The Usual Suspects: Cholesterol and Triglyceride

Lipid Structure
Cholesterol: Membranes
Bile Acids
Steroid Hormones
Protein modification
H
O
Fatty Acids: Fuel, Triglycerides: FA for Fuel, Prostanoids
Prostanoids Protein modification
COOH COO

COOH COO

COOH COO

H
O Phospholipid: Lecithin Membranes
+
Glycerol
H
O
2ndCOO
Messengers
COO
H
O
+ N OPOO
 Structure of a
Typical Lipoprotein
Free cholesterol
(surface and core)

Phospholipid Triglyceride
(amphipath at (core only)
surface only)

Apolipoprotein Cholesteryl ester

(amphipath at (core only)
surface only)
Lipoprotein classes and sub-Classes

0.95
 VLDL Chylomicron

VLDL
Remnants
Density (g/ml)

1.006
IDL

Chylomicron
1.02 Remnants
LDL

1.06
HDL2 Directly atherogenic
1.10 Lp(a) (found in plaque)
HDL3
pre-β2 HDL
1.20
pre-β1 HDL
1000
5 10 20 40 60 80

Particle Size (nm)

 Substrates for Triacylglycerol Synthesis
Plasma

NEFA
Glucose Triglycerides
Acyl-CoA Synthetase
Glc-6-Pase
Acyl-CoA CoA Multiple steps

Glucose-6-P
CPT I Acyl-CoA
PEPCK

Hepatocyte
Pyruvate kinase Mitochondria
Acyl-Carnitine
Fatty Acid Synthase
CPT II
PEP Malonyl-CoA
Acyl-CoA
Acyl-CoA Carboxylase
Krebs Citrate Acetyl-CoA
Pyruvate Cycle
ATP Citrate Lyase
Acetyl-CoA CO2
Pyruvate Citrate
HMG-CoA Synthase
Ketone Bodies
PEP = phosphoenolpyruvate
Beta-oxidation
PEPCK = PEP carboxylase CPT = Carnitine palmitoyl transferase
 Structures of Fatty Acids
O
C 16:0 (palmitic)
HO
O
C cis-18:1 -6 (oleic)
HO

O trans-18:1 -6 (elaidic)

C
HO
O
C 18:2 -6 (linoleic)
HO
O (alpha
C 18:3 -3
HO linolenic)
O 20:5 -3 (EPA)
C
HO
 Exogenous (dietary) lipid metabolism
Muscle and adipose tissue Fatty acids

Lipoprotein lipase Lipoprotein Lipase

Apo C-II enhances
and apo C-III inhibits
LPL activity

Bloodstream Chylomicron
Remnant
Plasma
chylomicron
Apo B and apo E are
ligands for LDL
receptor
LDL (apo B,E)
receptor clears
Chylomicron
Remnants
I
N LDL
T receptor
E
S
T
I
Lymphatic
N chylomicron Hepatocyte
E Liver
Xenical blocks diatary
fat digestion
 Endogenous (hepatic) lipid metabolism
Muscle and adipose tissue Fatty acids

Lipoprotein lipase Lipoprotein Lipase

And hepatic lipase Apo C-II enhances
and apo C-III inhibits
LDL LPL activity
Bloodstream

IDL
Apo B and apo E are
ligands for LDL
receptor
LDL (apo B,E)
receptor clears VLDL,
IDL & LDL

LDL
receptor

VLDL
Hepatocyte
Liver
 Clinical Hypertriglyceridaemia

Condition Features
Secondary Relatively common (obesity, diabetes,
renal impairment, liver disease, drugs)

Polygenic Accounts for the majority of cases

Familial HTG TG predominates. CVD risk varies
Predisposes to massive HTG
Familial Overproduction of apo B lipoproteins
Combined H/L TG and TC vary with age and weight
Massive HTG Lipoprotein Lipase deficiency or
saturation. Risk of pancreatitis
Therapy for Hypertriglyceridaemia

Intervention Features

Diet, Exercise Relatively responsive

Alcohol restriction Often sufficient in heavy intake
Manage 2o causes Diabetes, renal
Fibrates Effective in high TG, low HDL
Statins Mild TG and HDL benefit
Fish oils (eg 6gm/d) Benefits TG rather than HDL
Niacin Effective, but increases glu, urate.
Future DGAT 2 Inhibitors?
Bile acid resins Contraindicated. Increase TG
NORMAL CHOLESTEROL ABSORPTION
1,300 mg/day

400 mg/day

Oil phase
NORMAL CHOLESTEROL ABSORPTION
1,300 mg/day

400 mg/day

Oil phase

Plant sterols compete

with cholesterol here
NORMAL CHOLESTEROL ABSORPTION
1,300 mg/day

400 mg/day

17,400 mg/day
Oil phase

850 mg/day

Ezetimibe competes
with cholesterol here
NORMAL CHOLESTEROL ABSORPTION
1,300 mg/day

400 mg/day

Oil phase
17,400 mg/day

850 mg/day
 Intracellular cholesterol sensing by SREBPs
(Sterol Regulatory Element Binding Proteins)
Membrane fluidity
reflects intracellular
SCAP or SREBP activating protein
Nucleus
cholesterol. Low levels
SCAP SREBP allow cleavage to active
WD Reg bHLH SRE form which binds
ER
nuclear receptor to
Cytosol bHLH
control gene expression.
Lumen
• SREBP-2 controls
cholesterol synthesis
Sterols
and sterol metabolism
• SREBP-1c is the
Golgi
Apparatus
WD Reg bHLH bHLH major isoform in liver
ZN++ and is a key regulator
of fatty acid &
S1P S2P triglyceride synthesis
Serine protease Metalloproteinase
Other nuclear receptors:
FXR, LXR.
 LDL Receptor activity reflects
intracellular cholesterol homeostasis

Cholesterol delivery via LDL-R

alters intracellular membrane
cholesterol and SREBP, which
a) Reduces synthesis via
HMGCoA Reductase
b) Reduces LDL-R synthesis
c) Increases storage as ester
d) Reduces counter-regulatory
*[SREBP] = sterol regulatory element-binding protein.
PCSK9 1. Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29:431-438..
 The Role of HDL in
Reverse Cholesterol Transport
Bile ABCG1
&SR-B1
Spheroidal HDL Pre-β HDL UC
Liver
LCAT
ABCA1
UC
Hepatic lipase, PL&UC
SR-BI endothelial
lipase

CETP SR-A
LDL
Receptor t ion Macrophage
x ida
O

VLDL/LDL
ABCA1, ATP-binding cassette protein A1; CETP, cholesterol ester transfer protein; FC, free cholesterol; LCAT,
lecithin:cholesterol acyltransferase; SR-A, scavenger receptor class A; SR-BI, scavenger receptor class B type I.

Adapted with permission from Cuchel C et al. Arterioscler Thromb Vasc Biol. 2003;23:1710–1712
 Clinical Hypercholesterolaemia

Condition Features
Secondary Relatively uncommon, but potent
(hypothyroidism, nephrotic syndrome,
primary biliary cirrhosis)
Polygenic Accounts for the majority of cases

Familial Prevalent, Accelerates CVD.

Hyperchol’aemia Due to defects in genes related to LDL-R
Familial Overproduction of apo B lipoproteins
Combined H/L TG and TC vary with age and weight
Increased HDL ?OK if LDL not raised?
Therapy for Hypercholesterolaemia

Intervention Features

Diet Plant sterols, avoid sat & trans FA

Manage 2o causes Rarer, but potent: (Thyroid, liver,
renal.)
Statins First line for LDL reduction
Ezetimibe 2nd line. Neutral for TG & HDL
Niacin Improves LDL, TG, HDL & Lp(a)
Bile acid resins Colesevalam better tolerated

Future PCSK9 Inhibitors, MTP inhibitors?

Apo B antisense oligonucleotides
 Triglyceride and Cholesterol:
Why are they linked?

• Most lipoproteins have TG and/or CE in their core

• Hepatic Triglyceride rich lipoproteins are precursors of
cholesterol-rich LDL
• Cholesterol-ester transfer protein allows all
triglyceride-rich lipoproteins to modify the composition
of cholesterol-rich HDL and LDL. As a result,
hypertriglyceridaemia is associated with reduced HDL
cholersterol as well as “small dense LDL”.
• The major gene regulators for lipid metabolism affect
both TG and Chol
•
 Key Regulators of Genes in
Fatty Acid and Triglyceride Metabolism
Bile Acids SHP = Short Heterodimer Partner

SHP

LXR SREBP-1c FXR

Acetyl CoA Carboxylase Fatty

Acid Synthase
Spot 14
Acyl CoA
NEFA Unsaturated Saturated
PPAR

L-FABP HNF-4α

RXR
Fatty acid metabolism
Transport Apolipoproteins
Oxidation Pyruvate kinase
Fatty Acid Binding Protein Glucose-6-phosphalase
Ketogenesis Transferin
Bile Acids
Adapted from Pegorier JP et al. J Nutr 2004;134:2444S-9S
Key Regulators of Genes in Lipid Metabolism

Sterol Regulatory Element Binding Protein (SREBP2)

Peroxisome proliferated
Synthesis activator receptors PPARs Delivery
Acquisition
Cellular
Cholesterol
Homeostasis
Excretion

Hepatobiliary Intestine
Farnesoid X Receptor (FXR) Liver X Receptor (LXR)
Link to mixed HL cases
 Case MC
Patient is a 43 year-old male with a strong family history of
premature CVD who presents for initial evaluation.

He has a 10 year history of dyslipidaemia and hypertension, for

which he has received beta blockers in the past. More recently
he has been on an ARB/diuretic combination. Three months
prior to this visit a fasting lipid profile showed:

Total Cholesterol: 5.7 mmol/L Triglyceride: 2.8 mmol/L

HDL-C: 0.7 mmol/L LDL-C: 3.6 mmol/L

He has managed to lose 3 kg and today results include:

Total Cholesterol: 6.9 mmol/L Triglyceride: 1.8 mmol/L

HDL-C: 0.8 mmol/L LDL-C: 5.2 mmol/L
 Questions concerning Mr M.C.

• 1) Is ethnicity an independent risk factor for CVD? Yes / No?

• 2) In the absence of any symptoms or signs of hypothyroidism,

would you perform thyroid function tests? Yes / No?

• 3) His brother’s lipids include LDL = 5.4 mmol/l,

TG = 1.9 mmol/l, HDL = 0.9 mmol/l . What is the most likely
cause of MC’s lipid abnormality?

• A) Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome

• B) Polygenc dyslipidamia
• C) Familial Combined Hyperlipidaemia
• D) Familial Hypercholesterolaemia
• E) Lipids aren’t really an issue in this patient
 Patient is a 43 year-old male with a strong family history of
premature CVD who presents for initial evaluation.
He has a 10 year history of dyslipidaemia. Hypertension, for
which he has received beta blockers in the past. More recently
he has been on an ARB/diuretic combination. Three months
prior to this visit a fasting lipid profile showed:

TC: 5.7 Triglyceride: 2.8 mmol/L HDL-C: 0.7 mmol/L LDL-C: 3.6 mmol/L
He has managed to lose 3 kg and today results include:
TC: 6.9 Triglyceride:1.8 mmol/L HDL-C: 0.8 mmol/L LDL-C: 5.2 mmol/L

• 1) Is ethnicity an independent risk factor for CVD? Yes / No?

• 2) In the absence of any symptoms or signs of hypothyroidism,
would you perform thyroid function tests? Yes / No?
• 3) His brother’s lipids include LDL = 5.4 mmol/l,
TG = 1.9 mmol/l, HDL = 0.9 mmol/l . What is the most likely
cause of MC’s lipid abnormality?
• A) Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome
• B) Polygenc dyslipidamia
• C) Familial Combined Hyperlipidaemia
• D) Familial Hypercholesterolaemia
• E) Lipids aren’t really an issue in this patient
 Is ethnicity an independent risk factor for
CVD? Yes / No?

• YES:

• NO:
 Is ethnicity an independent risk factor for CVD?
Case for a qualified “Yes”.
Same risk factors, different pattern
 INTERHEART, Karthikeyan et al 2009,

INTERHEART, Joshi et al 2007

2) In the absence of any symptoms or signs
of hypothyroidism, would you perform thyroid
function tests? Yes / No?

• Yes:

• No:
2) In the absence of any symptoms or signs
of hypothyroidism, would you perform thyroid
function tests? The case for “Yes”
 What is the most likely cause of MC’s lipid
abnormality?
• A) Dyslipidaemia secondary
to Insulin resistance and
the Metabolic Syndrome

• B) Polygenc dyslipidamia

• C) Familial Combined
Hyperlipidaemia
• D) Familial
Hypercholesterolaemia
• E) Lipids aren’t really an
issue in this patient
 What is the most likely cause of MC’s lipid
abnormality? The case for “C”, maybe “A” or “B”
Condition Features
Secondary Relatively uncommon, but potent
(hypothyroidism, nephrotic syndrome,
primary biliary cirrhosis)
Polygenic Accounts for the majority of cases

Familial Prevalent, Accelerates CVD.

Hyperchol’aemia Due to defects in genes related to LDL-R
Familial Overproduction of apo B lipoproteins
Combined H/L TG and TC vary with age and weight
Increased HDL ?OK if LDL not raised?
 Case MC (continued)
Mr MC started statin, therapy, Atorvastatin 20 mg/ day,
but unfortunately he had and inferior AMI still 4 months
later. His discuharge medication include:
Atorvastatin 20mg, Metoprolol 20 mg, Aspirin 100mg,
and his previous ARB/diuretic. Follow-up 2 months later
reveals: 2 kg weight loss, BP 118 / 78, Fasting tests:
• Glu 5.3 mmol/l, TC 4.4 mmol/l TG 4.2 mmol/l,
• HDL 0.7 mmol/l, LDL 1.8 mmol/l
 Case MC: Further questions:
• Should you stop his beta blocker? Yes / No?

• Do you trust the LDL-C result? Yes / No?

• Is it practical to try to manage Mr M.C’s lipid profile to
target levels? Yes / No?
• What is the next lipid-lowering drug that you would add to
his therapy?
a) Ezetimibe
b) Niacin
c) I would increase Atorvastatin to 80 mg but I wouldn’t
give anything other than a statin
d) Fenofibrate
e) Fish Oil
Mr MC started statin, therapy, Atorvastatin 20 mg/ day, but
unfortunately he had and inferior AMI still 4 months later. His
discuharge medication include:
Atorvastatin 20mg, Metoprolol 20 mg, Aspirin 100mg, and his
previous ARB/diuretic. Follow-up 2 months later reveals: 2
kg weight loss, BP 118 / 78, Fasting tests:
• Glu 5.3 mmol/l, TC 4.4 mmol/l TG 4.2 mmol/l,
• HDL 0.7 mmol/l, LDL 1.8 mmol/l
• Should you stop his beta blocker? Yes / No?
• Do you trust the LDL-C result? Yes / No?
• Is it practical to try to manage Mr M.C’s lipid profile to target
levels? Yes / No?
• What is the next lipid-lowering drug that you would add to his
therapy?
a) Ezetimibe b) Niacin c) Increase Atorvastatin to 80 mg but
don’t give anything other than a statin
d) Fenofibrate e) Fish Oil
 Should you stop his beta blocker?

• Yes

• No
 Should you stop his beta blocker?
The case for “no”

Some β-blockers decrease HDL and increase triglycerides.25 In

spite of this, BHAT data showed that propranolol improves
survival after MI.26 Low-dose metoprolol CR/XL alone or in
combination with a statin resulted in significant slowing of the
progression of carotid artery’s intima-media thickness over a 3-
year period.27

M Gheorghiade et al Circulation.2002; 106: 394-398
 Do you trust the LDL-C result?

• Yes

• No
 Do you trust the LDL-C result?
The case for “No”

Discussion of the effect of Cholesterol ester transfer protein will

Explain why LDL-C underestimates risk when TG is elevated
Is it practical to try to manage Mr M.C’s lipid
profile to target levels?

• Yes

• No
Is it practical to try to manage Mr M.C’s lipid
profile to target levels? The case for “Yes”

Combination therapy is safe and effective, but yet to

be supported by clinical endpoint data.
What is the next lipid-lowering drug that you
would add to his therapy?

a) Ezetimibe
b) Niacin
c) I would increase Atorvastatin to 80 mg but I wouldn’t
give anything other than a statin
d) Fenofibrate
e) Fish Oil
What is the next lipid-lowering drug that you
would add to his therapy?
The case for “d” or “b”, possibly “c” or “e”
anticipate “residual risk” module
 Case GS
Patient is a 47 year-old female who has been gaining weight
for several years.

She has a 10 year history of mildly elevated triglyceride. She

received therapeutic lifestyle counseling but she has been
largely non-compliant. Three months prior to this visit, a
fasting lipid profile showed: Total Cholesterol: 5.5mmol/L
Triglyceride: 2.4mmol/L HDL-C: 1.0mmol/L
LDL-C:3.6 mmol/L

Now she has symptoms of hyperglycaemia and repeat fasting

glucose confirms Type 2 diabetes
 Case GS
Review of Symptoms: Thirst, polyuria, folliculitis,
Weight unchanged (increased 2kg, then lost when
polyuria commenced
BP 118/72 Pulse 72 Wt 85kg Ht 175cm Waist 93
cm BMI 27.8: Physical examination unremarkable

Current fasting lipid results surprise you:

Total Cholesterol: 8.5mmol/L
Triglyceride: 7.4mmol/L
HDL-C: 1.0mmol/L
LDL-C: unable to be calculated
 Questions:
How could you obtain an LDL-C result?
a) Friedewald equation
b) Abusive phonecall to lab
c) “Direct method” involving detergents
d) Ultracentifugation
e) Subtract HDL-C from Total cholesterol
Which class or classes of
lipoproteins would you expect to be increased?
f) Chylomicrons and LDL
g) VLDL and LDL
h) VLDL and HDL
i) IDL and chylmicron “remnants”
j) Why bother? It doesn’t matter
Which combination of extra tests would
be most useful?
a) LDL size+HDL subfractions b) Lipid EPG+ApoE phenotype
c) LDL subfractions HDL size d) Lp(a)+ homocysteine
e) Routine fasting lipids are the only lipid tests that are ever
required
She has a 10 year history of mildly elevated triglyceride. She
received therapeutic lifestyle counseling but she has been
largely non-compliant. Three months prior to this visit, a fasting
lipid profile showed: Total Cholesterol: 5.5mmol/L Triglyceride:
2.4mmol/L HDL-C: 1.0mmol/L LDL-C:3.6 mmol/L Now she
has symptoms of hyperglycaemia and repeat fasting glucose
confirms Type 2 diabetes. Symptoms: Thirst, polyuria, folliculitis,
Weight increased 2kg, then lost when polyuria commenced
BP 118/72 Pulse 72 Wt 85kg Ht 175cm Waist 93 cm
BMI 27.8: Physical examination unremarkable
Current fasting lipid results surprise you: TC: 8.5mmol/L
TG: 7.4mmol/L HDL-C: 1.0mmol/L
LDL-C: unable to be calculated
• How could you obtain an LDL-C result?
• Which class or classes of lipoproteins would you expect
to be increased?
• Which combination of extra tests would be most useful?
How could you obtain an LDL-C result?
a) Friedewald equation
b) Abusive phonecall to lab
c) “Direct method” involving detergents
d) Ultracentifugation
e) Subtract HDL-C from Total cholesterol
 How could you obtain an LDL-C result?
The case for “d”, but “c” is misleading

Lab Tests Online:

“Direct LDL-C is ordered whenever calculation of LDL cholesterol will
not be accurate because the person's triglyceridesare significantly
elevated. It may be ordered by a doctor when prior test results have
indicated high triglycerides. In some laboratories, this direct LDL test
will automatically be performed when the triglyceride levels are too high
to calculate LDL-C. This saves the doctor time by not needing to order
another test, saves the patient time by not needing to have a second
blood sample drawn, and speeds up the time to provide the test result.”

Ultracentrifuge gives absolute result. Detergent methods assume LDL

Which class or classes of lipoproteins would
you expect to be increased?
a) Chylomicrons and LDL
b) VLDL and LDL
c) VLDL and HDL
d) IDL and chylmicron “remnants”
e) Why bother? It doesn’t matter
Which class or classes of lipoproteins would
you expect to be increased?
The case for “b”(orange) or “d” (green)
 Which combination of extra
tests would be most useful?

• a) LDL size+HDL subfractions

• b) Lipid EPG+ApoE genotype/phenotype

• c) LDL subfractions HDL size

• d) Lp(a)+ homocysteine

• e) Routine fasting lipids are the only lipid tests that are
ever required
 Which combination of extra
tests would be most useful?
The case for “b”

CM β preβ α

ApoE isoforms
 Case GS (continued)
The patient subsequently complied with diet and started on
Simvastatin 40 mg daily and other treatment, which she
tolerates without difficulty. Current Medications:
1. Metformin 850 mg bid
2. Enalapril  10 mg q day
3. ASA 81 mg q day
4. Simvastatin 40 mg q day

Subsequent results include: LEPG – Broad beta pattern present

Apo E Genotype: Apo E2:E2
 Questions

This implies the accumulation of which lipoprotein class(es)?

a) VLDL + LDL b) IDL and Chylomicron “remnants”

Which lipid-lowering drug is the ideal treatment for this situation?

a) Simvastatin b) Nicotinic Acid c) Questran d) Fibrate
e) Fish oil f) Ezetimibe
Which lipid-lowering therapy is strongly CONTRAindicated? a)
Simvastatin b) Nicotinic Acid c) Questran d) Fibrate
e) Fish oil f) Ezetimibe
Would you stop his statin therapy? Yes / No
Do you agree with the use of low-dose aspirin in this patient? Yes / No
The patient subsequently complied with diet and started on
Simvastatin 40 mg daily and other treatment, which she
tolerates without difficulty. Current Medications:
1. Metformin 850 mg bid
2. Enalapril  10 mg q day
3. ASA 81 mg q day
4. Simvastatin 40 mg q day

Subsequent results include: LEPG – Broad beta pattern present

Apo E Genotype: Apo E2:E2

This implies the accumulation of which lipoprotein class(es)?

Which lipid-lowering drug is the ideal treatment for this situation?
Which lipid-lowering therapy is strongly CONTRAindicated?
Would you stop his statin therapy?
Do you agree with the use of low-dose aspirin in this patient?
The patient asks you about his risk of Alzheimers’ Disease. Is it increased?
 This implies the accumulation of which
lipoprotein class(es)?
a) VLDL + LDL

b) IDL and Chylomicron “remnants”

This implies the accumulation of which
lipoprotein class(es)? The case for “b”
 Which lipid-lowering drug is the ideal
treatment for this situation?
a) Simvastatin

b) Nicotinic Acid

c) Questran

d) Fibrate

e) Fish oil

f) Ezetimibe
 Which lipid-lowering drug is the ideal
treatment for this situation? The case for “d”
 Which lipid-lowering therapy is strongly
CONTRAindicated?
a) Simvastatin

b) Nicotinic Acid

c) Questran

d) Fibrate

e) Fish oil

f) Ezetimibe
Which lipid-lowering therapy is strongly
CONTRAindicated? The case for “c”

Hepatocyte
Heterodimerization
with RXR

Acetyl CoA

SREPB-1c

Bile Duct FA, TG

MDRP2/
3 FXR
VLDL
Phospholipids (TG levels)
 Do you agree with the aspirin dose?
Comment on the role of aspirin in this patient.

• Yes
• No
 Do you agree with the aspirin dose?
Comment on the role of aspirin in this patient.
Evidence and opinion tending towards “no”?