1. Rolf Gebhardt University of Leipzig Medical Faculty Institute of Biochemistry Garlic & Health: WP 5.2 Effect of Garlic Compounds on Cellular Signalling

2. Topics: 1.Interference of Garlic Compounds with Cholesterol-Biosynthesis -Role of AMPK 2.Structure and Function of AMPK -Interaction with other signalling pathways 3.New Results on the Effects of DADS and Conclusions

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

4. 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

5. 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

6. 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)

7. 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

8. Topics: 1.Interference of Garlic Compounds with Cholesterol-Biosynthesis -Role of AMPK 2.Structure and Function of AMPK -Interaction with other signalling pathways 3.New Results on the Effects of DADS and Conclusions

9. AMPK AMPKK PP2A AMP Nutrients Stress Exercise Glucose transport ß-Oxidation of fatty acids Fatty acid synthesis Cholesterol synthesis Apoptosis Transcription Creatine kinase Signalling RAF-1 ? (after Kemp et al. TIBS 2000;24:22-25) 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

10. Subunit structures of AMPK P P Catalytic domain Autoregulatory domain Subunit-targeting domain AMPK-a1 AMPK-a2 PPP P AMPK-b1 AMPK-b2 CßS AMPK-g1 AMPK-g2 AMPK-g3 (according to Kemp et al., TIBS 1999;24:22-25)

11. Basic properties of AMPK: 2. Regulated by a) the AMP-level (AMP/ATP ratio) b) various phosphorylation events 3. Present not only in the cytosol but also in the nucleus (--> may participate in controlling of transcription factors) 4. Participates in a variety of signalling events: e.g. leptin signalling biogenesis of mitochondria AMPK knockouts are diabetic! 1. Acts as a „fuel gauge“ in every cell and tissue (organs with high AMPK activity: liver, heart, muscle - lowest in hormone secreting glands)

12. Mediation by AMPK of chronic energy depletion-induced mitochondrial biogenesis GPA ATP / AMP + CaMK IV PGC-1a mitochondrial biogenesis + AMPK

13. Influence of ß-guanidinopropionic acid (GPA) and DADS on mitochondrial MTT reduction in muscle cells Lowering of the cellular energy charge - increases mitochon- drial MTT-reducing capacity

14. Influence of ß-guanidinopropionic acid (GPA) and DADS on mitochondrial cytochrome c content in muscle cells Lowering of the cellular energy charge - increases the sensi- tivity towards DADS GPA and DADS - synergistically increase mitochondrial cyto- chrome c content

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

16. 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

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

18. 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

19. 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:

20. Summary and Questions: 1.DADS seems to activate AMPK under conditions associated with high levels of AMP (hypoxia, high fructose, various drugs) 3.Do garlic organosulfur compounds act on AMPK also by influencing phosphorylation of this enzyme? 2.Which garlic organosulfur compounds besides DADS have a similar effect on AMPK activity?