The Usual Suspects: Cholesterol and Triglyceride

Lipid StructureCholesterol: Membranes

Bile AcidsSteroid HormonesProtein modification

Fatty Acids: Fuel, Prostanoids

Glycerol

Triglycerides: FA for Fuel, ProstanoidsProtein modification

Phospholipid: Lecithin Membranes2nd Messengers

HO

COOH

COOH

COOH

HO

HO

HO

+

COO

COO

COO

COO

COO

OPOON+

Structure of a Typical Lipoprotein

Free cholesterol (surface and core)

Phospholipid (amphipath at surface only)

Triglyceride (core only)

Cholesteryl ester (core only)

Apolipoprotein (amphipath at surface only)

Lipoprotein classes and sub-Classes

1.20

1.10

1.06

1.02

1.006

0.95

5 10 20 40 60 80 1000

ChylomicronRemnants

VLDL

LDL

HDL2

HDL3

Particle Size (nm)

Den

sity

(g/m

l)

Chylomicron

VLDLRemnants

Lp(a)

IDL

Directly atherogenic (found in plaque)

pre-β2 HDL

pre-β1 HDL

Substrates for Triacylglycerol Synthesis

Glucose

Glc-6-Pase

PEP

Pyruvate

NEFA

Acyl-CoA SynthetaseCoA

MitochondriaCPT I

Acyl-CarnitineCPT II

Acyl-CoA

Acetyl-CoA

Pyruvate

Ketone BodiesHMG-CoA Synthase

Krebs Cycle

CO2

CPT = Carnitine palmitoyl transferase

Glucose-6-P

PEPCKPyruvate kinase

ATP Citrate Lyase

Acetyl-CoA

Acyl-CoA Carboxylase

Malonyl-CoA

Fatty Acid Synthase

Acyl-CoA

Triglycerides

Beta-oxidation

Hepatocyte

PEP = phosphoenolpyruvate PEPCK = PEP carboxylase

Citrate

Acyl-CoA

Citrate

Plasma

Multiple steps

Structures of Fatty Acids

CHO

O

CHO

O

CHO

O

CHO

O

CHO

O

16:0 (palmitic)

cis-18:1 -6 (oleic)

trans-18:1 -6 (elaidic)

18:2 -6 (linoleic)

18:3 -3(alpha linolenic)

CHO

O 20:5 -3 (EPA)

Liver

Lymphatic chylomicron

Muscle and adipose tissue

Lipoprotein lipase Lipoprotein Lipase

Plasma chylomicron

Fatty acids

Bloodstream

LDLreceptor

Hepatocyte

ChylomicronRemnant

Exogenous (dietary) lipid metabolism

Apo C-II enhances and apo C-III inhibits

LPL activity

Apo B and apo E are ligands for LDL

receptorLDL (apo B,E) receptor clears ChylomicronRemnants

Xenical blocks diatary fat digestion

INTESTINE

Liver

VLDL

Muscle and adipose tissue

Lipoprotein lipase Lipoprotein LipaseAnd hepatic lipase

IDL

Fatty acids

Bloodstream

LDLreceptor

Hepatocyte

LDL

Endogenous (hepatic) lipid metabolism

Apo C-II enhances and apo C-III inhibits

LPL activity

Apo B and apo E are ligands for LDL

receptorLDL (apo B,E)

receptor clears VLDL, IDL & LDL

Clinical Hypertriglyceridaemia

Condition Features

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

Familial Overproduction of apo B lipoproteinsCombined H/L TG and TC vary with age and weight

Polygenic Accounts for the majority of cases

Familial HTG TG predominates. CVD risk variesPredisposes to massive HTG

Massive HTG Lipoprotein Lipase deficiency or saturation. Risk of pancreatitis

Therapy for Hypertriglyceridaemia

Intervention Features

Diet, Exercise Relatively responsive

Fibrates Effective in high TG, low HDL

Alcohol restriction Often sufficient in heavy intake

Manage 2o causes Diabetes, renal

Statins Mild TG and HDL benefitFish oils (eg 6gm/d) Benefits TG rather than HDLNiacin Effective, but increases glu, urate.

Bile acid resins Contraindicated. Increase TGFuture DGAT 2 Inhibitors?

400 mg/day

1,300 mg/day

NORMAL CHOLESTEROL ABSORPTION

Oil phase

400 mg/day

1,300 mg/day

NORMAL CHOLESTEROL ABSORPTION

Plant sterols competewith cholesterol here

Oil phase

400 mg/day

1,300 mg/day

17,400 mg/day

850 mg/day

NORMAL CHOLESTEROL ABSORPTION

Ezetimibe competeswith cholesterol here

Oil phase

400 mg/day

1,300 mg/day

850 mg/day

NORMAL CHOLESTEROL ABSORPTION

Oil phase 17,400 mg/day

Intracellular cholesterol sensing by SREBPs (Sterol Regulatory Element Binding Proteins)

WD Reg

WD

bHLH

bHLH bHLH

bHLH

Reg

S1P S2P

ZN++

Serine protease Metalloproteinase

Golgi Apparatus

Lumen

Sterols

Cytosol

SREBPSCAPNucleus

ERSRE

SCAP or SREBP activating protein

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

• SREBP-1c is the major isoform in liver and is a key regulator of fatty acid & triglyceride synthesis

Other nuclear receptors: FXR, LXR.

LDL Receptor activity reflects intracellular cholesterol homeostasis

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

Cholesterol delivery via LDL-R alters intracellular membrane cholesterol and SREBP, whicha) Reduces synthesis via

HMGCoA Reductaseb) Reduces LDL-R synthesisc) Increases storage as esterd) Reduces counter-regulatory

PCSK9

LCAT

Hepatic lipase, endothelial

lipase

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.

PL&UC

Bile

SR-BI

Macrophage

VLDL/LDL

CETP

Spheroidal HDL Pre-β HDL

SR-A

Oxidation

The Role of HDL inReverse Cholesterol Transport

LDLReceptor

LiverABCA1UC

ABCG1 &SR-B1

UC

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)

Familial Overproduction of apo B lipoproteinsCombined H/L TG and TC vary with age and weight

Polygenic Accounts for the majority of cases

Familial Prevalent, Accelerates CVD.Hyperchol’aemia Due to defects in genes related to LDL-R

Increased HDL ?OK if LDL not raised?

Therapy for Hypercholesterolaemia

Intervention Features

Diet Plant sterols, avoid sat & trans FAManage 2o causes Rarer, but potent: (Thyroid, liver,

renal.) Statins First line for LDL reductionEzetimibe 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

LXR

RXR

PPAR

FXR

HNF-4α

SREBP-1c

NEFAAcyl CoA

Apolipoproteins Pyruvate kinase

Glucose-6-phosphalase Transferin

Bile Acids

Unsaturated Saturated

Fatty acid metabolismTransport

Oxidation Fatty Acid Binding Protein

Ketogenesis

L-FABP

Acetyl CoA Carboxylase Fatty Acid Synthase

Spot 14

SHPBile Acids

Adapted from Pegorier JP et al. J Nutr 2004;134:2444S-9S

SHP = Short Heterodimer Partner

Key Regulators of Genes in Lipid Metabolism

Cellular Cholesterol

Homeostasis

Hepatobiliary IntestineLiver X Receptor (LXR)

Sterol Regulatory Element Binding Protein (SREBP2)

Farnesoid X Receptor (FXR)

Peroxisome proliferated activator receptors PPARsSynthesis Delivery

Acquisition

Excretion

Link to mixed HL cases

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:

Case MC

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

• 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/LHe 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

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, Joshi et al 2007

 INTERHEART, Karthikeyan et al 2009,

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)

Familial Overproduction of apo B lipoproteinsCombined H/L TG and TC vary with age and weight

Polygenic Accounts for the majority of cases

Familial Prevalent, Accelerates CVD.Hyperchol’aemia Due to defects in genes related to LDL-R

Increased HDL ?OK if LDL not raised?

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 (continued)

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) Niacinc) I would increase Atorvastatin to 80 mg but I wouldn’t

give anything other than a statind) Fenofibratee) Fish Oil

• 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 statind) 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

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 willExplain 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

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/LTriglyceride: 7.4mmol/LHDL-C: 1.0mmol/LLDL-C: unable to be calculated

Case GS

Questions:How could you obtain an LDL-C result?a) Friedewald equationb) Abusive phonecall to labc) “Direct method” involving detergentsd) Ultracentifugatione) Subtract HDL-C from Total cholesterol

Which class or classes of lipoproteins would you expect to be increased?

f) Chylomicrons and LDLg) VLDL and LDLh) VLDL and HDLi) 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)+ homocysteinee) Routine fasting lipids are the only lipid tests that are ever required

• 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?

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?a) Friedewald equationb) Abusive phonecall to lab

c) “Direct method” involving detergentsd) 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 LDLc) 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

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 presentApo E Genotype: Apo E2:E2

Case GS (continued)

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

Questions

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?

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 presentApo E Genotype: Apo E2:E2

a) VLDL + LDL

b) IDL and Chylomicron “remnants”

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

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

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?

Which lipid-lowering drug is the ideal treatment for this situation? The case for “d”

a) Simvastatin

b) Nicotinic Acid

c) Questran

d) Fibrate

e) Fish oil

f) Ezetimibe

Which lipid-lowering therapy is strongly CONTRAindicated?

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

Hepatocyte

Bile Duct

Acetyl CoA

SREPB-1c

FA, TG

VLDL (TG levels)

MDRP2/3

Heterodimerization with RXR

Phospholipids

FXR

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”?