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Effects on lipid levels and lipid metabolism under more human-like conditions.

Effect of garlic powders and garlic active compounds on lipid metabolism and atherosclerosis in APOE*3-Leiden mice. Sonia M.S. Espirito Santo, Bart J.M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H. Offerman, Louis M. Havekes and Hans M.G. Princen

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Effects on lipid levels and lipid metabolism under more human-like conditions.

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  1. Effect of garlic powders and garlic active compounds on lipid metabolism and atherosclerosis in APOE*3-Leiden mice Sonia M.S. Espirito Santo, Bart J.M. van Vlijmen, Rien Buytenhek, Wim van Duyvenvoorde, Erik H. Offerman, Louis M. Havekes and Hans M.G. Princen Gaubius Laboratory, TNO-PG, Leiden, the Netherlands EU project

  2. Aim of the study: Effects of garlic on lipid metabolism and atherosclerosis • Effects on lipid levels and lipid metabolism under more human-like conditions. • Lipid levels, cholesterol synthesis and cholesterol absorption. • Effects on fast atherosclerosis development and other processes that might be related with atherosclerosis. • Inflammation, blood pressure, platelet activation and atherosclerosis analysis.

  3. Lipid metabolism LDL LIVER LDL receptor SYNTHESIS UPTAKE HMG-CoA reductase other receptors CHOLESTEROL ACAT STORAGE 7  -hydroxylase CHOLESTERYL ESTERS 27-hydroxylase CONVERSION VLDL SECRETION BILE ACIDS BILIARY CHOLESTEROL BILE CHYLOMICRON REMNANTS INTESTINE DIETARY CHOLESTEROL CHYLOMICRONS

  4. Atherosclerosis Shear stress Inflammation Oxidation Intima Media Fibrous cap Monocytes LDL Thrombus Endothelium oxLDL O2 Lipid core Collagen- degrading proteinases

  5. Contradictory effects attributed to garlic • Soluble garlic extracts inhibit cholesterol biosynthesis in primary rat hepatocytes and human HepG2 cells. R. Gebhardt, Lipids, 1993, 28(7):613-9. • Lichtwer garlic powder reduces plasma lipid levels and blood pressure in hypercholesterolemic rats. M Ali et al., Prostaglandins Leukot Essent Fatty Acids, 2000, 62(4):253-9. • Allicin showed no effects on plasma lipid levels and decreased aorta atherosclerotic lesions in C57Black/6 mice fed a high fat/ cholesterol diet. D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519. • Garlic powder showed no effects on plasma lipid levels in patients with moderated hypercholesterolemia. B.W. McCrindle et al, Arch Pediatr Adolesc Med, 1998, 152(11): 1089-94 and C.D. Gardner et al, Atherosclerosis, 2001, 154(1):213-2. • Garlic oil showed no effects on plasma lipid levels, cholesterol synthesis and cholesterol absorption in patients with moderated hypercholesterolemia. H.K. Berthold et al, JAMA, 1998, 17(23):1900-2.

  6. Aim of the study: Effects of garlic on lipid levels and lipid metabolism (1) Study the effect of 2 garlic powders and 2 garlic active compounds on plasma lipid levels, cholesterol synthesis and intestinal absorption under more human-like conditions using hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

  7. APOE*3-Leiden transgenic mouse as a model for hyperlipidemia and atherosclerosis studies • Transgenic mice carrying a mutant form of human APOE*3 gene. • Dominant dysfunctional APOE*3-Leiden results in defect in chylomicron/ VLDL clearance and more human-like lipoprotein profile. • Highly responsive to feeding cholesterol-rich diets. • Develops atherosclerosis varying from fatty streaks to more human-like complicated lesions.

  8. Atherosclerosis in APOE*3-Leiden mice Monocytes Adherence of monocytes to the activated vessel wall

  9. Atherosclerosis in APOE*3-Leiden mice Media not affected foam cells intima { media Aorta with regular fatty streak

  10. Atherosclerosis in APOE*3-Leiden mice cholesterol clefts necrosis fibrosis { intima media Affected media Severe plaque in aorta

  11. Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: Selection of compounds • Lichtwer LI111 garlic powder: Reference dry garlic powder. • Morado de Cuenca 200 (Mor200): Garlic powder rich in organic sulfur-containing compounds. • Diallyl disulfide (DADS): Isolated garlic compound rich in sulfur. • Allicin: Reference isolated garlic compound reported to result in 43% reduction of fatty streaks in C57Black/6 mice (D. Abramovitz et al., Coronary Artery Disease, 1999, 10: 515-519)

  12. 80 Effect of garlic on lipid metabolism and atherosclerosis in APOE*3-Leiden mice: Experimental scheme 25 -2 0 Regular diet Western diet Atherogenic diet 6 14 Run in 8 10 12 16 18 20 22 2 4 Matching and start of the study 80 65 Weight and food intake 80 80 80 65 60 65 65 65 Blood sampling/ plasma isolation 80 80 65 65 65 65 65 60 80 Plasma lipid levels 80 65 80 65 65 65 65 Cholesterol lipoprotein profile p p p p p p Chol. synthesis/ Intestinal absorption p p p p p Marker for liver function p p p Liver lipid composition 15 60 Blood pressure 80 65 65 Platelet activation measurements 80 65 Inflammation markers 60 Atherosclerotic aorta lesions typing 60 5 Atherosclerotic aorta lesions area 60 5 Compound biostability p p p p p Inflammation and atherosclerosis related parameters General parameters Lipid metabolism related parameters

  13. Food intake Regular diet Western diet Atherogenic diet g/ mouse/ day Control 3.6 3.2 3.3 * LI111 2.6 3.3 3.0 * Mor 200 2.8 3.4 3.0 * DADS 2.4 3.0 2.6 Allicin 3.6 3.3 3.0 Effect of garlic on food intake under 3 dietary conditions *P< 0.05

  14. Body weight Regular diet Western diet Atherogenic diet g Control 20.9 23.3 23.2 LI111 21.0 22.0 22.6 Mor 200 21.5 23.2 22.1 * * DADS 21.0 20.6 20.3 Allicin 20.5 22.6 23.4 Effect of garlic on body weight under 3 dietary conditions *P< 0.05

  15. Effect of garlic on liver function under 3 dietary conditions Plasma alanine amino transferase Regular diet Western diet Atherogenic diet U/L Control 33.4 30.3 175 t LI111 104 33.2 34.4 t Mor 200 94.1 32.9 30.3 DADS 29.9 41.8 203 Allicin 25.2 28.2 171 tP=0.051

  16. Plasma cholesterol levels Regular diet Western diet Atherogenic diet mM Control 3.1 9.3 63 LI111 2.9 9.3 57 * Mor 200 3.0 9.1 44 DADS 2.9 8.3 70 * Allicin 3.5 9.2 73 Effect of garlic on plasma cholesterol levels under 3 dietary conditions *P< 0.05

  17. Effect of garlic on cholesterol lipoprotein distribution under 3 dietary conditions Regular diet Western diet Atherogenic diet  Control  LI111  Morado 200 VLDL 4 0.4 12 LDL HDL 3 0.3 8 0.2 2 4 0.1 1 Cholesterol (mM) 0.0 0 0 1 5 9 13 17 21 1 5 9 13 17 21 1 5 9 13 17 21  Control  DADS  Allicin 0.4 12 4 0.3 3 8 0.2 2 4 0.1 1 0.0 0 0 1 5 9 13 17 21 1 5 9 13 17 21 1 5 9 13 17 21 *P< 0.05 Fraction number

  18. squalene 1.5 lathosterol lanosterol 1.0 0.5 Concentration (µmol sterol / mmol cholesterol) 0.0 Control LI111 Mor 200 DADS Allicin 40 campesterol b-sitosterol 30 20 10 0 Control LI111 Mor 200 DADS Allicin Effect of garlic on markers of cholesterol synthesis and intestinal absorption under 3 dietary conditions Regular diet Markers of cholesterol synthesis Markers of intestinal absorption *P< 0.05

  19. Effect of garlic on liver lipid levels under fast atherogenic conditions Liver lipid composition Free cholesterol Triglycerides Cholesteryl esters mg lipid/ g protein 19.4 43.7 154 Control * * * 15.9 31.5 130 LI111 * * * 15.3 35.0 127 Mor 200 * 17.8 49.7 171 DADS 17.3 37.8 146 Allicin *P< 0.05

  20. Conclusions of the study of the effects of garlic on lipid levels and lipid metabolism (1) • Under more human-like hyperlipidemic conditions (8-12 mM): • No beneficial effects of garlic powders and garlic isolated compounds were observed on lipid levels, cholesterol synthesis and intestinal absorption in APOE*3-Leiden mice. • Under fast atherosclerosis conditions (45-70 mM): • Morado 200 showed a plasma cholesterol-lowering effect in APOE*3-Leiden mice. • LI111 and Morado 200 had a cholesterol and triglyceride liver storage lowering effect in APOE*3-Leiden mice.

  21. Aim of the study: Effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2) Study the effect of 2 garlic powders and 2 garlic active compounds on inflammation processes, blood pressure, platelet activation and fast atherosclerosis development using hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

  22. 8 250 p=0.001 p<0.001 200 6 * 150 * SAA (µg/ ml) vWF (%PP) 4 100 2 50 0 0 LI111 Mor 200 DADS LI111 Mor 200 DADS Allicin Control Allicin Control Effect of garlic on Serum Amyloid A and von Willebrand factor concentrations under fast atherogenic conditions *P< 0.05

  23. Effect of garlic on blood pressure and platelet activation under fast atherogenic conditions • No effects of garlic powders and garlic isolated compounds were observed on the percentage of activated platelets in blood under all dietary treatments. • No influence of garlic powders and garlic isolated compounds were found on blood pressure under regular and Western dietary conditions by cuff-tail method.

  24. 400 400 p = 0.017 p = 0.011 300 300 Plaque area of the aortic root (µm2 x 1000) 200 200 100 100 0 0 LI111 Mor 200 DADS Allicin Control Control Effect of garlic on plaque area of the aortic root under fast atherogenic conditions *P< 0.05

  25. Conclusions of the study of the effects of garlic on fast atherosclerosis development and other atherosclerosis related processes (2) Under fast atherogenic conditions: • LI111 and Morado 200 showed an improved liver function. • Morado 200 had a lowering effect on plasma cholesterol levels. • Morado 200 and Allicin showed a lowering effect on serum amyloid A concentration, suggesting a decreased liver specific inflammation. • LI111 and DADS treated groups showed a larger plaque area.

  26. Future mild atherosclerosis development study under more human-like conditions Study the effect of 2 garlic powders of the same strain with a high and a low content of sulfur-containing compounds and allicin as a positive control on inflammation related parameters and on mild human-like atherosclerosis development conditions by means of a hyperlipidemic and atherosclerosis-susceptible APOE*3-Leiden transgenic mice.

  27. Future mild atherosclerosis development study under more human-like conditions: Experimental scheme 24 Western diet -2 In weeks 0 22 6 14 Run in Matching and start of the study 60 Weight and food intake 60 60 60 60 Blood sampling/ plasma isolation 60 60 60 60 Plasma lipid levels 60 60 60 Cholesterol lipoprotein profile p p p 60 60 Marker for liver function p 60 60 Liver lipid composition 60 SAA and vWf 60 60 Fibrinogen, t-PA and soluble ICAM 60 60 Atherosclerotic aorta lesions typing 5 55 Atherosclerotic aorta lesions area 55 5 Moncytes adherence 55 5 Compound biostability p p Inflammation and atherosclerosis development related parameters General parameters Lipid metabolism related parameters

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