat cells store large lipid droplets of triglycerides and cholesterol ester. Leptin (167aa) synthesised and secreted, peptide hormone, binds to receptors in hypothalamic nuclei “satiety center.” Regulates energy.Signals a decrease in appetite. Prooxidant. Adiponectin (244aa), most abundant protein in adipocyte, (similar structure to TNFα) & released into the blood. Higher in females. Antioxidant.  adipocyte oxidative stress (insulin activates NADPH oxidase (Nox4)   body weight,  mitoch.fatty acid oxidation,  gluconeogenesis,  insulin resistance. Insulin causes lipogenesis and fatty acid release ( fatty liver). Plasma adiponectin decr. & leptin incr. in obesity (promotes breast cancer). Brown fat (babies) mitochondria make heat. 1

2. Adipocyte dysfunction & Metabolic disease Obesity due to overnutrition (high fat or sugar diet ) & inactivity causes metabolic disease. Insulin resistance & diabetes mellitus Hypertension Hyperlipidemia, nonalcoholic steatohepatitis (NASH), alcoholic liver disease, chronic hepatitis, liver cancer Therapy:  calories  exercise,taurine,salicylate,thiazolidinediones, Research : how to increase adiponectin levels J.Gastroenterol(2008)43,811-822,Clinical Chemistry (2008)54,945-55 2

3. Fatness increases cancer risk Fatness cancer rate may exceed cancer from smoking soon. Breast cancer, esophagus, colorectal, pancreas, ovary. gall bladder,endometrium, liver (after cirrhosis) NASH. NOT prostate,bladder, mouth, lung, skin, cervix, nasopharynx,skin cancer. Associated with energy-dense foods,fast food,sugary drinks,sedentary living,TV/computers. 2007 WCRF/AICR report 3

4. Figure 1 Following chronic alcohol ingestion, endotoxin is released from certain intestinal bacteria. Endotoxin moves from the gut into the bloodstream and the liver where it activates Kupffer cells- a type of immune cell (resident liver macrophages) - by interacting with CD14 causing nuclear factor kappa B (NFκB) production.This generates superoxide radicals (O2) and various signaling molecules (the cytokine TNF–α) which injures hepatocytes. (Alcohol Res Health. 2003; 27(4):300-6.)

5. 5 2) High plasma cholesterol and atherosclerosis A.Clinical chemistry B.Fat Absorption C.Liver cell synthesis of LDL and HDL D.Cholesterol Synthesis E.Drug Therapy F.Fibroblasts and other extrahepatic tissues for membrane biosynthesis G.Incr. heart attacks,strokes,atheroscelerosis H. Genetic Disorders

6. 6 Lipoproteins, Cholesterol and Atherosclerosis A) Clinical chemistry - Lipoproteins Conjugated proteins in which the prosthetic group are lipids: Lipoproteins responsible for the transport/distribution of lipids: - Lipid hormones - Lipids absorbed by the intestine - Fat-soluble vitamins

7. 7 Percent contribution of saturated fat and cholesterol from fats/oils, meats, dairy products and eggs in the US diet. Biochim. et Biophys. Acta 1529 (2000) 310-320 Plasma cholesterol >6.2mM (change diet); 5.5-6.2mM (borderline); <5.5mM normal

8. 8 A lipoprotein: Horton Fig 17-5

9. 9

10. 10 B) Stage 1 - Fat Absorption Chylomicrons Found in lymph draining the intestine not hepatic portal systems Largest ones are microscopically visible (diameter 500 nm) (floats upon centrifugation) Responsible for the lipemic (milky turbidity) of the blood following food digestion and disappears at 5 hours Contains 1% protein - formed by intestinal cell Triglycerides (apo AI and II, B)

11. 11 B48 M.W. = 300,000 (chylomicrons, chylomicron remnants) Dietary cholesterol  chylomicron & HDL formed in intestinal epithelial cell  remnant  lymph vessel  taken up by adipose cells & extrahepatic tissues

12. 12 C) Stage 2 : LDL activity and function LDL(apoB100) synthesised by liver moves cholesterol to the tissues (taken up by the apoB100 receptor of tissues). LDL carries 75% plasma cholesterol and HDL carries 25%.

13. 13 Liver cell synthesis of LDL,VLDL and HDL B-100 A,C,E i.e. LDL, VLDL, HDL

14. 14 Electron micrograph of a part of a liver cell actively engaged in the synthesis and secretion of very low density lipoprotein (VLDL). The arrow points to a vesicle that is releasing its content of VLDL particles. 10 Liver mitochondrial fatty acid oxidation inhibited by some drugs causing FATTY LIVER

15. 15 F) Stage 3- Fibroblasts and other extrahepatic tissues - cholesterol taken up for membrane biosynthesis Extrahepatic tissues obtain cholesterol from plasma LDL & NOT by synthesis STEPS: a) ApoB100 protein of LDL binds to receptor in coated pits b) Receptor-LDL complex is internalised by endocytosis c) Vesicles containing LDL fuse with lysosomes (proteases, esterases) LDL Protein amino acids LDL Cholesterol esters cholesterol + fatty acid LINOLEATE LDL receptor returns to plasma membrane (10min. - turnover ever 24 hours) proteases esterases

16. 16 Steps (cont’d) d) Free cholesterol in the cell is used or stored Cholesterol Membrane biosynthesis + Linoleate Cholesterol ester i.e., store for cholesterol Acyl-CoA: cholesterol acyl transferase Regulation: When excess, the synthesis of new LDL receptors is stopped, therefore LDL not taken up by cells

17. 17 The LDL receptor Membrane spanning domain The LDL receptor consists of five domains with different functions: an LDL-binding domain, 292 residues; a domain bearing N-linked sugars, 350 residues; a domain bearing O-linked sugars, 58 residues; a membrane- spanning domain, 22 residues; and a cytosolic domain, 50 residues.

18. 18 Four Mutations affecting LDL receptors 1) no receptor is synthesised 2) receptors are synthesised but lack signals for transport  don’t reach plasma membrane 3) receptors reach cell surface but don’t bind LDL normally 4) receptors don’t cluster in coated pits

19. 19 Genetic disorders (cont.) e.g. Familial hypercholesterolemia (Type II) - Autosomal dominant trait 1:500  Cholesterol 680 mg/100 mL instead of 175 mg/100 mL  Die of heart disease before 20 years (homozygous)  Die of heart disease before 40 years (heterozygous– inherit one defective and one normal gene) 1.LDL receptor is unable to bind to coated pits  randomly distributed in membrane  LDL binds but can’t be absorbed by endocytosis 2. Faulty LDL receptor formed which can’t bind LDL

20. 20 Extrahepatic tissue(e.g.fibroblasts) take up cholesterol via LDL receptors and store it as cholesterol esters in lysosomes Voet et al., Fig 19-37

21. 21 Cholesterol Ester Synthesis Endoplasmic reticulum

22. 22 Acid proteases (cathepsins) Cholesterol ester esterase Nucleases Acid phospholipases Require acid pH Amino acids Cholesterol Nucleotides Fatty acids Stores dietary CoQ CoQ reduction maintains acid pH ACID pH H+H+ CoQ ROS Cyt b5 FAD NADH Autophagic vacuole Aged proteins, Nucleic acids, lipids LYSOSOMES recycle proteins,lipids,nucleic acids Arch Biochem Biophys. 375, 347-54, (2000).

23. 23 G) Atherosclerosis – cholesterol plaque formation An atherosclerotic plaque (marked by the arrow) blocks most of the lumen of this blood cell. The plaque is rich in cholesterol.

24. 24 Atherosclerosis mechanism: oxidised LDL taken up by macrophage scavenger receptors Liver

25. 25

26. 26 D) Stage 4 GOOD HDL : Reverse cholesterol transport and function 1. HDL is synthesised and secreted from the liver and the intestine. HDL contains 65% protein + free fatty acids, cholesterol, triacylglyceride and phospholipids. 2 Function: HDL picks up cholesterol released into the plasma from dying cells and from membranes undergoing turnover and returns it to the liver 3. HDL contains cholesterol, cholesterol ester, phospholipid and Lecithin:Cholesterol Acyl Transferase (LCAT) - synthesised in the liver that catalyses : LECITHIN + CHOLESTEROL  LYSOLECITHIN + CHOLESTEROL ESTER LCAT is activated by apo-A1 and deficiency in LCAT means that HDL can’t take up cholesterol from tissue, therefore  cholesterol and lecithin in tissue

27. 27 Raising HDL to decrease tissue cholesterol Anacetrapib (Merck drug) NEW Niacin best Fibrate drugs bile acid binding resins Exercise,  -3 fatty acids,red wine,orange juice,beans, soy,oat bran Decreases HDL trans fatty acids, high carbohydrates

28. 28 E) Cholesterol synthesis Step 1 Mitochondria Biochim. et Biophys. Acta. 1529 (2000) 89-102.

29. 29 Synthesis of isopentenyl pyrophosphate from mevalonate occurs in the PEROXISOMES Stryer Fig 27-12 Step 2 J Biol Chem. 271, 1784-8 (1996)

30. 30 Synthesis of squalene occurs in the peroxisomes then the e.r. Step 3 peroxisome Progress in Lipid Research 41,369-391(2002) Squalene synthaseDIMERIZATION e.r. peroxisome CoQ, heme A Dolichol-PP Prenylated proteins

31. 31 Synthesis of cholesterol occurs in the ER Step 4 Dehydrocholesterol Squalene er P450, O 2, NADPH cyclase Squalene epoxide Lanosterol CHOLESTEROL NADPH P450 reductase sunlight diet Unsat. FA acyl CoA Cholesterol ester Vitamin D 3 +cholesterol acyltransferase (ACAT)

32. 32 F) Drug therapy to decrease plasma cholesterol i) The STATINS inhibit cholesterol biosynthesis to decrease plasma LDL cholesterol and cut the risk of heart attacks and strokes by at least 33% even in people with normal cholesterol. Several million Canadians are taking statins. HMG CoA reductase inhibitors but can induce rhabdomyolysis (test for muscle/kidney damage) Lipitor (Atorvastatin;Pfizer); Zocor (Simvastatin prodrug; Merck) Crestor (Rosuvastatin; AstraZeneca) lowers risk of heart attack, death and stroke Merck: Lovastatin:40-80 mg/day, Squibb: Pravastatin: 20mg/day for female patients - best for persons with sleep disorders; need a hepatocyte enzyme to open up lactone ring. Mevinolin (Fungal), a competitive inhibitor of HMG CoA reductase, resembles 3-hydroxy-3- methyl-glutaryl CoA, the substrate. Pravastatin (from Penicillin) - hepatotoxic (rare), (intestinal metabolism inhibited by grapefruit juice) 3-hydroxy-3-methyl- glutaryl CoA Mevinolin

33. 33  cholesterol  LDL receptors (induced)  LDL uptake  LDL  risk of atherosclerosis Isopentenyl-PP Dimethylallyl-PP Geranyl-PP Farnesyl-PP Squalene synthase Squalene Cholesterol Decaprenyl-PP TYROSINE 4-OH-benzoate Decaprenyl- 4-OH benzoate transf. Decaprenyl-4-OH-benzoate COENZYME Q trans-prenyltransf. But Statins may also decrease plasma ubiquinone antioxidant Free Rad. Biol. Med. 29, 285-94 (2000) Lancet 356, 391-5 (2000) HMG-CoA Polyprenyl-PP Dolichol N-glycosylates secretory proteins = Export glycoproteins ER, Golgi Peroxisome

34. 34 ii) RESINS THAT BIND BILE ACID TO LOWER CHOLESTEROL Cholesterol is reabsorbed from intestine by forming complexes with bile acids. Liver then replaces bile acids by oxidising cholesterol (catalysed by CYP7A ). a) Prescription therapeutic resins bind bile acids and prevent cholesterol reabsorption: problem of constipation, ↓absorption of fat sol. vitamin A,D,E,K e.g. colestipol 20g/day taken mixed with juice or apple sauce cholestyramine colesevelam b) Nonprescription bulk forming laxatives (soluble fibres) Psyllium husks (metamucil) Ispaghula husks Oat bran (  -glucan binds bile acids ) Action of bile acid binders  cholesterol excretion  hepatic cholesterol 7  hydroxylase (CYP7A) activity which oxidises cholesterol to bile acids. (feedback inhibitor is normally bile acids)

35. 35 iii) Hypolipidemic ie antihyperlipidemic FIBRATE drugs CLOFIBRATE: 2g/day (also: Gemfibrozil) 1. ↑ lipoprotein lipase activity 2. ↑ fatty acid oxidation by inducing PEROXISOMES  serum triglycerides  serum triglyceride-rich lipoprotein 3.Antioxidant action prevent LDL oxidation

36. 36 PEROXISOME (numerous genetic diseases) Peroxisomal fatty acid  -oxidation forms H 2 O 2 which is removed by catalase that is also located in the peroxisomes. Medium-chain fatty acids (C8-18) prefer mitochondrial  -oxidation that doesn’t form H 2 O 2. Acyl carnitine Fatty acyl CoA Synthase Acetyl CoA O2O2 H2O2H2O2 oxidase* heat Shorter-chain fatty acid MITOCHONDRIA  -oxidation NADH + Acetyl CoA Cholesterol CoQ10* Bile acids H 2 O + O 2 catalase H 2 O 2 also formed by peroxisomal glycolate/glyoxylate oxidases, xanthine oxidase, uricase * Peroxisomes induced by peroxisome proliferators via a cytosolic receptor (PPAR) e.g., hypolipidemic drugs, e.g., clofibrate; plasticizers, e.g., phthalate (DEHP); endogenous steroids formed by the adrenal glands e.g., dehydroepiandrosterone. Ann Rev Biochem. 61, 157-97 (1992) Ann Rev Nutr. 14, 343-70 (1994) Long chain or  3 or branched fatty acids

37. 37 iv. NIACIN(Vitamin B 3 ) Deficiency (maize,indian millet) causes pellagra ( rough photosensitive skin, dementia,GI ). Flour now fortified with niacin ; B 1 thiamine; B 2 riboflavin tryptophan   niacin  nicotinamide  NAD  NADP  NADPH Niacin 1.5-3g/day ↓ plasma LDLcholesterol & triglycerides; best for ↑ HDL (not nicotinamide) but early hot flashes so use slow delivery pill, rare hepatotox or hyperglycemia

38. 38 NIACIN INHIBITS 1) DGAT (Synthesizes triglycerides, 2) Fatty Acyl CoA Synthase AND 3) Blocks HDL uptake, 4) Prevents LDL Oxidation Daniel Meyers Current Opinion in Lipidology 2004, 15: 659-665

39. 39 v.Blocking intestinal cholesterol permease Ezetimibe, a new drug that blocks cholesterol uptake by inhibiting intestinal sterol permease (packaged with a statin). Plant sterols eg sitosterol, a natural method

40. 40 Non prescription ways of preventing cholesterol absorption Plant sterol/stanols HO H HH SITOSTEROL (a sterol) HO H HH SITOSTANOL (a stanol) Cholesterol lowering action of plant sterols in the diet Plant sterol not absorbed by gut (2g/day) so inhibits gut absorption of cholesterol from diet. “functional margarine”) e.g. Becel pro-activ.in Loblaws

41. 41 vi Natural ways of binding dietary cholesterol to prevent absorption Chitosan (shellfish exoskeleton) (LIBRACOL is polychitosamine: amine groups bind cholesterol) Policosanol (sugar cane wax or rice wax alcohol ie. Octacosanol) CH 3 (CH 2 ) 26 CH 2 OH)

42. 42 DIETARY WAYS OF DECREASING THE ATHEROSCLEROSIS RISK: 1.  cholesterol and saturated fatty acids  plant stanols (2g/margarine day) 2.  polyunsaturated fatty acids which  cholesterol oxidation to bile acids  LDL catabolism  cholesterol excretion into intestine 3. smoking, obesity, lack of exercise, low Ca 2+ 4.high HDL in premenopausal women protects but not after menopause. CONCLUSIONS

43. 43 Dietary mechanisms to decrease cholesterol are additive (e.g., use in patients resistant or intolerant to statins). 1. Decrease intestinal bile acids by binding them to viscous fibres, e.g., oats (  -glucan), barley, psyllium (metamucil), egg plant,ochra. Glucan is also a soluble fibre & an antioxidant which prev. oxidn of PUFA & cholesterol. Amer.J.Clin.Nutrition 75(2002)834-9. 2. Competitive inhibition of cholesterol absorption from the gut, e.g., plant sterols margarine, almonds, flaxseed. 3. Increase LDL receptor-mediated LDL cholesterol uptake and degradation, e.g., soy proteins, soy milk. 4. Decrease oxidized LDL using antioxidants, e.g., almonds (Vit E), soy proteins (isoflavones). Dr David Jenkins, Risk factor modification center, St. Michael’s Hospital, Toronto Metabolism 51(12):1596-1604 (2002)

44. 44 Biochemistry. 31, 4737-49, (1992) G) BILE ACID SYNTHESIS BY LIVER Endoplasmic reticulum (except CYP27) Hepatocyte death

45. 45 Then efflux into bile and stored in gall bladder. Then released by bile duct into upper-small intestine (ileum). Then metabolised (deconjugation (CO 2 ), dehydroxylation) by anaerobic bacteria of colon to deoxycholate, lithocholate, urodeoxycholate. Then actively reabsorbed and recirculates via liver 8 times / day. Ga) BILE ACID SYNTHESIS BY LIVER (cont)

46. Gb) Drugs that can induce Cholestasis Cholestasis accounts for most liver transplants Bile acids are synthesised by the liver to help absorb fat or fat soluble vitamins (Vit E, K and D). Drugs that slow/block bile flow from the liver to the gallbladder & gut causes liver failure. e.g. chlorpromazine,prochlorperazine, penicillin (ampicillin),estradiol, nitrofurantoin, sulindac Symptoms dark urine, pale stool, jaundice,fever/rash persistent itching. Stop taking the drug. 46

47. Gc)Bile acids are endogenous toxins that can cause liver failure by radicals ! Bile acids are cytotoxic to hepatocytes. Reduces FeIII which reduces H 2 O 2 to form hydroxyl radicals and reactive oxygen species. Radicals oxidise nucleic acids,protein and unsaturated lipids to form other radicals. Antioxidants & blockers of mitochondrial permeability transition prevent apoptosis Hepatolog.33,616-26(2001),Chem.Res.Toxl22,1984-91(2009),J.Cellular Biochem.110,1219-25(2010) 47

48. THE END Don’t memorize slides 29,30,31,38,41,45 48