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

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

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)

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

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 fertilization: comparison of French harvest Qalitatively similar results in both years

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

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

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

Regulation of HMGCoA Reductase Mechanism Regulators Transcription: SREBP-1,-2Insulin, Cholesterol, LXROxysterols Protein degradationMevalonate, Calpain-Inh. Protein-Phosphorylation AMP-dependent kinaseAMP, low energy charge Protein kinase CHormones Calmodulin-dep. kinaseCalcium, hormones

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

Effect of fructose on hepatocellular ATP level Fructose

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

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

Basic properties of the indirect influence on HMGCoA reductase: 1. Regulation in harmony with cellular requirements: i.e. in cases of dysregulation 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) 2. Increased sensitivity only when needed

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

Flavonoids in Garlic: University of Leipzig Literature Results Starke & Hermann, 1976only trace amounts of quercetin and Michahelles, 1974kaempferol in peeled garlic Kaneta et al., 1980 Leighton et al., 1992 Carotenuto et al., 1996Kaempferol-glycosides in Allium ursinum (no quantitative data) Miean & Mohamed, 2001detailed HPLC analysis, but poorly described plant material Myricetin693 mg/kg dry weight Quercetin 47 Luteolin not detectable Apigenin217

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

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