1

Sensitive In vitro Inhibition of Cholesterol Biosynthesis by Diallyl disulfide and Related Garlic Compounds

Rolf Gebhardt, Institute of Biochemistry, Medical Faculty, University of Leipzig, D-04103 Leipzig, Germany

Running title:

Sensitive In vitro Inhibition of Cholesterol

Correspondending author:

Prof. Dr. Rolf Gebhardt Institute of Biochemistry Medical Faculty University of Leipzig Liebigstr. 16 D-04103 Leipzig Germany Tel.: Fax: +49 341 9722101 +49 341 9722109

Email: rgebhardt@medizin.uni-leipzig.de

ABSTRACT The efficiency of several garlic-derived compounds to reduce cholesterol

3 biosynthesis in cultured hepatocytes was compared. Allicin, dially disulfide (DADS) and allyl mercaptan were found to directly inhibit HMGCoA reductase activity only at high concentrations. At low concentrations, these compounds seemed to inhibit cellular cholesterol biosynthesis via an indirect mechanism. Using peptide phosphorylation assays, evidence was collected that this indirect effect involves enhanced phosphorylation of HMGCoA reductase through AMP-activated kinase. In the case of DADS, sensitivity of this inhibition increased with increasing cytoplasmic levels of AMP as induced by incubation with fructose, hypoxia or exposure to KCN. In the case of high fructose (20 mM), the the EC 50-values of DADS for stimulating AMPK activity and inhibiting cholesterol biosynthesis were approx. 2.1 and 3.5 M, respectively. Adenine 9--D-arabinofuranoside, an inhibitor of AMPK, counteracted the synergistic interplay between DADS and AMP. As derived from structural studies, an allyl group combined with a disulfide bond or a free SH group seem necessary for these effects. These findings may have considerable implications on how garlic might influence (hepato)cellular cholesterol biosynthesis as AMPK activation is associated with various types of cellular stress.

Garlic (Allium sativum L.) has received considerable attention from traditional medicine in many cultures for thousands of years. Since their discovery, garlicderived organosulfur compounds have been claimed responsible for many biological functions ascribed to garlic (Koch and Lawson, 1996). Various studies in vivo as well as in vitro have dealt with the influence of these compounds on plasma lipid levels and on cellular cholesterol biosynthesis (for review see Koch and Lawson, 1996; Gebhardt, 1997; Yeh and Liu, 2001). In most studies the concentrations reported to affect hepatocellular cholesterol biosynthesis appeared very high in the

4 upper micromolar or even in the lower millimolar range (Sendl et al., 1992; Liu and Yeh, 2000) rising some doubt as to the physiological relevance of these findings. Sometimes, high concentration of the compounds resulted in stimulation rather than inhibition (Sendl et al., 1992). Detailed comparative analysis, however, revealed that different garlic-derived organosulfur compounds affect this pathway at various steps and at considerably different concentrations (Gebhardt and Beck, 1996; Sendl et al., 1992; Gebhardt et al., 1994; Yeh and Yeh, 1994; Liu and Yeh, 2000). For instance, it was shown that allicin and to some extent DADS can inhibit lanosterol 14- demethylase leading to the accumulation of lanosterol at the expense of other sterols, particularly cholesterol (Gebhardt and Beck, 1996). Another target in the downstream pathway for cholesterol biosynthesis recently found to be affected was squalene monooxygenase (Gupta and Porter, 2002) which seemed to be inhibited through reactions with sulfhydryl groups. In both cases EC 50-values varied over a wide range (65 M to 400 M) for different organosulfur and -seleno compounds (Gebhardt and Beck, 1996; Gupta and Porter, 2002). A similar mechanism as for squalene monoxygenase was proposed for the inactivation of HMGCoA reductase by DADS at concentrations around 100-200 M (Kumar et al., 1991). In contrast, allicin and DADS were reported to inhibit an early step in the biosynthetic pathway (most likely HMGCoA reductase) at lower concentrations (Gebhardt and Beck, 1996), particularly when incubated in the presence of palmitate (Gebhardt, 1995).

HMGCoA reductase can be regulated at various levels including transcription, translation, phosphorylation/ dephosphorylation and degradation of the enzyme (Goldstein and Brown, 1990). Inactivation by phosphorylation is carried out by AMPactivated kinase (AMPK), an enzyme activated in many cases of cellular stress

5 associated with an increase in AMP levels (Kemp et al., 1999; Hardie et al., 1998). Since palmitate via palmitoyl-S-CoA and AMPK-kinase also activates AMPK (Hardie et al., 1998), the possibility emerged that HMGCoA reductase might be indirectly inhibited by the synergism between DADS and palmitate (Gebhardt, 1995) through enhanced phosphorylation by AMPK. In the present study we have tried to address these questions and to focus on possible sensitive mechanisms as they appear more relevant under physiological conditions. In particular, the possibility of phosphorylation of HMGCoA reductase was investigated using various culture conditions leading to elevated intracellular concentrations of AMP. Likewise, it was our intention to compare preexisting organosulfur compounds of garlic as well as early and long lasting metabolites with respect to their possible influence on these mechanisms.

MATERIALS AND METHODS

Materials. Diallyl disulfide (DADS), di-n-propyl disulfide, and allyl mercaptane (AM) were purchased from Aldrich (Steinheim, Germany). S-allyl cystein was a kind gift of Dr. J. Auger (Tour, France) and allicin was provided by Prof. K.G. Wagner (Braunschweig, Germany). The purity of the compounds was checked by HPLC. The SAMS peptide (Davies et al., 1989) was kindly provided by Dr. H. Echner (University of Tbingen, Germany)

Isolation and cultivation of rat hepatocytes. Rat hepatocytes were isolated from male Sprague-Dawley rats (220-300 g) according to the two-step collagenase

6 technique described (Gebhardt et al., 1990). After inocculation in Williams Medium E supplemented with 10% newborne calf serum, cells were maintained in serum-free medium as described in detail elsewhere (Gebhardt et al., 1990; Gebhardt and Beck, 1996). Cultures were kept in a humidified incubator in an atmosphere of 95% air and 5 % CO2 at 37C.

Incubation of rat hepatocytes with test material. Confluent cultures were used for all experiments. The culture medium was removed and fresh medium was added containing the compounds to be tested in the appropriate dilutions and trace amounts of [14C]acetate (18.5 KBq/mL (0.5 Ci/mL)) (Gebhardt, 1993). After the incubation period indicated in the legend to figures and tables (up to 2 h), the medium was removed and the cell-layer was washed twice with saline and further processed as described (Gebhardt and Beck, 1996; Gebhardt, 1993). For some experiments hypoxic conditions were used during the incubation period. For this purpose medium was saturated with nitrogen before added to the cultures which were subsequently incubated in a gas-tight chamber under an atmosphere of 95% nitrogen and 5% CO2. A block of oxidative phosphorylation was exerted by adding 15 mM KCN to the culture medium.

Determination of acetate incorporation into cholesterol. The incorporation of [14C]acetate into non-saponifiable neutral lipids was determined after saponification of the cell homogenates with 0.5 M KOH in EtOH and subsequent efficient separation on Extrelut7-columns (large-pore kieselgur) as described elsewhere (Gebhardt, 1993; Gebhardt, 1991; Gebhardt, 1998). Thereafter, the eluate was used for scintillation counting (Gebhardt and Beck, 1996). The sterol composition was

7 analysed by silver-ion thin-layer chromatography or HPLC as described (Gebhardt and Beck, 1996; Gebhardt, 1993) to assure that cholesterol accounted for more than 90% of the incorporated radioactivity.

Measurement of AMPK activity. AMPK activity was measured using the SAMS peptide following the procedure of Davies et al., 1989. Briefly, cell homogenates prepared by scraping at least 7.5 x 10 6 cells in 5 x homogenization buffer followed by sonication were subjected to precipitation with poly(ethylene glycol) 6000 at 2.5% and 6% (mass./vol.) according to Moore et al., 1991, for enrichment of the enzyme. After resuspension in 1 x homogenization buffer aliquots were used for measurements in the synthetic peptide assay.

Enzyme Measurements and other Methods.

Determination of the activity of

HMGCoA reductase was performed as described by Gebhardt, 1991. For determination of cellular ATP and AMP levels, hepatocyte cultures were shockfrozen in liquid nitrogen and stored at -80 C until assayed. Immediately prior to the assay, perchloric acid (55% v/v) was added to the plates (approx. 500 l for 105 cells). Cells were scraped form the plates, sonicated and centrifuged in a microfuge at 10000g for 10 min. The supernatant was neutralized with 2 M KOH, kept on ice for 1 h and centrifuged again. ATP and AMP were determined enzymatically [19a]. Protein content in the cell homogenate was determined according to Lowry et al., 1951.

RESULTS The influence of several garlic-derived organosulfur compounds, namely allicin, diallyl disulfide (DADS), allyl mercaptan (AM) and S-allyl cysteine, on the activity of HMGCoA reductase, one of the key enzymes of hepatic cholesterol biosynthesis, and on total cellular cholesterol biosynthesis was compared in cultured rat hepatocytes. In addition, some structurally related compounds (diallyl sulfide and dipropyl disulfide) were tested. Direct inhibition of enriched HMGCoA reductase activity (determined by adding the compounds during the assay) occurred only at very high concentrations of these compounds (table 1). On the other hand, acetate incorporation into cholesterol (determined within a 2 h incubation period) was inhibited at much lower concentrations by all garlic-derived compounds except Sallyl cysteine. For allicin and DADS, EC 50-values were determined to 17 and 65 M, respectively. Together with the fact that exchange of mevalonate for acetate as a precursor completely omitted inhibition of cholesterol biosynthesis (Gebhardt and Beck, 1996) these results point to a time-dependent indirect inhibition of HMGCoA reductase by allicin, DADS and AM. The related compounds diallyl sulfide and dipropyl disulfide did not exert a similar influence on cholesterol biosynthesis (table 1). In order to investigate whether phosphorylation of HMGCoA reductase by AMPdependent kinase might be involved in the indirect inhibition exerted by DADS, the cellular level of AMP was modulated by incubating the hepatocytes with high concentrations of fructose (20 mM). As shown in fig. 1 this resulted in a rapid but transient loss of ATP associated with an increase in AMP (table 2). Consequently, the activity of AMPK measured directly using the SAMS peptide was upregulated

9 under these conditions (fig. 2). In agreement with published results [Moore et al., 1991] exposure of the cultured hepatocytes to 20 mM fructose also resulted in a marked inhibition of cholesterol biosynthesis (table 2). DADS neither influenced cellular AMP levels (table 2) nor the activity of AMPK (fig. 2). However, DADS at concentrations that barely affected cholesterol biosynthesis when present alone, synergistically enhanced the inhibition caused by fructose (table 2). Coincubation of fructose with DADS also increased AMPK activity (fig. 2), while the AMP concentration was not further elevated at 5 min (table 2) or at later times (not shown). Obviously, the synergistic effect of fructose and DADS on AMPK activity lead to a prolongation of AMPK stimulation (fig. 2). Inhibtion of AMPK by adenine 9--D-arabinofuranoside, a precursor of ara-ATP, counteracted this synergistic interaction (table 2). These results suggest that DADS most likely sensitized AMPK towards AMP by an as yet unknown mechanism. Careful analysis of the concentration-dependence of this effect on AMPK activity and cholesterol biosynthesis revealed that the sensitivity of both parameters towards DADS was considerably increased in the presence of fructose (fig. 3A, B). Thus, the EC50-values of DADS for stimulating AMPK activity and inhibiting cholesterol biosynthesis were approx. 2.1 and 3.5 M, respectively. This means a more than 18-fold reduction of the EC 50-value (cf. Table 1). At rather high concentrations (>500 M) DADS decreased the activity of AMPK (not shown). Whether this is due to direct inhibition of this protein kinase is not known. Other conditions resulting in an increase of cellular AMP like transient hypoxia and inhibition of oxidative phosphorylation also gave rise to an increased sensitivity towards DADS. Exchange of the culture medium by medium that was saturated with nitrogen and incubation of the cells under an oxygen-free atmosphere for 2 h led to

10 an increase in cellular AMP and AMPK activity associated with a decreased cholesterol biosynthesis (table 3). Addition of DADS reinforced the stimulation of AMPK and further decreased cholesterol biosynthesis. The effect of hypoxia was reversible when hepatocyte cultures were subsequently maintained under the normal atmosphere of 95% air and 5% CO 2 indicating that downregulation of cholesterol biosynthesis was not due to cell damage (not shown). A similar synergistic effect was observed when cells were incubated with KCN with and without the addition of DADS (table 3). Cell viability under these conditions was assured by staining with Trypan blue and the MTT assay for the whole incubation period (not shown). Again, the influence of DADS was counteracted by adenine 9-D-arabinofuranoside (table 3). Plotting cholesterol biosynthesis against AMPK activity determined under the various conditions mentioned above revealed a strong negative correlation between these parameters (fig. 4). This further supports the view that modulation of AMPK activity is indeed the relevant step by which DADS is influencing cholesterol biosynthesis.

Allicin and AM seemed to react similarly to DADS. However, the synergistic effect was significant even at lower concentrations (down to 1 M) in the case of allicin (not shown). AM was much less effective and the synergistic effect occurred at higher concentrations (EC50-value approx. 82 M). Neither diallyl sulfide nor dipropyl disulfide exerted any synergistic influence.

DISCUSSION

The results presented herein provide information relevant for several aspects of a possible influence of garlic organosulfur compounds on cholesterol biosynhesis. First, our findings clearly demonstrate that direct inhibition of HMGCoA reductase by such compounds occurs only at high concentrations (EC 50 values >> 250 M) rendering such an influence rather unlikely under conditions of normal garlic consumption. On the other hand, some of the garlic-derived organosulfur compounds inhibit cholesterol biosynthesis at much lower concentrations than might be expected from their direct inhibition of isolated HMGCoA reductase. Obviously, the structural elements required for a potent action seem to be (a) an allyl group, since a propyl group is inactive, and (b) a difulfide bond or, less active, a free SHgroup. Thus, diallyl sulfide which does not met condition (b) is almost inactive. Likewise, S-allyl cysteine, a genuine garlic compound that has been claimed to influence hepatic cholesterol biosynthesis (Yeh and Liu, 2001), is at least an order of magnitude less potent than, for instance, DADS. Second, the sensitive inhibition exerted by DADS, allicin and, to some extent, AM

appears the result of an activation of AMPK presumably a sensitization against AMP. This is particularly obvious under conditions associated with an elevation of intracellular AMP levels such as exposure to fructose, hypoxia and inhibition of oxidative phosphorylation. AMPK activation has been shown in many studies to provide a means for a coordinated down-regulation of HMGCoA reductase and acetyl-CoA carboxylase by reversible phosphorylation (Carling et al., 1987; Moore et al., 1991; Witters et al., 1994; Munday and Hemmingway, 1999). This mechanism is activated by cellular stress leading to a breakdown of ATP and helps the cells to shut down energy-consuming pathways in order to favour recovery (Sato et al., 1993; Hardie et al., 1998; Kemp et al., 1999). As suggested by our data such stress which rises intracellular AMP levels is necessary for DADS and some related compounds to potently inhibit cholesterol biosynthesis by synergistically enhancing AMPK activity. Obviously, the same structural features of these molecules as outlined above seem to be required for this action. The molecular mechanism of the sensitization, however, remains to be established. Third, since cholesterol and fatty acid biosynthesis are likewise subject to regulation by AMPK, specific garlic compounds render possible the coordinated modulation of cholesterol and lipid synthesis to harmonize with general requirements of the body. This may concern both, early (allicin) or lte metabolites (DADS, AM). In particular, this mechanism may prevent complete shut-off of these pathways in response to garlic compounds which is of considerable importance for vital functions such as synthesis of steroid hormones and terpene intermediates (Gebhardt, 1997). In this respect it is interesting that AMPK shows highest expression and activities in skeletal muscle, liver, kidney, brain, mammmary gland, but less in adrenal gland and no expression in testis [19,30]. Furthemore, since the

phosphorylation/dephosphorylation cycle of HMGCoA reductase shows a diurnal variation (Davies et al., 1992), the influence of garlic material may also vary in a time-dependent manner.

The results of this study have important implications for garlic =s ability to influence blood lipid levels. Unless high concentrations of potent garlic-compounds come into play (which seems not to be the case during normal consumption of garlic products), garlic can only reinforce not counteract the body =s own regulation of cholesterol biosynthesis. As a consequence, garlic therapy may be helpful mainly in moderate pathological situations when normal regulatory mechanisms of cholesterol biosynthesis are not functioning correctly or show thresholds deviating from normal. This fact may be one reason why inconsistent results were obtained in clinical studies (Orekhov and Grunwald, 1997; Steiner et al., 1996; Neil et al., 1996; Bordia et al., (1998; Berthold et al., 1998; Isaacsohn et al., 1998; Superko and Krauss, 2000). Until now, the described mechanisms and conditions that may affect the action of garlic compounds have not yet adequately considered in the design of such studies. Additionally, intensive studies seem necessary to establish whether the regulatory details described herein for rat hepatocytes are also valid for human cells. Preliminary results with HepG2 cells argue in favor of this assumption (unpublished observation).

Acknowledgements The author would like to thank Mrs. A. Hanika and F. Struck for excellent

technical assistence. These studies were supported in part by the European Commission (grant No. QLK-CT-1999-0498).

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TABLE 1 Comparison of the efficiency of several garlic-derived and related organosulfur compounds concerning inhibition of enriched HMGCoA reductase and cholesterol biosynthesis in cultured rat hepatocytes.

Test compounds

EC50-valuesa

HMGCoA reductaseb

Cholesterol biosynthesisc

Allicin 300

>

17 2

Diallyl disulfide

> 250 65 7

Allyl mercaptan

> 1500 450 20

S-allyl cysteine

n.d. > 1500

Diallyl sulfide

> 1000 735 48

Dipropyl disulfide

> 1500 > 1000

Values were determined from dose-response studies that were

performed in triplicate. The concentration range was 1 to 2000 M for the test compounds, except for allicin (1000 M).
b

HMGCoA reductase was enriched and measured as described [17]. Cholesterol biosynthesis was determined from acetate incorporation into

cholesterol during a 2h incubation period as described in Methods.

TABLE 2 Effect of fructose and diallyl disulfide on AMP levels, AMP-activated protein kinase and cholesterol biosynthesis in cultured rat hepatocytes.

Condition

AMP (nmoles/mg protein)

level

AMPK activity (%) Cholesterol biosynthesis

Control 0.34 0.25a 100

100

Fructoseb 5.78 0.84*

523 71* 54 12*

Diallyl disulfidec 0.34

0.50

157 33 92 6

Fructose + Diallyl disulfide

4.85 0.94*

686 59*,+ 25 7*,+ Fructose + Diallyl disulfide + araAd 0.72* 194 26*,+ 93 5*,+ 4.56

Values represent means standard deviations obtained after 5 min (AMP level and AMPK activity) or 30 min

(cholesterol biosynthesis) in 3 independent experiments.

b

Concentration: 20 mM Concentration: 25 M Concentration: 500 M

* Significantly different from controls, P < 0.01

+

Sigificantly different from fructose alone, P < 0.01

TABLE 3 Effect of hypoxia and diallyl disulfide on AMP levels, AMP-activated protein kinase and cholesterol biosynthesis in cultured rat hepatocytes.

Condition

DADSa

AMP

level (moles/g protein) AMPK activity (%) Cholesterol

biosynthesis (%)

Control

0.41 0.22b 100

100

low oxygenc

4.75 0.61*

408 49* 72 17*

low oxygenc

+ 4.59 0.68*

566 60* 59 11*

KCld

2.58 0.31 346 52 79 9

KCl

+ 2.73 0.33 424 41 63 15

KCl + araAe

+ 2.46 0.28*

170 27*,+ 89 8*,+

Concentration: 25 M Values represent means standard deviations obtained after 1 h in 3 independent experiments. incubation under nitrogen (see Methods) Concentration: 15 mM Concentration: 500 M

* Significantly different from controls, P < 0.01

+

Sigificantly different from fructose alone, P < 0.01

Legends to figures Fig. 1. Hepatocellular ATP content in response to incubation with

fructose. Hepatocytes were incubated for up to 120 min in the absence (open circles) or presence (closed circles) of 20 mM fructose. ATP and cellular protein were determined as described in Methods. Values represent means of duplicate determinations.

Fig. 2.

Effect of fructose and DADS on hepatocellular AMPK activity.

Hepatocytes were incubated with (closed symbols) and without (open symbols) 20 mM fructose in the absence (circles) or presence (squares) of 25 M DADS for the times indicated. After cell harvest, AMPK activity was measured with the SAMS peptide assay as described in Methods. Values represent means of triplicate determinations from a representative

culture. *, statistically different from values with fructose alone, P < 0.01.

Fig. 3. Concentration dependence of the effect of DADS on (A) AMPK activity and (B) acetate incorporation in the presence (closed circles) and absence (open circles) of 20 mM fructose. AMPK activity and acetate incorportaion were determined after 10 and 30 min, respectively. Values represent means SD of duplicate determinations from 3 different cultures. *, statistically different from values with fructose alone, P < 0.01.

Fig. 4. Correlation between AMPK activity and cholesterol biosynthesis under various experimental conditions. Values of both parameters determined under the conditions listed in Tables 2 and 3 in the absence (closed symbols) or presence (open symbols) of DADS are depicted. Values represent means SD from 3 different cultures. The correlation coefficient was determined as r = -0.96 (P < 0.001).

Fig. 1
20

ATP (nmoles/mg protein)

16

12

0 0 30 60 90 120

Time (min)

Fig. 2

15

AMPK (nmoles/min/mg protein)

* 12

* 9

0 0 10 20 30

Time (min)

Fig. 3 A

700 600 500 400 + Fruct.

* * *

AMPK activity (%)

200 - Fruct. 100 0 0.1 1 10

DADS concentration ( M)

B
inhibition of acetate incorporation (%) B
100

80 * 60 + Fruct. 40 *

20 - Fruct. 0 0.1 1 10

DADS concentration ( M)

Fig. 4

Cholesterol Biosynthesis (%)

100

80

60

40

r = -0.96

20

0 0 200 400 600 800

AMPK Activity (%)