Rolf Gebhardt

University of Leipzig

Medical Faculty

Institute of Biochemistry

Garlic & Health: WP 5.2Effect of Garlic Compounds

on Cellular Signalling

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 ofseveral garlic-derived organosulfur compounds

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

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

Correlation between AMPK activity andinhibition 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 ofhepatocytes towards DADS

Lowering of the cellular energy charge

- increases the sensi- tivity 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

AMPK

AMPKK PP2A

AMP

Nutrients StressExercise

Glucosetransport

ß-Oxidationof fatty acids

Fatty acidsynthesis

Cholesterolsynthesis Apoptosis

Transcription

Creatinekinase

SignallingRAF-1

?

(after Kemp et al. TIBS 2000;24:22-25)

Central Role of AMP-dependent kinase (AMPK) as amediator of stress signals, e.g. low energy charge

Energy consuming pathways e.g. fatty acid andcholesterol synthesis are shut down

Subunit structures of AMPK

P

P

Catalyticdomain

Autoregulatorydomain

Subunit-targetingdomain

AMPK-a1

AMPK-a2

P P P

P

AMPK-b1

AMPK-b2

CßSCßSCßSCßS

CßSCßSCßSCßS

CßS CßSCßSCßS

AMPK-g1

AMPK-g2

AMPK-g3

(according to Kemp et al., TIBS 1999;24:22-25)

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 signallingbiogenesis of mitochondriaAMPK 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)

Mediation by AMPK of chronic energy depletion-induced mitochondrial biogenesis

GPA

ATP / AMP

+

CaMK IV PGC-1a

mitochondrial biogenesis

+

AMPK

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

Con DADS GPA GPA+DADS0

20

40

60

80

100

120

140

160

Abs

orba

nce

(%)

Condition

a

a,b

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

Con DADS GPA GPA+DADS0

20

40

60

80

100

120

140

160

180

200

220

Den

sity

(%

)

Condition

a

a,bGPA and DADS

- synergistically increase mitochondrial cyto- chrome c content

Effects of antidiabetic drugs

Rosiglitazone

MetforminInsulin sensitivity

blood glucose level

- peripheral glucoseuptake-fatty acid oxidation-fatty acid esterification- hepatic glucoseproduction

Role of AMPK as a mediator of antidiabetic drugs

Rosiglitazoneor DNP

ATP / AMP +

+

AMPK

antidiabetic effects

Metforminor hyperosmolarstress

+

+

?

Phosphorylationat Threonine 172

Influence of insulin and DADS on glucose production in normal rat hepatocytes

Insulin:

- lowers glucose production

DADS:

- has no influence in normal rats

Con DADS Insulin Ins+DADS fa-/fa-0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

300

320

340

a

Glu

cose

pro

duct

ion

(%

)

Condition

a

a

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

Con DADS Insulin Ins+DADS Sprague-D0

20

40

60

80

100

120

a

Glu

cose

pro

duct

ion

(%)

Condition

a

a

a,b

a

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:

Summary and Questions:

1. DADS seems to activate AMPK under conditionsassociated with high levels of AMP (hypoxia, high fructose, various drugs)

3. Do garlic organosulfur compounds act on AMPK also by influencing phosphorylationof this enzyme?

2. Which garlic organosulfur compounds besidesDADS have a similar effect on AMPK activity?