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

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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

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

Cholesterol biosynthesis: 3.Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis -Sulfur fertilization -Environmental influences 1.Influence of Garlic Organosulfur Compounds: -Mechanistic studies 4.Consequences and Conclusions 2.Influence of flavonoids on cholesterol synthesis

Cholesterol Biosynthesis is highly regulated at various steps among others HMGCoA reductase is a key enzyme Many important biomolecules are derived from intermediates of cholesterol biosynthesis -->the biosynthetic pathway should never be completely inhibited

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

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

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

Effect of fructose and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg)(%) (%) Control Fructose Diallyl disulfide Fructose + DADS Fruct. + DADS + araA

Effect of hypoxia, KCl and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg)(%) (%) Control low oxygen low oxygen + DADS KCl KCl + DADS KCl + DADS + araA

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

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

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

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

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

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

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

Garlic Powder: reinvestigation Garlic Powder: expected influence Inhibition of cholesterol biosynthesis Concentration (mg/ml) Cholesterol Biosynthesis (%) Hepatoc. HepG2 fresh Garlic 1

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

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

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

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

Influence of fertilization on garlic properties Influence of high sulfur fertilization: French harvest 2001 Too much sulfur may reduce potency again

Environmental influence on garlic properties Comparison of French and Spanish harvest 2001 Only stimulation is found with spanish garlicToo much sulfur may reduce potency again

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

Properties of acetate uptake:

Influence of garlic on acetate uptake:

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?

Current extensions: 1.Studies on greenhouse garlic of Odile 2.Studies on garlic from the core collection of Rina 100% S- TS S++

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

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

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:

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

Garlic & Health: WP5 Cholesterol and cytokines University of Leipzig Aim:Interaction of garlic constitutents with vessel wall Inhibition of Matrix Metalloproteinase-2 Production by DADS, AM and SAC in HCA- endothelial cells

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