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

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:

caloriesexercise,taurine,salicylate,thiazolidinediones,• Research : how to increase adiponectin levels • J.Gastroenterol(2008)43,811-822,Clinical Chemistry (2008)54,945-55

2

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

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

2) High plasma cholesterol and atherosclerosis

A. Clinical chemistryB. Fat AbsorptionC. Liver cell synthesis of LDL and HDLD. Cholesterol SynthesisE. Drug TherapyF. Fibroblasts and other extrahepatic tissues for

membrane biosynthesisG. Incr. heart attacks,strokes,atheroscelerosisH. Genetic Disorders

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

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

A lipoprotein:Horton Fig 17-5

9

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)

Particle Size(nm)

Electroph. Origin

Chylomicron >75 - IntestineVLDL 25-75 A2 LiverLDL BAD 19-26 B VLDLHDL GOOD 7-19 A1 Liver, intestine

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

C) Stage 2 : LDL activity and functionLDL(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%.

1. Intestine

2. Liver(Synthesis)

chylomicrons

VLDL

tissues for oxidation

adipose tissuefor storage

13

Liver cell synthesis of LDL,VLDL and HDL

B-100A,C,E

i.e. LDL, VLDL, HDL

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

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 pitsb) Receptor-LDL complex is internalised by endocytosisc) 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

Steps (cont’d)d) Free cholesterol in the cell is used or stored

Cholesterol Membrane biosynthesis

+ Linoleate Cholesterol esteri.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

The LDL receptor

Membrane spanningdomain

The LDL receptor consistsof five domains withdifferent functions: anLDL-binding domain, 292residues; a domain bearingN-linked sugars, 350 residues; a domain bearingO-linked sugars, 58 residues; a membrane-spanning domain, 22residues; and a cytosolicdomain, 50 residues.

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

Genetic disorders (cont.)

e.g. Familial hypercholesterolemia (Type II) - Autosomal dominant trait 1:500Cholesterol 680 mg/100 mL instead of 175 mg/100 mLDie 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 pitsrandomly distributed

in membrane LDL binds but can’t be absorbed by endocytosis 2. Faulty LDL receptor formed which can’t bind LDL

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

Cholesterol Ester Synthesis

Endoplasmic reticulum

22

Acid proteases (cathepsins)Cholesterol ester esteraseNucleasesAcid phospholipasesRequire acid pH

Amino acidsCholesterolNucleotidesFatty acids

Stores dietary CoQCoQ reduction maintains acid pH

ACID pHACID pHACID pHACID pH

H+CoQ

ROS Cyt b5

FAD NADH

Autophagic vacuoleAged proteins,Nucleic acids,

lipids

LYSOSOMES recycle proteins,lipids,nucleic acids

Arch Biochem Biophys. 375, 347-54, (2000).

23

G) Atherosclerosis – cholesterol plaque formation

An atherosclerotic plaque(marked by the arrow)blocks most of the lumen ofthis blood cell. The plaqueis rich in cholesterol.

24

B100

NH2

LDL

B100NH2

OOH

LIPID PEROXIDE

(H2O2/Fe or Cu)

LEUKOCYTES, ENDOTHELIAL CELLS

ANTIOXIDANTe.g. PROBUCOL (500mg/day) -SCISSION

B100

NH2

+ H

O

H

O

MALONDIALDEHYDE

B100

NH

HC CH

CHO

SCHIFF BASE

MODIFIED LDL

Don't bind to apo B LDL receptoror peripheral cellsBUT recognised by scavenger receptor of macrophage

macrophageaccumulates chol.

transformation

Forms atherosclerotic plaque Foam Cell

Atherosclerosis mechanism: oxidised LDL taken up by macrophage scavenger receptors

M.W.ELECTROPH. MOBILITYFLUORESC.

Liver

25

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 cellsand 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

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

E) Cholesterol synthesis

Step 1 Mitochondria

Biochim. et Biophys. Acta. 1529 (2000) 89-102.

Acetoacetyl CoA + Acetyl CoA + H2O

3-Hydroxy-3-methylglutaryl CoA (HMG CoA)

Mevalonate

synthase

NADPH

NADP+

rate-limitingstep

CHOLESTEROL or STATIN: feedbackinhibition bycholesterol

thiolase

Acetyl CoA+

*Acetoacetate

*-OH butyrate

NADH

*acetone (breath)

HMG CoA reductase

CH2 OH

CH2

C CH3OH

CH2

COO

Lyase

NAD+

COO

CH2

C CH3OH

CH2

C S CoA

O

29

Synthesis of isopentenyl pyrophosphate frommevalonate occurs in the PEROXISOMES

Stryer Fig 27-12

Step 2

J Biol Chem. 271, 1784-8 (1996)

COO-

CH2

CHO CH3

CH2

CH2OH

COO-

CH2

CHO CH3

CH2

CH2O P O-

O-

O

COO-

CH2

CHO CH3

CH2

CH2O P O

O-

O

P

O-

O-

O

CH2

C CH3

CH2

CH2O P O

O-

O

P

O-

O-

O

ATP ADP

ATP

ADP + Pi + CO2

Mevalonate 5-phosphomevalonate

5-pyrophosphomevalonate

Isopentenylpyrophosphate

ATP

ADP

phosphotransferase

kinase

(pyrophosphate)

decarboxylase

30

Synthesis of squalene occurs in the peroxisomesthen the e.r.Synthesis of squalene from dimethylallyl pyrophosphate, an isomer of isopentenyl pyrophosphate. The joining of two C15 units to form squalene is a tail-to-tail condensation, in contrast with the preceding condensations, which are head-to-tail.

Step 3

peroxisome

Progress in Lipid Research 41,369-391(2002)

Squalene synthase DIMERIZATIONe.r.

peroxisome

H3C C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-

CH2

C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-CH2

CH

C

CH3

H3C

H3C C CH2

CH2

CH2

O P

O-

O

O

P

O

O-

O-

PPi

H3C C CH2

CH2

CH2

O P

O-

O

O

P

O

O-

O-

PPi

CH2

C

CH3

C

H

CH2

O P

O-

O

O

P

O

O-

O-CH2

CH

C

CH3

CH2

CH2

CH

C

CH3

H3C

Dimethylallyl pyrophosphate

Geranyl pyrophosphate

Farnesyl pyrophosphate

H3C C CH

CH3

CH2

H2C C

CH3

CH

CH2 CH2C

CH3

CH

H2C CH2

CH

C

CH3

CH3

2 2

Squalene

Farnesyl pyrophosphate + NADPH

NADP+ + 2PPi + H+

isopentenylpyrophosphate

isopentenylpyrophosphate

isomerase

prenyltransferase (head-to-tail)

prenyltransferase

CoQ, heme ADolichol-PPPrenylated proteins

31

Synthesis of cholesterol occurs in the ER

CH3

CH3H3C

CH3

CH3

CH3

CH3

C

CH3

CH3H3C

CH3

CH3

CH3

OCH3

C

CH3

CH3H3C

CH3

CH3

H3C CH3

HO

CH3

Step 4

Dehydrocholesterol

Squalene

er P450, O2, NADPH

cyclase

Squalene epoxide

Lanosterol

CHOLESTEROL

CH3

CH3H3C

HO

CH3

CH3

NADPHP450 reductase

sunlight

diet

Unsat. FA acyl CoAOC

O

R

Cholesterol ester

H2C

HO

CH3

H3C

H

CH3

H3C

Vitamin D3

+cholesterol acyltransferase(ACAT)

CH3

CH3H3C

HO

CH3

CH3

32

F) Drug therapy to decrease plasma cholesteroli) 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

OO-

OH

O

SCoA

OO-

OH

OH

O

O

33

cholesterol LDL receptors (induced) LDL uptake LDL risk of atherosclerosis

Endogenous synthesisof ubiquinone and cholesterol. Formationof mevalonate is therate limiting step insynthesis.

Isopentenyl-PP

Dimethylallyl-PP

Geranyl-PP

Farnesyl-PP

Squalene synthase

Squalene

Cholesterol

Decaprenyl-PP

TYROSINE

4-OH-benzoate

Decaprenyl-4-OHbenzoatetransf.

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, GolgiPero

xis

om

e

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,Ke.g. colestipol 20g/day taken mixed with juice or apple sauce cholestyramine colesevelamb) Nonprescription bulk forming laxatives (soluble fibres)Psyllium husks (metamucil)Ispaghula husksOat 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

iii) Hypolipidemic ie antihyperlipidemic FIBRATE drugs

CLOFIBRATE: 2g/day(also: Gemfibrozil)

1. ↑ lipoprotein lipase activity2. ↑ fatty acid oxidation by inducing PEROXISOMES serum triglycerides serum triglyceride-rich lipoprotein3.Antioxidant action prevent LDL oxidation

Cl O C

CH3

CH3

C

O

O C2H5

36

PEROXISOME (numerous genetic diseases)Peroxisomal fatty acid -oxidation forms H2O2 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 H2O2 .

Acylcarnitine

Fatty acyl CoA

SynthaseAcetyl CoA

O2 H2O2

oxidase*

heat

Shorter-chain fatty acid

MITOCHONDRIA-oxidation

NADH+

Acetyl CoA

CholesterolCoQ10*

Bile acidsH2O + O2

catalase

H2O2 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

iv. NIACIN(Vitamin B3)

• Deficiency (maize,indian millet) causes pellagra (rough photosensitive skin, dementia ,GI).

Flour now fortified with niacin ; B 1 thiamine; B2 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

NIACIN INHIBITS 1) DGAT (Synthesizes triglycerides, 2) Fatty Acyl CoA SynthaseAND 3) Blocks HDL uptake, 4) Prevents LDL Oxidation

Daniel Meyers Current Opinion in Lipidology 2004, 15: 659-665

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

Non prescription ways of preventing cholesterol absorption Plant sterol/stanols

HO

H

H H

SITOSTEROL(a sterol)

HO

H

H H

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

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) CH3(CH2)26CH2OH)

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 Ca2+

• high HDL in premenopausal women protects but not after menopause.

CONCLUSIONS

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)

44Biochemistry. 31, 4737-49, (1992)

G) BILE ACID SYNTHESIS BY LIVEREndoplasmic reticulum (except CYP27)

COO-OH

HOHOH

COO-OH

HOHOH

Cholesterol

cholesterol 7 hydroxylaseCYP7A

er, CYP12

erside chainoxidation

erthioesterase (ligase)acetylCoA

CHOLIC ACID(Bile acid, "detergent")

LIVER CIRRHOSIS AND DEATH

CYP27mitoch.

CYP7Ber

side chain oxidation

CHENODEOXYCHOLIC ACID

er, NADPHinhibited by bile acids or fasting; induced by cholesterol or thyroxine

er

Cholyl CoA

3

er, UGT

UDPGA

OGluc3

7

CHOLESTASISTOXICITY

CYTOSOL

blocked bile duct

Hepatocyte death

45

Then efflux into bile and stored in gall bladder.Then released by bile duct into upper-small intestine (ileum).Then metabolised (deconjugation (CO2), 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)

OHH

OH

OH NH

O

taurine

Cholyl CoA

N-acetyltransferase+ taurine

CYTOSOL

Glycocholate

SULT2A1

O3SO

sulfotransferase(SULT2A1)+ PAPS

3 TAUROCHOLATE

N-acetyltransferase+ glycine

sulfate

Cholic Acid

NHglycine

Endoplasmic reticulum

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

Gc)Bile acids are endogenous toxins that can cause liver failure by radicals !

• Bile acids are cytotoxic to hepatocytes .

• Reduces FeIII which reduces H2O2 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

THE END

• Don’t memorize slides 29,30,31,38,41,45

48