1. Rolf Gebhardt University of Leipzig Medical Fakulty Institute of Biochemistry Garlic & Health: WP 5.2 The interaction of Garlic with vessel wall and cholesterol biosynthesis

2. Cholesterol biosynthesis: 1.Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis -Sulfur fertilization -Environmental influences 3.Influence of Garlic Organosulfur Compounds: -Mechanistic studies 4.Consequences for Signal Transduction 2.Influence of Flavonoids on Cholesterol Synthesis

3. Garlic Powder: reinvestigation Garlic Powder: expected influence Inhibition of cholesterol biosynthesis 0.010.11 Concentration (mg/ml) 0 20 40 60 80 100 120 140 160 Cholesterol Biosynthesis (%) Hepatoc. HepG2 fresh Garlic 1

4. Garlic Powder: reinvestigation Unexpected Result !! Garlic Powder (Low Quality Garlic): Stimulation of cholesterol biosynthesis !

5. Field Study on Garlic Quality: 1.Controlled planting of four defined garlic varieties in two different european countries 2.Controlled variation of soil fertilisation 3.Standardized harvesting, drying and powder preparation 4.HPLC characterisation of organosulfur content by standardized techniques in two laboratories

6. Influence of fertilization on garlic properties Proper fertilization may change the response of hepatic cells with respect to cholesterol biosynthesis: Stimulation may turn into inhibition Inhibiting properties may be reinforced

7. Influence of fertilization on garlic properties Influence of high sulfur fertilization: French harvest 2001

8. Open Questions: 1.What compounds of garlic inhibit or stimulate hepatic cholesterol-biosynthesis? 2.What is the molecular mechanism of stimulation?

9. Structure of Favonoids used University of Leipzig All tested flavonoids belong to the flavonol group They differ only in the amount and/or position of one or two hydroxyl groups In the case of Luteolin and Kaempferol, several glycosides were also tested

10. University of Leipzig Comparison of the effects of Kaempferol, Luteolin and their glycosides in rat hepatocytes Kaempferol: sensitive stimulation of cholesterol biosynthesis high cytotoxicity (responsible for decline) Luteolin: inhibition of cholesterol biosynthesis (no significant stimulation) low cytotoxicity

11. University of Leipzig Comparison of the effects of Myricetin, Kaempferol and Luteolin in HepG2 cells Myricetin: stimulation of cholesterol biosynthesis Kaempferol: no significant effect Luteolin: inhibition of cholesterol biosynthesis

12. Cholesterol biosynthesis: 1.Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis -Sulfur fertilization -Environmental influences 3.Influence of Garlic Organosulfur Compounds: -Mechanistic studies 4.Consequences for Signal Transduction 2.Influence of Flavonoids on Cholesterol Synthesis

13. Comparison of different garlic-derived organosulfur compounds for inhibition of cholesterol biosynthesis

14. Inhibition of HMGCoA reductase and cholesterol biosynthesis Test Compound EC 50 values HMGCoACholesterol reductasebiosynthesis Allicin> 300 17 Diallyl disulfide> 250 65 Allyl mercaptan> 1500 450 S-allyl cysteine n.d. > 1500 Diallyl sulfide> 1000 735 Dipropyl disulfide> 1500 > 1000

15. Effect of fructose and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg)(%) (%) Control0.34100 100 Fructose5.78523 54 Diallyl disulfide0.50157 92 Fructose + DADS4.85686 25 Fruct. + DADS + araA4.56194 93

16. Effect of hypoxia, KCl and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg)(%) (%) Control0.41100 100 low oxygen4.75408 72 low oxygen + DADS4.59566 59 KCl2.58346 79 KCl + DADS2.73424 63 KCl + DADS + araA2.46170 89

17. Correlation between AMPK activity and inhibition of cholesterol biosynthesis Correlation coefficient: r = - 0.96 for all conditions: - absence of DADS (closed symbols) - presence of DADS (open symbols)

18. Influence of fructose, i.e. high AMP on the sensitivity of hepatocytes towards DADS Lowering of the cellular energy charge - increases the sensi- tivity towards DADS 5- to 10-fold

19. Schematic view of the probable mode of action of several garlic-derived organosulfur compounds

20. Influence of garlic on acetate uptake:

21. Questions on acetate transport: 1.What transport system is involved? (alkyl monocarboxylate carrier?) 3.What is the molecular mechanism of stimulation? 2.What compounds are responsible for stimulation?

22. Central Role of AMP-dependent kinase (AMPK) as a mediator of stress signals, e.g. low energy charge Energy consuming pathways e.g. fatty acid and cholesterol synthesis are shut down

23. Effects of antidiabetic drugs Rosiglitazone Metformin Insulin sensitivity blood glucose level - peripheral glucose uptake -fatty acid oxidation -fatty acid esterification - hepatic glucose production

24. Role of AMPK as a mediator of antidiabetic drugs Rosiglitazone or DNP ATP / AMP + + AMPK antidiabetic effects Metformin or hyperosmolar stress + + ? Phosphorylation at Threonine 172

25. Influence of insulin and DADS on glucose production in normal rat hepatocytes Insulin: - lowers glucose production DADS: - has no influence in normal rats

26. Influence of insulin and DADS on glucose production in obese Zucker (fa-/fa-) rat hepatocytes Insulin: - slightly lowers glucose production DADS: - reinforces the insulin effect in obese rat hepatocytes

27. Conclusion: 1. DADS influences glucose production in diabetic rats, but not in normal rats 2. DADS acts only in synergy with insulin Does DADS increase sensitivity of diabetic hepatocytes for insulin? Question:

28. Interactions with vessel wall: 3.Effects of Garlic Extracts on MMPs and TIMPs 1.Influence of Garlic Organosulfur Compounds on matrix metalloproteinases (MMPs): -Studies in HUVEC - Studies in HCAEC 2.Influence of Organosulfur Compounds on Tissue Inhibitors of MMPs

29. MMP TIMP ArteriosclerosisFibrosis ArthritisDiabetic Nephro- Rheumatoid Arthritis pathy InflammationGlomerulosclerosis Tumour-GrowthScleroderma -Metastasis -Angiogenesis Ulcers Parodontosis Morphogenesis, Differentiation, Wound healing, etc. Pathological Implications of MMP-TIMP-Imbalance:

30. Spectrum of matrix metalloproteinases and TIMP-1 in cultured HUVEC cells. After 9 h of cultivation. Induction of MMPs and TIMP-1 in HUVEC cells 9 / 24 h after PMA, forskolin and TNF-alpha

31. Inhibition of MMP-2 Production in HUVEC by DADS, AM and SAC Activity assayELISA MMP-1 and MMP-3 are not affected by DADS, AM and SAC in HUVEC cells

32. Inhibition of MMP-9 and TIMP-1 Production by DADS, AM and SAC MMP-9 (activity assay) TIMP-1 (ELISA) - Unstimulated HUVEC cells do not secrete MMP-9 - In the case of TIMP-1 unstimulated cells respond similarly HUVEC stimulated with PMA, forskolin and TNF-alpha

33. Inhibition of MMP-2 Production by different garlic extracts (MOR-200; MES-200; PRI-200; PRI-0) (unstimulated HCAEC cells) RESULTS: - Inhibtion is stronger in HCAEC than in HUVEC cells - only slight differences between garlic extracts with different fertilization

34. Summary: DADS is able to inhibit the production of several MMPs and of TIMP-1 by endothelial cells HCAEC cells are more sensitive to DADS than HUVEC cells AM and SAC are less potent than DADS Garlic powders do not affect HUVEC cells but exert an inhibition of MMP production in HCAEC cells so far no significant influence of garlic fertilization Final conclusion: Garlic preparations may potentially influence the MMP-TIMP balance in certain endothelia

35. New Studies on Properties of different Garlic Varieties: 1.No significant influence on cholesterol biosynthesis (sometimes only slight stimulation) (Studies on garlic from the core collection of Rina) 2.No detectable radical scavenging activity 3.Interesting anti- and pro-oxidant properties in hepatocytes exposed to t-butylhydroperoxide Preliminary results:

36. Influence of different garlic varieties on malon dialdehyde production:

37. Classification of different Garlic Varieties: 1 Anti = 0 << Pro(IX-45-00, IX-52-00, IX-97-00, IX-124-00, IX-158-00, IX-229-00) GroupPropertiesVarieties 2 Anti = 0 = Pro(IX-41-00, IX-107-00, IX-129-00) 3 Anti > 0 < Pro(IX-29-00, IX-32-00, IX-227-00) 4 Anti > 0 ~ Pro(IX-35-00, IX-95-00, IX-100-00, IX-102-00, IX-113-00, IX-144-00, IX-147-00) 5 Anti >> 0 = Pro(IX-50-00, IX-58-00, IX-123-00, IX-127-00)

38. Lack of correlation between alliin content and MDA production in different garlic varieties.

39. Deliverables: 1.DH2: Short report on properties and effects of garlic flavonoids 2.DH6: Publication on the molecular interaction of garlic compounds on AMP-dependent kinase pathway 3.DH7: Influence of garlic constituents on MMP and TIMP gene expression 4.DH11: Interference of garlic organosulfur compounds and flavonoids on signal transduction 5.DH12: Influence of different garlic feeding on markers for tissue remodeling - w P14 6.DH18: Effect of garlic compounds on several parameters in the ApoE mouse (Leiden) - w P14 cancelled

40. Many Thanks to: Katrin Meyer Sanja Pavlica Ludwig Nissler Dr. Sebastian Zellmer Dr. Gayane Buniatian Doris Kellert Frank Struck Chris Kik All Partners from the Plant Part and the Health Part