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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:
caloriesexercise,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.)
2) High plasma cholesterol and
atherosclerosis
A.
B.
C.
D.
E.
F.
G.
H.
Clinical chemistry
Fat Absorption
Liver cell synthesis of LDL and HDL
Cholesterol Synthesis
Drug Therapy
Fibroblasts and other extrahepatic tissues for
membrane biosynthesis
Incr. heart attacks,strokes,atheroscelerosis
Genetic Disorders
5
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
6
Plasma cholesterol >6.2mM (change diet); 5.5-6.2mM (borderline); <5.5mM normal
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
7
A lipoprotein:
Horton Fig 17-5
8
9
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)
Chylomicron
VLDL
LDL BAD
HDL GOOD
Particle Size Electroph.
(nm)
>75
25-75
A2
19-26
B
7-19
A1
Origin
Intestine
Liver
VLDL
Liver, intestine
10
Dietary cholesterol  chylomicron & HDL formed in intestinal epithelial cell
 remnant  lymph vessel taken up by adipose cells & extrahepatic tissues
B48 M.W. = 300,000
(chylomicrons,
chylomicron remnants)
11
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%.
1. Intestine
2. Liver
(Synthesis)
chylomicrons
VLDL
tissues for oxidation
adipose tissue
for storage
12
Liver cell synthesis of LDL,VLDL and HDL
B-100
A,C,E
i.e. LDL, VLDL, HDL
13
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.
Liver mitochondrial fatty acid oxidation inhibited by some drugs causing
14
FATTY LIVER
10
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
proteases
LDL Cholesterol esters
amino acids
esterases
cholesterol + fatty acid
LINOLEATE
LDL receptor returns to plasma membrane
(10min. - turnover ever 24 hours)
15
Steps (cont’d)
d) Free cholesterol in the cell is used or stored
Cholesterol
+ Linoleate
Acyl-CoA: cholesterol
acyl transferase
Membrane biosynthesis
Cholesterol ester
i.e., store for cholesterol
Regulation:
When excess, the synthesis of new LDL
receptors is stopped, therefore LDL not
taken up by cells
16
The LDL receptor
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 membranespanning domain, 22
residues; and a cytosolic
domain, 50 residues.
Membrane
spanning
domain
17
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
18
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
19
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
20
Cholesterol Ester Synthesis
Endoplasmic reticulum
21
LYSOSOMES recycle proteins,lipids,nucleic acids
Autophagic vacuole
Aged proteins,
Nucleic acids,
lipids
FAD
Amino acids
Cholesterol
Nucleotides
Fatty acids
Acid proteases (cathepsins)
Cholesterol ester esterase
Nucleases
Acid phospholipases
Require acid pH
ROS
H+
NADH
Cyt b5
CoQ
ACID pH
Stores dietary CoQ
CoQ reduction maintains acid pH
22
Arch Biochem Biophys. 375, 347-54, (2000).
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.
23
Atherosclerosis mechanism: oxidised LDL taken up by macrophage
scavenger receptors
LEUKOCYTES, ENDOTHELIAL CELLS
NH 2
Liver
(H2O2/Fe or Cu)
NH 2
B100
B100
OOH
LDL
LIPID PEROXIDE
ANTIOXIDANT
e.g. PROBUCOL
(500mg/day)
HC
-SCISSION
C CHO
H
MODIFIED
NH SCHIFF
BASE
LDL
B100
M.W.
ELECTROPH. MOBILITY
FLUORESC.
NH 2
O
B100
+
O
H
H
MALONDIALDEHYDE
Don't bind to apo B LDL receptor
or peripheral cells
BUT recognised by scavenger
receptor of macrophage
macrophage
accumulates chol.
transformation
Forms atherosclerotic plaque
Foam Cell
24
25
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
26
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
27
E) Cholesterol synthesis
O
Step 1 Mitochondria
C
thiolase
Acetoacetyl CoA
+
Acetyl CoA
+
synthase
H2O
S
CoA
CH2
HO
C
CH3
CH2
COO
3-Hydroxy-3-methylglutaryl CoA
(HMG CoA)
NADPH
Lyase
+
NADP
NADH
NAD+
Acetyl CoA
+
*Acetoacetate
*acetone
(breath)
*-OH butyrate
Biochim. et Biophys. Acta. 1529 (2000) 89-102.
HMG CoA
reductase
H2C
OH
CH2
ratelimiting
step
CHOLESTEROL or
STATIN: feedback
inhibition by
cholesterol
HO
C
CH3
CH2
COO
Mevalonate
28
COO-
Step 2
Synthesis of isopentenyl
pyrophosphate from
mevalonate occurs in the
PEROXISOMES
ATP
CH2
HO
C
COO-
ADP
CH3
CH2
HO
C
CH3
phosphotransferase
CH2
CH2
O
CH2 OH
CH2 O
P
O-
O-
Mevalonate
5-phosphomevalonate
ATP
kinase
COO-
ADP
CH2
HO
C
CH3
CH2
O
CH2 O
P
O
O
O-
P
O-
O-
(pyrophosphate)
5-pyrophosphomevalonate
ATP
decarboxylase
ADP + Pi + CO2
CH2
C
Stryer Fig 27-12
J Biol Chem. 271, 1784-8 (1996)
CH3
CH2
O
CH2 O
P
O-
O
O
P
O-
O-
29
Isopentenyl
pyrophosphate
Step 3
CH3
H 3C
C
Synthesis of squalene
occurs in the peroxisomes
then the e.r.
O
C
C
H2
O
P
O
O-
P
O-
H
O-
isomerase
Dimethylallyl pyrophosphate
CH2
peroxisome
CH3
H 3C
C
O
C
H2
C
H2
isopentenyl
pyrophosphate
prenyltransferase (head-to-tail)
CH3
C
H
O
H 3C C C C O P O P OH2 H2
OO-
PPi
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.
O
O
C
C
C
H2
O
O
P
O
O-
H
O-
P
O-
Geranyl pyrophosphate
CH2
peroxisome
H 3C C C C O
H2 H2
PPi
CH3
H 3C
C
C
H2
C
H2
C
C
H2
C
H2
C
P
O-
O-
O-
O
C
C
H2
O
P
O
O
O-
H
Farnesyl pyrophosphate
O-
P
O-
Farnesyl pyrophosphate + NADPH
e.r.
CoQ, heme A
Dolichol-PP
Prenylated proteins
P O
CH3
C
H
isopentenyl
pyrophosphate
O
prenyltransferase
CH3
C
H
O
Squalene synthase
DIMERIZATION
NADP+ + 2PPi + H+
CH3
H 3C
C
CH3
C
H
Progress in Lipid Research 41,369-391(2002)
C
H2
H 2C
C
CH3
CH3
C
H
CH2
H 2C
2
Squalene
C
H
C
C
H2
CH2
2
C
H
C
30
CH3
H3C
CH3
Step 4
CH3
CH3
Squalene
C
CH3
er P450, O2, NADPH
CH3
CH3
CH3
H3C
Synthesis of cholesterol
occurs in the ER
CH3
CH3
Squalene epoxide
C
CH3
cyclase
O
CH3
CH3
CH3
H3C
CH3
CH3
CH3
CH3
Lanosterol
HO
H3C
diet
CH3
H3C
CH3
H3C
CH3
C
H3
CH3
CH3
sunlight
Dehydrocholesterol
CH
3
H2C
HO
H3C
H
HO
NADPH
P450 reductase
Vitamin D3
CH3
H3 C
CH3
CH3
CH3
CHOLESTEROL
HO
Unsat. FA acyl CoA
+cholesterol acyltransferase
31
Cholesterol ester
(ACAT)
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.
O
O
-
-
O
O
3-hydroxy-3-methylglutaryl CoA
HO
OH
HO
O
SCoA
Mevinolin
O
O
Mevinolin (Fungal), a competitive inhibitor of HMG CoA reductase, resembles 3-hydroxy-3methyl-glutaryl CoA, the substrate.
32
Pravastatin (from Penicillin) - hepatotoxic (rare), (intestinal metabolism inhibited by grapefruit juice)
Peroxisome
But Statins may also decrease plasma ubiquinone antioxidant
HMG-CoA
TYROSINE
4-OH-benzoate
ER, Golgi
Decaprenyl4-OH
benzoate
transf.
Decaprenyl-PP
Isopentenyl-PP
Endogenous synthesis
of ubiquinone and
Dimethylallyl-PP cholesterol. Formation
of mevalonate is the
rate limiting step in
synthesis.
Geranyl-PP
Farnesyl-PP
Polyprenyl-PP
trans-prenyltransf.
Squalene
synthase
Decaprenyl-4-OH-benzoate
Squalene
COENZYME Q
Free Rad. Biol. Med. 29, 285-94 (2000)
Lancet 356, 391-5 (2000)
Dolichol
N-glycosylates secretory
proteins = Export glycoproteins
 cholesterol
 LDL receptors (induced)
33
Cholesterol  LDL uptake  LDL
 risk of atherosclerosis
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 7a hydroxylase (CYP7A) activity which oxidises
cholesterol to bile acids. (feedback inhibitor is normally bile acids)
34
iii) Hypolipidemic ie antihyperlipidemic FIBRATE drugs
CLOFIBRATE: 2g/day
(also: Gemfibrozil)
Cl
1. ↑ lipoprotein lipase activity
2. ↑ fatty acid oxidation by inducing
CH3 O
O C
C O
C2H5
CH3
PEROXISOMES
 serum triglycerides
 serum triglyceride-rich lipoprotein
3.Antioxidant action prevent LDL oxidation
35
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 .
Long chain or 3 or branched fatty acids
Acyl
carnitine
MITOCHONDRIA
-oxidation
Synthase
Acetyl CoA
oxidase*
Fatty acyl CoA
O2
heat
H2
Shorter-chain fatty acid
NADH
+
Acetyl
CoA
O
2
catalase
H2O + O2
Cholesterol
CoQ10*
Bile acids
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)
36
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
37
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
38
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
39
Non prescription ways of preventing cholesterol absorption
Plant sterol/stanols
Cholesterol lowering
action of plant sterols
in the diet
H
H
H
HO
SITOSTEROL
(a sterol)
Plant sterol not absorbed by
gut (2g/day) so inhibits gut
absorption of cholesterol
from diet.
“functional margarine”)
H
e.g. Becel pro-activ.in Loblaws
H
H
HO
SITOSTANOL
(a stanol)
40
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)
41
CONCLUSIONS
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+
4. high HDL in premenopausal women protects
but not after menopause.
42
Dietary mechanisms to decrease cholesterol are additive
(e.g., use in patients resistant or intolerant to statins).
1.
2.
3.
4.
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.
Competitive inhibition of cholesterol absorption from the gut, e.g.,
plant sterols margarine, almonds, flaxseed.
Increase LDL receptor-mediated LDL cholesterol uptake and
degradation, e.g., soy proteins, soy milk.
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)
43
G) BILE ACID SYNTHESIS BY LIVER
Endoplasmic reticulum (except CYP27)
OH
cholesterol 7a-hydroxylase
CYP7A
Cholesterol
CYP27
mitoch.
COO-
er, NADPH
inhibited by bile acids HO
or fasting;
induced by cholesterol
or thyroxine
7
OH
H
er, CYP12
er
side chain
oxidation
CYP7B
er
side chain
oxidation
er
OH
COOblocked
bile duct
CHENODEOXYCHOLIC
ACID
3
HO
OH
H
er, UGT
UDPGA
er
thioesterase (ligase)
acetylCoA
CHOLIC ACID
(Bile acid, "detergent")
3
CHOLESTASIS
TOXICITY
Cholyl CoA
Hepatocyte death
Gluc O
LIVER
CIRRHOSIS
AND DEATH
Biochemistry. 31, 4737-49, (1992)
CYTOSOL
44
Ga) BILE ACID SYNTHESIS BY LIVER (cont)
Endoplasmic reticulum
CYTOSOL
Cholic Acid
Cholyl CoA
sulfotransferase
(SULT2A1)
+ PAPS
SULT2A1
N-acetyltransferase
+ taurine
N-acetyltransferase
+ glycine
O
taurine
OH
NH
Glycocholate
glycine
NH
3
O3SO
TAUROCHOLATE
sulfate
HO
OH
H
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.
45
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