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Rolf Gebhardt

Garlic & Health: WP 5.2 Effect of Garlic Compounds on Cellular Signalling. Rolf Gebhardt. University of Leipzig Medical Faculty Institute of Biochemistry. Topics:. 1. Interference of Garlic Compounds with Cholesterol-Biosynthesis - Role of AMPK. 2. Structure and Function of AMPK

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Rolf Gebhardt

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

  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) (%) (%) 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

  5. 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

  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. Signalling ? RAF-1 ß-Oxidation Energy consuming pathways e.g. fatty acid and cholesterol synthesis are shut down of fatty acids Glucose transport Creatine Fatty acid kinase synthesis Cholesterol Transcription Apoptosis synthesis Central Role of AMP-dependent kinase (AMPK) as a mediator of stress signals, e.g. low energy charge (after Kemp et al. TIBS 2000;24:22-25) Stress Nutrients Exercise AMP PP2A AMPKK AMPK

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

  11. Basic properties of AMPK: 1. Acts as a „fuel gauge“ in every cell and tissue (organs with high AMPK activity: liver, heart, muscle - lowest in hormone secreting glands) 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!

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

  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 GPA and DADS - synergistically increase mitochondrial cyto- chrome c content Lowering of the cellular energy charge - increases the sensi- tivity towards DADS

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

  16. Rosiglitazone or DNP + + + ATP / AMP + AMPK ? Metformin or hyperosmolar stress antidiabetic effects Role of AMPK as a mediator of antidiabetic drugs 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. Question: Does DADS increase sensitivity of diabetic hepatocytes for insulin? Conclusion: 1. DADS influences glucose production in diabetic rats, but not in normal rats 2. DADS acts only in synergy with insulin

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

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