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Rolf Gebhardt

Garlic & Health: WP 5.2 Effect of Garlic on Cholesterol Biosynthesis. Rolf Gebhardt. University of Leipzig Medical Fakulty Institute of Biochemistry. Topics:. 1. Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis - Sulfur fertilization - Environmental influences.

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Rolf Gebhardt

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  1. Garlic & Health: WP 5.2 Effect of Garlic on Cholesterol Biosynthesis Rolf Gebhardt University of Leipzig Medical Fakulty Institute of Biochemistry

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

  3. Methods: 1. Use of human HepG2 cells or primary cultures of rat hepatocytes 2. Determination of HMGCoA reductase activity 3. Incorporation of radiolabeled acetate into steroids, mainly cholesterol (> 90%) 4. Studies on HMGCoA reductase regulation (phosphorylation of model peptides)

  4. 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

  5. 160 140 120 100 Hepatoc. 80 Cholesterol Biosynthesis (%) 60 40 HepG2 20 fresh Garlic 1 0 0.01 0.1 1 Concentration (mg/ml) Garlic Powder: reinvestigation Garlic Powder: expected influence Inhibition of cholesterol biosynthesis

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

  7. 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

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

  9. Qalitatively similar results in both years Influence of fertilization on garlic properties Influence of fertilization: comparison of French harvest 2000 - 2001

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

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

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

  13. Topics: 1. Differential Effects of Garlic Extracts on Cholesterol-Biosynthesis - Environmental influences 2. Influence of Garlic Organosulfur Compounds: - Mechanistic studies 3. Consequences and Conclusions

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

  15. Inhibition of HMGCoA reductase and cholesterol biosynthesis Test Compound EC50 values HMGCoA Cholesterol reductase biosynthesis 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

  16. Regulation of HMGCoA Reductase Mechanism Regulators Transcription: SREBP-1,-2 Insulin, Cholesterol, LXR Oxysterols Protein degradation Mevalonate, Calpain-Inh. Protein-Phosphorylation AMP-dependent kinase AMP, low energy charge Protein kinase C Hormones Calmodulin-dep. kinase Calcium, hormones

  17. 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

  18. Effect of fructose on hepatocellular ATP level Fructose

  19. Effect of fructose and DADS on AMP-dep. kinase fructose + DADS fructose

  20. Effect of fructose and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg) (%) (%) Control 0.34 100 100 Fructose 5.78 523 54 Diallyl disulfide 0.50 157 92 Fructose + DADS 4.85 686 25 Fruct. + DADS + araA 4.56 194 93

  21. Effect of hypoxia, KCl and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP AMPK Cholesterol biosynthesis (nmoles/mg) (%) (%) Control 0.41 100 100 low oxygen 4.75 408 72 low oxygen + DADS 4.59 566 59 KCl 2.58 346 79 KCl + DADS 2.73 424 63 KCl + DADS + araA 2.46 170 89

  22. 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)

  23. 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

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

  25. Basic properties of the indirect influence on HMGCoA reductase: 1. Regulation in harmony with cellular requirements: i.e. in cases of dysregulation 2. Increased sensitivity only when needed 3. No complete shut-off of cholesterol biosynthesis (no loss of important intermediates) 4. Restricted to organs with high AMPK activity: (liver, heart, muscle - no hormone secreting glands)

  26. Summary: 1. Sulfur fertilisation seems to influence the potential of garlic extracts to inhibit or stimulate hepatic cholesterol-biosynthesis 2. The nature of stimulatory compounds is still unknown 3. Garlic organosulfur compounds may sensitively inhibit cholesterol biosynthesis under stress-like conditions

  27. Flavonoids in Garlic: Literature Results Starke & Hermann, 1976 only trace amounts of quercetin and Michahelles, 1974 kaempferol in peeled garlic Kaneta et al., 1980 Leighton et al., 1992 Carotenuto et al., 1996 Kaempferol-glycosides in Allium ursinum (no quantitative data) Miean & Mohamed, 2001 detailed HPLC analysis, but poorly described plant material Myricetin 693 mg/kg dry weight Quercetin 47 Luteolin not detectable Apigenin 217 University of Leipzig

  28. Structure of Favonoids used 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

  29. 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

  30. 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 University of Leipzig

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

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