Garlic Health WP 5 2 Effect of Garlic

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 Interaction with other signalling pathways 3. New Results on the Effects of DADS and Conclusions

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

Effect of fructose and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP (nmoles/mg) AMPK (%) Cholesterol biosynthesis (%) Control 0. 34 100 Fructose 5. 78 523 54 Diallyl disulfide 0. 50 157 92 Fructose + DADS 4. 85 686 25 Fruct. + DADS + ara. A 4. 56 194 93

Effect of hypoxia, KCl and DADS on AMP levels, AMP-dep. kinase and cholesterol biosynthesis Condition AMP (nmoles/mg) AMPK (%) Cholesterol biosynthesis (%) Control 0. 41 100 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 + ara. A 2. 46 170 89

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)

Influence of fructose, i. e. high AMP on the sensitivity of hepatocytes towards DADS Lowering of the cellular energy charge - increases the sensitivity towards DADS 5 - to 10 -fold

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

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 PP 2 A AMPKK Signalling RAF-1 Energy consuming pathways e. g. fatty acid and cholesterol synthesis are shut down ? AMPK ß-Oxidation of fatty acids Glucose transport Creatine kinase Fatty acid synthesis Cholesterol synthesis Apoptosis Transcription

Subunit structures of AMPK (according to Kemp et al. , TIBS 1999; 24: 22 -25) Autoregulatory domain Catalytic domain AMPK-a 1 Subunit-targeting domain P AMPK-a 2 AMPK-b 1 P P AMPK-b 2 P AMPK-g 1 CßS CßS AMPK-g 2 CßS CßS AMPK-g 3 CßS CßS

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!

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

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

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

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

Role of AMPK as a mediator of antidiabetic drugs Rosiglitazone or DNP + ATP / AMP + Phosphorylation at Threonine 172 AMPK + + Metformin or hyperosmolar stress ? antidiabetic effects

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 Question: Does DADS increase sensitivity of diabetic hepatocytes for insulin?

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?