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

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

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 inhibition Inhibiting properties may be reinforced

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

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

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

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

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 sensi- tivity towards DADS 5- to 10-fold

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

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?

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

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

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

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

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

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:

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

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:

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

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

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

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

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

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:

Influence of different garlic varieties on malon dialdehyde production:

Classification of different Garlic Varieties: 1 Anti = 0 << Pro(IX-45-00, IX-52-00, IX-97-00, IX , IX , IX ) GroupPropertiesVarieties 2 Anti = 0 = Pro(IX-41-00, IX , IX ) 3 Anti > 0 < Pro(IX-29-00, IX-32-00, IX ) 4 Anti > 0 ~ Pro(IX-35-00, IX-95-00, IX , IX , IX , IX , IX ) 5 Anti >> 0 = Pro(IX-50-00, IX-58-00, IX , IX )

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

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

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