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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.” - PowerPoint PPT Presentation
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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
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