Role of glycated LDL in diabetic atherosclerosis

Prof. Sanaa Gazareen, MD
Head of Diabetes, Endocrinology & Obesity Unit-
Menoufia University
Visiting Prof. USC-California & Harvard-MA USA
sgazareen@yahoo

• Patients with DM are 2-8 times more likely to
experience future cardiovascular events.
• LDLc is an important factor in atherogenesis.
Howard BV, Rodriguez BL, Bennett PH, et al. Prevention Conference VI: Diabetes and Cardiovascular disease: Writing Group I:
epidemiology. Circulation. May 7 2002;105(18):e132-7.
Malmberg K, Yusuf S, Gerstein HC, et al. Impact of diabetes on long-term prognosis in patients with unstable angina and non-Qwave
myocardial infarction: results of the OASIS (Organization to Assess Strategies for Ischemic Syndromes) Registry. Circulation.
2000;102:1014-1019

Pathogenic mechanisms involved in the initiation and
progression of atherosclerosis in patients with DM
AGE, advanced glycation end products, CRP, C-reactive protein; HDL, high-density lipoprotein; IL-1b, interleukin-1b; IL-6, interleukin-
6; LDL, low-density lipoprotein; PAI-I, plasminogen activator inhibitor-I; ROS, reactive oxygen species; SAA, serum amyloid A protein;
TF, tissue factor; TG, triglycerides; tPA, tissue-type plasminogen activator.
Bion di-Zoccai et al (2003, Journal of the American College of Cardiology

Prevalence of Dyslipidemia is high in Type 2 Diabetes
Control of Lipids
Patients With
Diabetes, %
Patients Without
Diabetes, %
P Value
LDL-C
> 100 mg/dL
HDL-C
< 40 mg/dL (men)
< 50 mg/dL (women)
Triglycerides
> 150 mg/dL
74.7 75.7 NS
63.7 40.0 < .001
61.6 25.5 < .001
N = 498 adults (projected to 13.4 million) aged > or = 18 years with diabetes
representative of the US population and surveyed within the cross-sectional
National Health and Nutrition Examination Survey 1999-2000.
Jacobs MJ, et al. Diabetes Res Clin Pract. 2005;70:263-269.

IN DM: LDLc
Routine LAB. Measurement of LDL?
Is LDLc really a single entity??

LDL Subfractions
• Larger LDL subfractions (1, 2, 3) are considered type A
and are less frequently associated with coronary artery
disease.
• Small, dense LDL subfractions (4-7, non type A) are
generally considered more atherogenic. There is an
increased prevalence of non-type A LDL subfractions in
Type 2 diabetes and insulin resistance syndrome 1 .
lipoproteins of > 70 nm have limited transcytosis past the
endothelium
1. Austin MA, Hokanson JE, Brunzell JD. Characterization of low-density lipoprotein
subclasses: methodologic approaches and clinical relevance. Curr Opin Lipidol 1994;5:395-
403.
2. Younis NN, Soran H, Sharma R, et al. Small-dense LDL and LDL glycation in metabolic
syndrome and in statin-treated and nonstatin-treated type 2 diabetes. Diab Vasc Dis Res
2010; 7:289–295

The primary atherogenic lipoprotein is LDL (small dense LDL)
lipoproteins of > 70 nm have limited transcytosis past the endothelium
Monocyte
Nascent
chylomicron Nasce
t VLDL
Vessel Lumen
Adhesion
Molecules
Χ
Χ
LDL
Artery wall
MCP-1
LDL
Endothelium
Modified LDL
Taken up by
Macrophage
Intima
Foam Cell
Macrophage
Modified LDL is more taken by macrophages then non modified LDL Steinberg D e
al. N Engl J Med 1989;320:915-924.

Oxidized LDL pathway during atherogenesis
Oxidative modification of LDL has been most widely studied and is currently believed to be
involved in atherosclerosis
1- Mackness MI, Durrington PN, Mackness B. How high-density lipoprotein protects against the effects of lipid peroxidation. Curr
Opin Lipidol 2000; 11:383–388. 2- Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of lowdensity
lipoprotein that increase its atherogenicity. N Engl J Med 1989; 320:915–924

LDL Oxidation
• Although LDL oxidation may be part of the
explanation for the participation of LDL in
atherogenesis, it is unsatisfactory as the sole
explanation. This is not least because of the
notable lack of success of antioxidant therapy
to prevent atherosclerosis
Kris-Etherton PM, Lichtenstein AH, Howard BV, et al. Antioxidant vitamin
supplements and cardiovascular disease. Circulation 2004; 110:637–641

? What about
other LDL Modification

Naturally occurring atherogenic modifications of lipoproteins include oxidation, glycation
& glycoxidation
glycoxidation
Modified LDL is considered to be a risk factor for the development and progression of atherosclerosis.
glycated LDL is present in the circulation at much higher concentration than oxidized
LDL, even in non-diabetic people & can stimulate foam cell formation.
Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest 1991;
88:1785–1792.& Tanaka, Akira Atherosclerosis, Volume 52 Springer Protocols – Jan 1, 2001

Glycated Vs Oxidized LDL in non diabetic subjects
Oxidized LDL
• does not exist in the
circulation because of the
presence of many
antioxidizing agents .
• LDL cannot be oxidized
until it has entered the
arterial wall from the
circulation
Glycated LDL
• The only modified LDL that
exists in the circulation . It
has been shown to be more
easily oxidized than native
LDL
• Thus glycation of LDL may
be the initial step in the
development of
atherosclerosis.
Tanaka, Akira : Atherosclerosis, Volume 52 Springer Protocols – Jan 1,
2001

Small dense-LDL is the most glycated LDL subfraction
Atherosclerosis 2008; Advance online publication
• Even in non-diabetic people,, small dense LDL may be
rendered more atherogenic because it is more abundantly
glycated than more buoyant LDL."

Glycation process in DM
reactive sugars, such as glucose and its [alpha]-
oxoaldehyde metabolites can induce Glycation
The non-enzymatic glycation of LDL is a naturally occurring chemical modification of lysine residues of apo-B of LDL by
blood glucose. involving the formation of a Schiff's base , followed by Amadori rearrangement and the formation of a
stable ketoamine link to generate [alpha]-1-deoxy-1-amino-D-fructopyranose. Glycation can be induced by reactive
sugars, such as glucose and its [alpha]-oxoaldehyde metabolites that can be derived both through glucose degradation or
through other pathways, such as the polyol pathway Diabetes Metab 2001; 27:535–542. Chinese Medical Journal 2009

Is glycation of LDL in patients with Type 2 DM a preoxidative
condition?
Moro E, Alessandrini P, Zambon C, Pianetti S, Pais M,
Cazzolato G, Bon GB
Results
• In Type 2 diabetic patients, LDL were more
glycated & more susceptible to oxidation.
Diabet Med. 1999 Aug;16(8):663-9.

It was suggested that the earliest event is glycation of
LDL, whilst oxidative events (glycoxidation or direct
oxidation of lipid or proteins) only occur at later time
points. This may facilitate the formation of foam cells
and the vascular complications of diabetes
Eur J Biochem. 2003 Sep;270(17):3572-82.
Glycation and glycoxidation of low-density lipoproteins by glucose and lowmolecular
mass aldehydes. Formation of modified and oxidized particles.
Knott HM, Brown BE, Davies MJ, Dean RT.

Factors Influencing Plasma Level of Glycated apoB
• Plasma glyc-apoB correlated with SD-LDL-apoB ( p <
0.0001 in DM & , p < 0.001 in MS), but not with
HbA1c. Thus SD-LDL concentration is a stronger
determinant of plasma glyc-apoB than glycaemia.
Younis NN, Soran H, Sharma R, et al. Small-dense LDL and LDL glycation in
metabolic syndrome and in statin-treated and nonstatin-treated type 2 diabetes.
Diab Vasc Dis Res 2010; 7:289–295

Schematic overview of lipoprotein metabolic pathways in
type 2 diabetes
↑ TG
↑ ApoB
↓ HDLc
↔ LDLc
Lancet 1997 Jul ; 350 suppl 1: S 120-3

“
Glycated LDL “Ultra-bad Cholesterol”
Glycation of LDL Decreases the uptake and degradation of LDL by the high-affinity LDL receptor
Increases uptake by macrophages ( foam cells)
Glycated LDL is present in the circulation under physiological conditions and is present at
higher concentrations in DM, polygenic hyperlipidaemia and familial hypercholesterolaemia.
Curr Opin Lipidol. 2011 Aug;22(4):254-61.
Susceptibility of LDL and its subfractions to glycation. Soran H, Durrington PN

Consequence of LDL Glycation (Role in diabetic atherosclerosis)
• ability to regulate cholesterol transferase activities (HMG-coA & acyl–coenzyme A)
• the uptake and degradation of LDL by the high-affinity LDL receptor
• uptake by macrophages ( foam cells)
• prone to oxidation
• arterial smooth muscle cell proliferation (PKC, phospholipase C)
• Inflamatory expression of IL6, CD40, fibronectin
• Glycated LDL is prothrombogenic ( (tPA) & PAI-1)
• endothelial dysfunction (altered PGI 2 , peroxinitrite)
L.

• Prevention and treatment

First Joint ESC/EAS Guidelines for the
Management of Dyslipidaemias 2011
Clinicians should aim for LDL cholesterol levels :

DM Control : DIET, EXERCISE, Drugs
To improve lipid profile in patients with DM, recommend lifestyle modification (A), focusing on
– Reduction of saturated fat, trans fat, cholesterol intake
– Increased n-3 fatty acids, viscous fiber, plant stanols/sterols
– ADA. VI. Prevention, Management of Complications. Diabetes Care 2011;34(suppl 1):S29

Role of Metformin in preventing
atherosclerosis
Metformin :
• increases LDL particle size
• decreases plasma concentrations of remnant lipoprotein
cholesterol thought to reflect atherogenicity of cholesterol
ester–rich chylomicrons and VLDL
• decreases the plasma concentration of methylglyoxal
( important for glycation) thus may help preventing glycation
• decreases oxidative stress and related oxidation of LDL.
) Naila Rabbani etal Glycation of LDL by Methylglyoxal Increases Arterial Atherogenicity A Possible
Contributor to Increased Risk of Cardiovascular Disease in DiabetesDiabetes April 2010 vol. 59 no. 4
1038-1045)

Treatment of Diabetic Dyslipidemia
– 1. Statins
– 2. Bile Acid Sequestrants
– 3. Fibric Acid Derivatives
– 4. Nicotinic Acid:
– 5. Estrogens
– 6. n-3 Polyunsaturated Fatty Acids
– 7. Statins Plus Bile Acid Sequestrants
– 8. Statins Plus Fibric Acid Derivatives
– 9. Statins Plus Fish Oil
– 10. Statins Plus Estrogens
– 11. Statins Plus Nicotinic Acid

Recommendations:
Dyslipidemia/Lipid Management
• Statin therapy should be added to lifestyle therapy,
regardless of baseline lipid levels, for diabetics:
– with overt CVD (A)
– without CVD who are >40 years of age and have one or
more other CVD risk factors (A)
ADA. VI. Prevention, Management of Complications. Diabetes Care 2011;34(suppl 1):S29.

-Plasma glyc-apoB correlated with SD-LDL-apoB but not
with HbA1c.
-Statin-induced changes in Small Dense-LDL level may be
important in decreasing apoB glycation in DM.
‣ This may explain the small effect of improving glycaemia
relative to statin treatment in reducing atherosclerosis
risk in type 2 diabetes.
Younis NN, Soran H, Sharma R, et al. Small-dense LDL and LDL glycation in
metabolic syndrome and in statin-treated and nonstatin-treated type 2 diabetes.
Diab Vasc Dis Res 2010; 7:289–295

Drugs that can protect against glycation
• Metformin and buformin (biguanides)
• Pioglitazone
• Aspirin (acetylsalicylic acid)
• Ibuprofen
• Diclofenac
• Amino acids
• Polyamines
• Carnosine
• Glutathione (gamma-glutamylcysteinyl-glycine)
• Aminoguanidine
• Penicillamine
• Pyridoxamine
• Aminoguanidine-pyridoxal
adduct
• Pentoxifylline
• Thiamine pyrophosphate
• OPB-9195 ( (+/-)-2-isopropylidinehydrazono-4-
oxo-thiazolidin-
• 5-ylacetanilide] )
• ALT-946 (N-(2-
acetamidoethyl)hydrazinecarboximidamide
hydrochloride),
• Lipoic acid
• Olmesartan and other angiotensin II type I
receptor antagonists
• Captopril and other ACE inhibitors
• Pyruvate
• Dilazep
• Anserine
• Cysteine,
• Cysteamine
• Sulphite
• 3-mercaptopropionate
J.J. Harding, E. Ganea / Biochimica et Biophysica Acta (2006)

. Aqueous extracts of ground pepper, cinnamon, rosemary,
ginger, and clove were analyzed and tested for anti-atherosclerotic
activity in vitro and in vivo using hypercholesterolemic zebrafish.
Cinnamon and clove extracts (at final concentration 10 μg/mL) had the
strongest anti-glycation and antioxidant activity in this study.
(JinS,ChoKH.Food chem Toxicol2011 Jul;49(7):1521-9. Epub 2011 Apr 5 )
. Aqueous extracts of turmeric and laurel showed potent inhibitory activity
against fructose-mediated glycation with antioxidant ability against lowdensity
lipoprotein (LDL) oxidation and radical scavenging activity
. )Jin s,HongJH, Turmeric and laurel aqueous extracts exhibit in vitro antiatherosclerotic
activity and in vivo hypolipidemic effects in a zebrafish model. 2011
Mar;14(3):247-56 )

Reversal of glycation
• glycine, taurine,
• spermidine and N-acetylcysteine
• glutathione,
• other aminothiolamines, carnosine and
anserine
J.J. Harding, E. Ganea / Biochimica et Biophysica Acta (2006)

Preventing the consequences of
glycation
• Alpha-crystallin, a molecular chaperone, is able
to protect almost completely a variety of
enzymes against glycation-induced inactivation in
vitro without interfering with the original
glycation reaction
• Some small organic molecules share some
protective properties with molecular chaperones
and are often referred to as ‘chemical
chaperones’ although a better term is ‘Small
Organic Stress Molecules’ or ‘SOS molecules
• of these trehalose and 6-aminohexanoic
J.J. Harding, E. Ganea / Biochimica et Biophysica Acta (2006)

CONCLUSION
• In DM Increased Plasma Glycated LDL
Concentration is A Marker of Atherogenic Risk
• Glycation of LDL is a pre-oxidative condition
• Routine Total LDL measurement is misleading
• Thus Lab. Measurement of glycated LDL can
help in proper management of diabetic
patients