The Usual Suspects: Cholesterol and Triglyceride

H
O
Fatty Acids: Fuel,
Prostanoids
COOH
+
Glycerol
COOH
COOH
H
O
H
O
H
O
Lipid Structure
Cholesterol: Membranes
Bile Acids
Steroid Hormones
Protein modification
Triglycerides: FA for Fuel, Prostanoids
Protein modification
COO
COO
COO
Phospholipid: Lecithin Membranes
COO 2 nd Messengers
COO
+ N OPOO

Structure of a
Typical Lipoprotein
Free cholesterol
(surface and core)
Phospholipid
(amphipath at surface only)
Triglyceride
(core only)
Apolipoprotein
(amphipath at surface only)
Cholesteryl ester
(core only)

Lipoprotein classes and sub-Classes
0.95
Chylomicron
VLDL
IDL
VLDL
Remnants
1.006
1.02
Chylomicron
Remnants
LDL
1.06
1.10
1.20
HDL
2
HDL3
5 preβ1 HDL
10
Lp(a) preβ2 HDL
20
Directly atherogenic
(found in plaque)
40 60
Particle Size (nm)
80
1000

Substrates for Triacylglycerol Synthesis
Plasma
Glucose
NEFA
Triglycerides
Glc-6-Pase
Acyl-CoA Synthetase
Acyl-CoA
CoA
Multiple steps
Glucose-6-P
PEPCK
PEP
Pyruvate kinase
Pyruvate
Pyruvate
CPT I
Acyl-Carnitine
CPT II
Acyl-CoA
Acyl-CoA
Mitochondria
Fatty Acid Synthase
Malonyl-CoA
Acyl-CoA Carboxylase
Krebs
Cycle
Citrate Acetyl-CoA
ATP Citrate Lyase
Acetyl-CoA CO
2
HMG-CoA Synthase
Ketone Bodies
Beta-oxidation
Citrate
PEP = phosphoenolpyruvate
PEPCK = PEP carboxylase
CPT = Carnitine palmitoyl transferase

O
C
HO
O
HO
C
O
HO
C
O
HO
C
O
HO
C
O
HO
C
Structures of Fatty Acids
16:0 (palmitic) cis-18:1

-6 (oleic) trans-18:1

-6 (elaidic)
18:2

-6 (linoleic)
18:3

-3
(alpha linolenic)
20:5

-3 (EPA)

Exogenous (dietary) lipid metabolism
Muscle and adipose tissue
Fatty acids
Lipoprotein lipase
Bloodstream
E
S
T
I
I
N
T
N
E
Lymphatic chylomicron
Xenical blocks diatary fat digestion
Plasma chylomicron
Lipoprotein Lipase
Apo C-II enhances and apo C-III inhibits
LPL activity
Chylomicron
Remnant
Apo B and apo E are ligands for LDL receptor
LDL (apo B,E) receptor clears
Chylomicron
Remnants
LDL receptor
Liver
Hepatocyte

Endogenous (hepatic) lipid metabolism
Muscle and adipose tissue
Lipoprotein lipase
Fatty acids
Lipoprotein Lipase
And hepatic lipase
LDL
Apo C-II enhances and apo C-III inhibits
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

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

Intervention
Diet, Exercise
Alcohol restriction
Manage 2 o causes
Fibrates
Statins
Fish oils (eg 6gm/d)
Niacin
Future
Bile acid resins
Features
Relatively responsive
Often sufficient in heavy intake
Diabetes, renal
Effective in high TG, low HDL
Mild TG and HDL benefit
Benefits TG rather than HDL
Effective, but increases glu, urate.
DGAT 2 Inhibitors?
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
17,400 mg/day
Oil phase
850 mg/day

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

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
PCSK9
*[ SREBP] = sterol regulatory element-binding protein.
1.
Goldstein JL, et al. Arterioscler Thromb Vasc Biol. 2009;29:431-438.
.

UC
The Role of HDL in
Bile
Reverse Cholesterol Transport
ABCG1
&SR-B1
Spheroidal HDL
Preβ HDL
Liver
UC
LCAT
ABCA1
SR-BI
Hepatic lipase, endothelial lipase
PL&UC
LDL
Receptor
CETP
SR-A
Macrophage
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

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

Intervention
Diet
Manage 2 o causes
Statins
Ezetimibe
Niacin
Bile acid resins
Future
Features
Plant sterols, avoid sat & trans FA
Rarer, but potent: (Thyroid, liver, renal.)
First line for LDL reduction
2 nd line. Neutral for TG & HDL
Improves LDL, TG, HDL & Lp(a)
Colesevalam better tolerated
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
NEFA
PPAR
L-FABP
RXR
Fatty acid metabolism
Transport
Oxidation
Fatty Acid Binding Protein
Ketogenesis
Adapted from Pegorier JP et al. J Nutr 2004;134:2444S-9S
Acyl CoA
Unsaturated Saturated
HNF-4 α
Apolipoproteins
Pyruvate kinase
Glucose-6-phosphalase
Transferin
Bile Acids

Key Regulators of Genes in Lipid Metabolism
Sterol Regulatory Element Binding Protein (SREBP2)
Synthesis
Peroxisome proliferated activator receptors PPARs
Delivery
Acquisition
Excretion
Hepatobiliary
Farnesoid X Receptor (FXR)
Intestine
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)
• B)
• C)
• D)
• E)
Dyslipidaemia secondary to Insulin resistance and the Metabolic Syndrome
Polygenc dyslipidamia
Familial Combined Hyperlipidaemia
Familial Hypercholesterolaemia
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)
• C)
• D)
• E)
Polygenc dyslipidamia
Familial Combined Hyperlipidaemia
Familial Hypercholesterolaemia
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)
Accounts for the majority of cases Polygenic
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 3year period.
27
M Gheorghiade et al Circulation.
2002; 106: 394-398

• Yes
Do you trust the LDL-C result?
• 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:
HDL-C:
LDL-C:
7.4mmol/L
1.0mmol/L 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?
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 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 triglycerides are 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)

• 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

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 “re m nants”

This implies the accumulation of which lipoprotein class(es )? The case for “b”

Which lipid-lowering drug is the ideal a) Simvastatin treatment for this situation?
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”
Heterodimerizatio n with RXR
Acetyl CoA
SREPB-1c
Hepatocyte
Bile Duct FA, TG
MDRP2/
3
Phospholipid s
FXR
VLDL
(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”?