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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3064-3070

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3064-3070.)
© 1997 American Heart Association, Inc.

Articles

Cafestol, the Cholesterol-Raising Factor in Boiled Coffee, Suppresses Bile Acid Synthesis by Downregulation of Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase in Rat Hepatocytes

Sabine M. Post; Elly C. M. de Wit; ; Hans M. G. Princen.

From the Gaubius Laboratory, TNO-PG, Leiden, The Netherlands (S.M.P., E.C.M.deW., H.M.G.P.)

Correspondence to Dr. Hans M.G. Princen, Gaubius Laboratory, TNO-PG, Zernikedreef 9, 2333 CK, Leiden, The Netherlands, POBox 2215, 2301 CE, Leiden, The Netherlands. E-mail jmg.princen{at}pg.tno.nl


*    Abstract
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 *Abstract
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Abstract Consumption of boiled coffee raises serum cholesterol levels in humans. The diterpenes cafestol and kahweol in boiled coffee have been found to be responsible for the increase. To investigate the biochemical background of this effect, we studied the effects of cafestol and a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) on bile acid synthesis and cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase in cultured rat hepatocytes.

Dose-dependent decreases of bile acid mass production and cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase activity were found, showing a maximal reduction of -91%, -79%, and -49% respectively, at a concentration of 20 µg/mL cafestol. The decrease in 7{alpha}-hydroxylase and 27-hydroxylase activity paralleled well the suppression of the respective mRNAs, being -79% and -77%, and -49% and -46%, respectively, at 20 µg/mL cafestol. Run-on data showed a reduction in 7{alpha}-hydroxylase and 27-hydroxylase gene transcriptional activity after incubation with cafestol. The mixture of cafestol/kahweol/isokahweol was less potent in suppression of bile acid synthesis and cholesterol 7{alpha}-hydroxylase. Cafestol (20 µg/mL) had no effect on lithocholic acid 6ß-hydroxylase mRNA, another enzyme involved in bile acid synthesis. LDL-receptor, HMG-CoA reductase, and HMG-CoA synthase mRNAs were significantly decreased by cafestol (-18%, -20%, and -43%, respectively).

We conclude that cafestol suppresses bile acid synthesis by downregulation of cholesterol 7{alpha}-hydroxylase and of, to a lesser extent, sterol 27-hydroxylase in cultured rat hepatocytes, whereas kahweol and isokahweol are less active. We suggest that suppression of bile acid synthesis may provide an explanation for the cholesterol-raising effect of cafestol in humans.


Key Words: bile acid synthesis • cholesterol 7{alpha}-hydroxylase • sterol 27-hydroxylase • rat hepatocytes • cafestol


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowDiscussion
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In Scandinavia, coffee consumption is associated with elevated levels of serum cholesterol1,2 and an increased risk of coronary heart disease.3 Scandinavians often drink coffee that is prepared by boiling ground coffee beans directly with water, instead of coffee prepared by the commonly used coffee filter. The method of brewing is particularly important for the cholesterol-raising effect and for the increase in triglyceride levels. Consumption of boiled coffee is associated with a hypercholesterolemic effect, whereas filtered coffee does not increase serum cholesterol levels.4–6 The diterpenes cafestol and kahweol, which are removed on filtering, were found to be responsible for the cholesterol-raising effect of boiled coffee.7,8 However, the mechanism by which coffee diterpenes cause an increase in serum cholesterol and lipid levels is not well understood. A potential site of action is the liver, which plays a pivotal role in the homeostasis of cholesterol.

Removal of low-density-lipoproteins (LDL) from circulation by the liver is crucial in controling plasma concentrations of LDL cholesterol in humans. In normal subjects, more than 70% of this removal takes place via the LDL-receptor.9 The liver plays an important role in synthesis of cholesterol, and the major regulatory and rate-limiting enzyme in this process is 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Furthermore, the liver is the sole organ synthesizing bile acids, and conversion of cholesterol into bile acids is the major route for elimination of cholesterol from the mammalian body.10,11 Modulation of bile acid synthesis has been shown to have an effect on serum cholesterol levels. Interruption of the enterohepatic circulation of bile acids by administration of bile acid-binding resins lowers LDL levels and the risk of coronary heart disease in humans.12,13 On the other hand, a low bile acid synthetic capacity was found to be an independent risk factor for the incidence of coronary heart disease, and subnormal levels of bile acid synthesis could be correlated to progression of atherosclerosis and coronary mortality in patients heterozygous for familial hypercholesterolemia.14 Additionally, an increase in serum levels of LDL and a decrease in bile acid synthetic capacity occur parallel with ageing.15 Animal studies show that genetic factors may influence the responsiveness to dietary cholesterol, as evident from changes in plasma cholesterol concentration, and that this is closely related to the bile acid synthetic capacity.16,17 The rate of bile acid synthesis is therefore considered to be an important regulator of cholesterol homeostasis.

The primary route in bile acid biosynthesis in rats and humans is initiated by 7{alpha}-hydroxylation of cholesterol catalyzed by the major rate-limiting enzyme cholesterol 7{alpha}-hydroxylase, which is located in the smooth endoplasmic reticulum. This pathway leads predominantly to the formation of cholate and chenodeoxycholate.18–20 An alternative pathway in bile acid synthesis is operational as well, a pathway which contributes considerably to total bile acid synthesis in humans21 and in cultured human and rat hepatocytes.22,23 This latter pathway is initiated by the enzyme sterol 27-hydroxylase, which is located in the inner mitochondrial membrane, leading predominantly to the formation of chenodeoxycholate.21–26

The coffee diterpenes (Fig 1Down) bear structural resemblance to sterols.27 Since sterols, like oxysterols, can have an inhibitory effect on cholesterol 7{alpha}-hydroxylase,28,29 bile acid synthesis may be influenced by these compounds. In the light of the effect of the coffee diterpenes on cholesterol levels in humans, we studied the effect of these compounds on bile acid synthesis in cultured rat hepatocytes and investigated the mechanism of action.



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  		Figure 1. Structure of cafestol. Kahweol has an additional double bond between C1 and C2.

Our data indicate that cafestol inhibits bile acid synthesis by decreasing cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Materials and Animals
Materials used for isolation and culturing of rat hepatocytes, and assaying cholesterol 7{alpha}-hydroxylase were obtained from sources described previously.30–32 [{alpha}-32P]dCTP (3000 Ci/mmol), [{alpha}-32P]UTP (400 Ci/mmol) and [4-14C]-cholesterol (60 mCi/mol) were obtained from the Radiochemical Center, Amersham. Cafestol and a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) were kindly provided by Dr. Huggett of the Nestec Research Center.

Male Wistar rats weighing 250 to 350 g were used throughout and were maintained on standard chow and water ad libitum. Two days before isolation of hepatocytes, rats were fed a diet supplemented with 2% cholestyramine (Questran, Bristol Myers BV) unless otherwise stated. For preparation of hepatocytes, animals were killed between 9 and 10 AM. Institutional guidelines for animal care were observed in all experiments.

Rat Hepatocyte Isolation and Culture
Hepatocytes were isolated by perfusion with 0.05% collagenase and 0.005% trypsin inhibitor and cultured as described previously.30-32 After a 4-hour attachment period, the cell medium was refreshed with 1.0 mL (6-well plates) or 2.5 mL (dishes) of Williams E medium supplemented with 10% fetal calf serum (FCS), and cells were incubated for a further 14 hours. Coffee diterpenes, dissolved in DMSO, were added to the culture medium of the cells after this period, at between 18 to 42 hours of culture age, unless otherwise stated. The final concentration of DMSO in the medium was 0.1% (v/v). After a 42-hour culture period, cells were harvested at the same time for measurement of cellular lipid, cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase activity, and determination of mRNA levels and transcriptional activity. Cell viability, after culturing with the coffee diterpenes, was assessed by ATP measurements33 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl bromide (MTT) assays. This assay depends on the cellular reduction of MTT (Sigma Chemical Co), by the mitochondrial dehydrogenase of viable cells, to a blue formazan product, which can be measured spectrophotometrically. The assay was performed essentially as described by De Vries et al.34 Briefly described, this meant that, parallel with the various incubations, cells were cultured on 12-wells plates (5x105 cells/well) in 0.5 mL medium containing coffee diterpenes. At the end of the incubation period, 55 µL of MTT solution (5 mg MTT/mL PBS) were added to each well for 2 hours. The medium was aspirated, and 1 mL 100% DMSO was added to solubilize the formazan crystals. Absorbance at 545 nm was measured immediately.

Quantitation of Mass Production of Bile Acids
Mass production of bile acids by rat hepatocytes was measured by gas-liquid-chromatography after a preincubation period of 8 hours (from 18 to 26 hours of culture age), during the following 24 hours culture period from 26 to 50 hours in the absence or presence of coffee diterpenes as described previously.30

Assay of Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase Enzyme Activity
Cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase activity in homogenates of cultured rat hepatocytes were measured as described previously.22,23,32 Microsomes were isolated as described previously.31 [14C]-labeled products were analyzed by thin layer chromatography, and the amount of [14C]-7{alpha}-hydroxycholesterol and [14C]-27-hydroxycholesterol were quantitated by scraping off and counting the spots containing this product, using the [14C]-cholesterol input as a recovery standard. Blank values, determined by running parallel incubations without a NADPH-generating system, were subtracted before calculating enzyme activity.

RNA Isolation, Blotting and Hybridization Procedures
Isolation of total RNA, and subsequent electrophoresis, Northern-blotting and hybridization techniques were performed as described previously.23,30 The following DNA fragments were used as probes in hybridization experiments: a 1.6 kb PCR-synthesized fragment of rat cholesterol 7{alpha}-hydroxylase cDNA, spanning the entire coding region,30 a 1.6 kb HindIII/XbaI fragment of rat sterol 27-hydroxylase cDNA, kindly provided by J.F. Strauss III.35 A 0.7 kb EcoRI fragment of pFR29-3 containing the cDNA for hamster lithocholic acid 6ß-hydroxylase, kindly provided by G.Gil,36 a 773 bp HindIII fragment of hamster HMG-CoA reductase cDNA,37 a 435 bp Pst I fragment of hamster HMG-CoA synthase cDNA,38 and a rat LDL-receptor cDNA.39 As controls, a 1.2 kb Pst I fragment of hamster ß-actin cDNA and a 1.2 kb Pst I fragment of rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA were used. The actin or GAPDH mRNA was used as an internal standard to correct for differences in the amount of total RNA applied onto the gel or filter. mRNA levels were quantitated by Phosphorimager (Fuji Fujix BAS 1000) analysis.

Nuclear Run-On Studies
Nuclear run-on studies were conducted essentially as described by Twisk et al.40

Hybridization
Target DNA, being 5 µg of plasmid material containing cDNA sequences of rat cholesterol 7{alpha}-hydroxylase, rat sterol 27-hydroxylase, hamster actin, rat GAPDH (see above), and the empty vector pUC19 were slot-blotted onto strips of Hybond-N+ filter (Amersham) and cross linked with 0.4 N NaOH for 30 minutes. The filters were preincubated for 30 minutes at 65°C in a sodium phosphate buffer as described above, and hybridized with the labeled RNA for 36 hours in the same buffer. Labeled RNA was generated by incorporation of [32P]-UTP into nascent RNA, using isolated nuclei from cells that had been cultured with or without cafestol for 24 hours of culture time. After hybridization, the various filters were washed once for 5 minutes and twice for 30 minutes in 2xSSC/1% SDS at 65°C, and exposed to a Fuji imaging plate type BAS-MP for 3 to 5 days. Quantitation of relative amounts of transcribed mRNA was performed using a Phosphorimager BAS-reader (Fuji Fujix BAS 1000) and the computer programs BAS-reader version 2.8 and TINA version 2.08c.

Measurement of the Mass of Intracellular Triglycerides, Cholesterol, and Cholesteryl Esters
After a 24-hour incubation period, with or without coffee diterpenes, cells were washed three times with cold phosphate-buffered saline (pH 7.4). Thereafter, cells were harvested by scraping, and homogenized. Samples were taken for measurement of protein content. Lipids were extracted from the cell suspension as described by Bligh and Dyer,41 after addition of cholesterol acetate (2 µg per sample) as an internal standard. The neutral lipids were separated by high-performance, thin-layer chromatography on silica-gel-60 precoated plates as described.42 Quantitation of the amounts was done by scanning the plates with a Shimadzu CS910 chromatograph scanner at 380 nm, and areas under the curves were integrated by using a data processor (Shimadzu).

Statistical Analysis
Data were analyzed statistically using a paired Student's t test with the level of significance selected to be P<.05. Values are expressed as mean±SEM.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effect of Coffee Diterpenes on Bile Acid Synthesis
Bile acid mass production was measured over a 24-hour incubation period after a preincubation of 8 hours as described in "Methods." Incubation of the rat hepatocytes with cafestol or a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) alone resulted in a dose-dependent decline in bile acid mass production, showing a 91±5% and 68±3% inhibition, respectively at 20 µg/mL (Fig 2Down). The main bile acids formed were cholic acid and ß-muricholic acid in a ratio of approximately 20:80, which did not change after incubation with coffee diterpenes. The concentrations used in these experiments at 20 µg/mL of cafestol or the mixture did not have adverse effects on cell viability as shown by measurements of MTT (95±4% and 102±7%, respectively) and of ATP (93±11% and 110±12%, respectively). Data are expressed as percentage of control and are means ± SEM of independent experiments using hepatocytes from 3 to 4 rats.



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  		Figure 2. Effect of cafestol or a mixture of cafestol/kahweol/isokahweol on mass production of bile acids. After an 8-hour preincubation period (from 18 to 26 hours of culture), rat hepatocytes were cultured for 24 hours (between 26 to 50 hours of culture) in the presence or absence of different concentrations (0 to 20 µg/mL) of the diterpenes cafestol (circles) or a mixture (triangles). Values shown are expressed as percentage of bile acid synthesis in control incubations and are means (± SEM) of independent experiments with hepatocytes from 3 to 4 rats. Absolute synthesis rate in the absence of coffee diterpenes was 2.06±0.31 µg/24 hours/mg cell protein. A significant difference between control and treated cells is indicated by an asterisk (*P<.05; **P<.005; ***P<.001).

Effect of Coffee Diterpenes on Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase Activity and mRNA Levels
To assess the level at which coffee compounds decrease bile acid mass production, enzyme activity and mRNA levels of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase were determined. Rat hepatocytes were cultured in the presence or absence of cafestol or the mixture. Fig 3ADown shows that there is a dose-dependent decrease in cholesterol 7{alpha}-hydroxylase activity with a maximal suppression of -79±3% at a concentration of 20 µg/mL of cafestol. The decrease in cholesterol 7{alpha}-hydroxylase activity paralleled well the decrease in mRNA, being -77±4% at a concentration of 20 µg/mL of cafestol. The 7{alpha}-hydroxylase mRNA levels of cells incubated with different concentrations of the mixture also showed a significant decline, but to a lesser extent, with a maximal suppression of -31±8% at 20 µg/mL of the mixture (Fig 3BDown). This suggests that cafestol is the most potent compound and that kahweol and isokahweol are less active. The suppressing effect of cafestol on cholesterol 7{alpha}-hydroxylase mRNA was rapid and detectable after 4 hours of incubation (-43±9%) with 10 µg/mL of cafestol (data not shown). In addition to the effect of cafestol on cholesterol 7{alpha}-hydroxylase mRNA and enzyme activity after cell incubation, the compound also had a direct inhibitory effect when added in cholesterol 7{alpha}-hydroxylase enzyme activity assays. Dose-dependent decreases in cholesterol 7{alpha}-hydroxylase activity were found in homogenates of freshly isolated rat hepatocytes with a maximal suppression of -86±2% at a concentration of 20 µg/mL cafestol. This was in agreement with experiments using isolated rat liver microsomes (Table 1Down).



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  		Figure 3. Effect of cafestol or a mixture of cafestol/kahweol/isokahweol on cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase enzyme activity and mRNA levels. Rat hepatocytes were incubated for 24 hours, from 18 to 42 hours of culture, in presence or absence of different concentrations of cafestol (3a) or a mixture (3b) (0 to 20 µg/mL). Cells were harvested after 24 hours of incubation to measure cholesterol 7{alpha}-hydroxylase (closed symbols) and sterol 27-hydroxylase (open symbols) activity (triangles) and mRNA levels (circles). Values shown are expressed as percentage of enzyme activity or mRNA levels in control cells and are means (± SEM) of independent experiments with hepatocytes from 4 to 8 rats. The amount of mRNA was corrected for differences in total RNA applied to the filter, using GAPDH mRNA as an internal standard. Absolute activities of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase in cell homogenates in the absence of diterpenes were 291±57 and 68±4 pmol/h/mg cell protein, respectively. A significant difference between control and treated cells is indicated by an asterisk (*P<.05; **P<.005; ***P<.001).


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  		Table 1. Effect of Cafestol on Cholesterol 7{alpha}-Hydroxylase Activity in Cell Homogenates and Microsomes

Next to the effect on cholesterol 7{alpha}-hydroxylase, cafestol also caused a significant (P < .05) and dose-dependent decrease in sterol 27-hydroxylase activity and mRNA levels, being -49±5% and -46±14%, respectively at concentrations of cafestol of 20 µg/mL (Fig 3AUp). Cafestol did not have a direct inhibitory effect on sterol 27-hydroxylase activity in homogenates of freshly isolated rat hepatocytes (data not shown). In contrast, mRNA levels of the lithocholic acid 6ß-hydroxylase and of the mRNAs of the house-keeping genes actin and GAPDH did not change significantly on incubation with 20 µg/mL of cafestol (data not shown).

Effect of Cafestol on the Transcriptional Activity of Cholesterol 7{alpha}-Hydroxylase and Sterol 27-Hydroxylase
To further examine the mechanism of suppression of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase mRNA level, nuclear run-on studies were conducted using nuclei isolated from rat hepatocytes that were incubated in presence or absence of cafestol for 24 hours. [{alpha}32P]-labeled total RNA was hybridized to cDNAs for rat cholesterol 7{alpha}-hydroxylase, rat sterol 27-hydroxylase, rat GAPDH, and hamster actin. The latter two served as transcriptional activity controls between the different samples and specific transcriptional activity is expressed relative to that of actin. After incubation with 10 µg/mL cafestol, there is a significant decrease in cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase transcriptional activity of -55±13% and -39±6%, respectively (Fig 4Down), well in line with the suppression of the respective mRNA levels at this concentration (-56±5 and -33±12%, respectively).



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  		Figure 4. Effect of cafestol on transcriptional activity of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase. Transcriptional activity of different genes in nuclei isolated from hepatocytes incubated with 10 µg/mL cafestol for 24 hours between 18 to 42 hours of culture time. Hepatocytes were harvested simultaneously with untreated cells after this period for preparation of nuclei. Transcriptional activity of the different genes was determined by nuclear run-on assays as described in "Methods." Data are expressed as transcriptional activity relative to that of actin, and as percentage of control cells (no cafestol added). Each value represents a mean±SEM of 3 to 4 independent experiments. A significant difference (P<.05) between control and treated cells is indicated by an asterisk.

Effect of Cafestol on Intracellular Lipids and mRNA Levels of the LDL-Receptor, HMG-CoA Reductase, and HMG-CoA Synthase
Since such a large decrease in bile acid synthesis may have consequences for the level of intracellular cholesterol, we determined the amount of free and esterified cholesterol in hepatocytes cultured for 24 hours with different amounts of cafestol. However, the amount of free and esterified cholesterol and of triglycerides did not change significantly on incubation with 20 µg/mL cafestol (data not shown). Probably these changes are too small to be detectable. Another sensitive measure to detect changes in the regulatory free cholesterol pool is measurement of LDL-receptor mRNA and mRNAs of enzymes involved in cholesterol synthesis, like HMG-CoA reductase and HMG-CoA synthase.43 Table 2Down shows that LDL-receptor and HMG-CoA reductase mRNA levels were mildly but significantly decreased (-18±8% and -20±5%, respectively) on incubation with 20 µg/mL of cafestol, whereas the mRNA of HMG-CoA synthase was clearly suppressed (-43±10%). These data indicate that inhibition of bile acid synthesis by cafestol leads to downregulation of genes involved in cholesterol synthesis and LDL-receptor-mediated uptake.


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  		Table 2. Effect of Cafestol on mRNA Levels of LDL-Receptor and HMG-CoA-Reductase and -Synthase


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we have investigated the effect of cafestol and a mixture of cafestol/kahweol/isokahweol (48:47:5 w/w) on bile acid synthesis. Cafestol suppressed bile acid synthesis by a direct inhibitory effect on cholesterol 7{alpha}-hydroxylase activity and by downregulation of mRNA of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase. The decrease in mRNA levels is due to a decline of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase gene transcription, as shown by nuclear run-on assays. Elevation of the initial level of bile acid synthesis by feeding the rats with chow supplemented with 2% cholestyramine prior to isolation of the hepatocytes22,23,30 was not found to be obligatory to observe downregulation of bile acid synthesis by coffee diterpenes. Similar results as reported in this paper were obtained using rats fed on control chow (data not shown). Simultaneous with the decline in bile acid synthesis LDL-receptor, HMG-CoA reductase, and HMG-CoA synthase mRNA levels were downregulated.

Cafestol suppressed bile acid synthesis more potently (2- to 4-fold) than the mixture at the same concentration. In fact, the mixture appeared to counteract the effects of cafestol on bile acid synthesis and cholesterol 7{alpha}-hydroxylase mRNA level. We conclude therefore that cafestol is the active compound and suggest that kahweol and isokahweol are less active or not active. In an intervention study in humans, Weusten-Van der Wouw et al,8 found that both oil from arabica beans, which contains both cafestol and kahweol, and robusta oil, which contains cafestol but negligible amounts of kahweol, increased serum cholesterol in healthy volunteers to a similar extent. The latter findings also suggest the involvement of cafestol in raising cholesterol levels, but do not really exclude an additional role for kahweol.

Two different modes of inhibition of bile acid synthesis were found in our study. On the one hand, cafestol suppressed bile acid synthesis by downregulation of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase gene transcription, which caused a decrease in mRNA levels and activity. Since cafestol resembles sterols, it is conceivable that inhibition of gene transcription by cafestol is regulated via, as yet unidentified, sequences within the cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase promoter. On the other hand, bile acid synthesis can be affected by a direct inhibitory effect of cafestol on cholesterol 7{alpha}-hydroxylase activity. In line with this, we found that bile acid mass production is inhibited to a stronger degree than can be explained by suppression of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase alone. In earlier studies, the magnitude of suppression of these parameters was comparable with mediators that do not have a direct inhibitory effect on enzyme activity in the assay, eg, bile acids and insulin.23,30,40 Based on the structural similarity, coffee diterpenes may act as direct inhibitors like oxysterols, which have been also reported to inhibit cholesterol 7{alpha}-hydroxylase activity.28,29 Whether these two different ways of inhibition are linked remains obscure. No evidence was obtained, however, that cafestol has a general effect on cytochrome P-450 enzymes involved in bile acid synthesis, since lithocholic acid 6ß-hydroxylase mRNA was not significantly affected on addition of cafestol.

Measurement of cellular lipid levels did not show significant rises in cellular free or esterified cholesterol after incubation with cafestol. High amounts of free cholesterol present in the membranes of cells44 probably overshadow changes in free cholesterol caused by downregulation of bile acid synthesis by cafestol. Furthermore, an excess of free cholesterol in hepatocytes is rapidly converted into cholesteryl esters42. Since the decrease in bile acid synthesis was not accompanied by an increase in cholesteryl esters, it is possible that inhibition of bile acid synthesis by cafestol leads to an enhanced production and secretion of very-low-density-lipoprotein (VLDL) particles or biliary cholesterol excretion to remove cholesterol from the cell. However, we found no increase in apolipoprotein B secretion after incubation with cafestol (data not shown). The latter data are in line with observations in CaCo-2 cells, which also did not show a difference in mass of intracellular cholesterol after incubation with cafestol and which showed decreased rates of secretion of cholesteryl esters and triacylglycerol in these cells, representative for secretion of chylomicrons, in the presence of cafestol.27

Another way to maintain intracellular cholesterol homeostasis during inhibition of bile acid synthesis by cafestol is by downregulation of cholesterol synthesis and LDL-receptor-mediated uptake. Indeed, a mild but significant suppression of the mRNA levels of the LDL-receptor and HMG-CoA reductase and a marked decrease in the HMG-CoA synthase mRNA level was found. It is well known that the regulatory free cholesterol pool plays an important role in this regulation and that this pool is small as compared with the total free intracellular cholesterol mass.42–45 Subtle increases in intracellular cholesterol have been shown to prevent processing of sterol regulatory element binding proteins (SREBP), and are shown to be involved in this downregulation of gene transcription of the LDL-receptor, HMG-CoA reductase, and HMG-CoA synthase genes.46,47 A modest decrease of LDL-receptor and HMG-CoA reductase mRNA levels by dietary cholesterol has also been shown in vivo in the rat.48 In addition, Molowa and Cimis showed that in the human hepatoma cell-line HepG2 both HMG-CoA reductase and LDL-receptor mRNA levels were only moderately downregulated by LDL as compared to extrahepatic cells.49 In different studies both in vitro50 and in vivo,48 it is demonstrated that HMG-CoA reductase can be regulated at different levels, showing only a small decrease in mRNA level, despite the fact that hepatic cholesterol synthesis is largely suppressed. HMG-CoA reductase and HMG-CoA synthase mRNA are coordinately regulated, showing a larger effect on the latter mRNA,38 which is also found in our study. Similarly, in the case of high suppression of hepatic cholesterol synthesis, only modest downregulation of LDL-receptor mRNA can be observed in rats,48 since the role of the LDL-receptor in controlling the cholesterol balance in rats is small in contrast to humans.45 On the other hand, based on the structural similarity of cafestol with oxysterols, a direct effect of cafestol on transcription of the HMG-CoA reductase, HMG-CoA synthase, and LDL-receptor genes cannot be excluded. The downregulation of LDL-receptor mRNA by cafestol in this study is comparable in magnitude to the decrease in LDL uptake in the human hepatoma cell-line HepG2 and in human skin fibroblasts reported by others.27

We conclude from this study that a decreased bile acid synthesis and downregulation of the LDL-receptor may form an explanation for the rise in serum cholesterol in humans after consumption of boiled coffee.


*    Acknowledgments
 
This work was supported by The Netherlands Heart Foundation (grant 94-049).

Received December 2, 1996; accepted July 26, 1997.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Thelle DS, Arnesen E, Førde OH. The Tromsø Heart Study. Does coffee raise serum cholesterol? N Eng J Med. 1983;308:1454–1457.[Abstract]
  2. Thelle DS, Heyden S, Fodor JG. Coffee and cholesterol in epidemiological and experimental studies. Atherosclerosis. 1987;67:97–103.[Medline] [Order article via Infotrieve]
  3. Tverdal A, Stensvold I, Solvall K, Foss OP, Lund-Larssen P, Bjartveit K. Coffee consumption and death from coronary heart disease in middle aged Norwegian men and women. BMJ. 1990;300:566–569.
  4. Aro A, Tuomilehto J, Kostiainen E, Uusitalo U, Pietinen P. Boiled coffee increases serum low density lipoprotein concentration. Metabolism. 1987;36:1027–1030.[Medline] [Order article via Infotrieve]
  5. Bak AAA, Grobbee DE. The effect on serum cholesterol levels of coffee brewed by filtering or boiling. N Eng J Med. 1989;321:1432–1437.[Abstract]
  6. Zock PL, Katan MB, Merkus MP, Van Dusseldorp M, Harryvan JL. Effect of a lipid-rich fraction from boiled coffee on serum cholesterol. Lancet. 1990;335:1235–1237.[Medline] [Order article via Infotrieve]
  7. Heckers H, Gobel U, Kleppel U. End of coffee mystery: diterpene alcohols raise serum low-density lipoprotein cholesterol and triglyceride levels. J Int Med. 1994;235:192–193.[Medline] [Order article via Infotrieve]
  8. Weusten-Van der Wouw MPME, Katan MB, Viani R, Huggett AC, Liardon R, Lund-Larsen P, Thelle DS, Ahola I, Aro A, Meyboom D, Beynen AC. Identity of the cholesterol-raising factor from boiled coffee and its effect on liver function enzymes. J Lipid Res. 1994;35:721–733.[Abstract]
  9. Bilheimer DW, Goldstein JL, Grundy SM, Starzl TE, Brown NS. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia N Eng J Med. 1984;311:1658–1664.[Abstract]
  10. Björkhem I. Mechanism of bile acid synthesis in mammalian liver. In: Danielsson H, Sjövall J, eds. Sterols and Bile Acids. Amsterdam: Elsevier Scientific Publishers; 1985:231–278.
  11. Carey M, Duane WC. Enterohepatic circulation. In: Arias I, Boyer JL, Jakoby WB, Schachter D, Shafritz DA, eds. The Liver: Biology and Pathology. 3rd ed. New York: Raven Press; 1994:719–768.
  12. Blankenhorn DH, Nessim SA, Johnson RL, Sanmarco ME, Azen SP, Cashin-Hemphill L. Beneficial effects of combined colestipol-niacin therapy on coronary venous bypass grafts. JAMA. 1987;257:3233–3240.[Abstract]
  13. Lipid Research Clinics Program. The lipid research clinics coronary primary prevention trial results. I. Reduction in incidence of coronary heart disease. JAMA. 1984;251:351–364.[Abstract]
  14. Miettinen TA, Gylling H. Mortality and cholesterol metabolism in familial hypercholesterolemia. Arterosclerosis. 1988;8:163–167.[Abstract/Free Full Text]
  15. Einarsson K, Nilsell K, Leijd B, Angelin B. Influence of age on secretion of cholesterol and synthesis of bile acids by the liver. N Eng J Med. 1985;313:277–282.[Abstract]
  16. Kirk EA, Moe GL, Caldwell MT, Lernmark JÅ, Wilson DL, LeBoeuf RC. Hyper- and hypo-responsiveness to dietary fat and cholesterol among inbred mice: searching for level and variability genes. J Lipid Res. 1995;36:1522–1532.[Abstract]
  17. Poorman JA, Buck RA, Smith SA, Overturf ML, Loose-Mitchell DS. Bile acid excretion and cholesterol 7{alpha}-hydroxylase expression in hypercholesterolemia resistant rabbits. J Lipid Res. 1995;34:1675–1685.[Abstract]
  18. Danielsson H, Einarsson K, Johansson G. Effects of biliary drainage on individual reactions in the conversion of cholesterol to taurocholic acid. Eur J Biochem. 1967;2:44–49.[Medline] [Order article via Infotrieve]
  19. Shefer S, Hauser S, Bekersky I, Mosbach EH. Biochemical site of regulation of bile acid synthesis in the rat. J Lipid Res. 1970;11:404–411.[Abstract]
  20. Myant NB, Mitropoulos KA. Cholesterol 7{alpha}-hydroxylase. J Lipid Res. 1977;18:135–153.[Medline] [Order article via Infotrieve]
  21. Axelson M, Sjövall J. Potential bile acid precursors in plasma: possible indicators of biosynthetic pathways to cholic and chenodeoxycholic acids in man. J Steroid Biochem. 1990;36:631–640.[Medline] [Order article via Infotrieve]
  22. Princen HMG, Meyer P, Wolthers BG, Vonk RJ, Kuipers F. Cyclosporin A blocks bile acid synthesis in cultured hepatocytes by specific inhibition of chenodeoxycholic acid synthesis. Biochem J. 1991;275:501–505.
  23. Twisk J, De Wit ECM, Princen HMG. Suppresion of sterol 27-hydroxylase mRNA and transcriptional activity by bile acids in cultured rat hepatocytes. Biochem J. 1995;305:505–511.
  24. Danielsson H. Formation and metabolism of 26-hydroxycholesterol. Ark Kemi. 1961;17:373–379.
  25. Anderson KE, Kok E, Javitt NB. Bile acid synthesis in man: metabolism of 7{alpha}-hydroxycholesterol 14C and 26-hydroxycholesterol-3H. J Clin Invest. 1972;51:112–117.
  26. Swell L, Gustafsson J, Schwartz CC, Halloran LG, Danielsson H, and Vlacevic ZR. An in vivo evaluation of the quantitative significance of several potential pathways to cholic and chenodeoxycholic acid synthesis. J Lipid Res. 1980;21:455–466.[Abstract]
  27. Ranheim T, Halvorsen B, Huggett AC, Blomhoff R, Drevon CA. Effect of a coffee lipid cafestol on regulation of lipid metabolism in CaCo-2 cells. J Lipid Res. 1995;36:2079–2089.[Abstract]
  28. Sampson WJ, Houghton JD, Bowers P, Suffolk RA, Botham KM, Suckling CJ, Suckling KE. The effect of 6-azacholest-4-en-3 beta-ol-7 one an inhibitor of cholesterol 7{alpha}-hydroxylase, on cholesterol metabolism and bile acid synthesis in primary rat hepatocytes. Biochim Biophys Acta. 1988;960:268–274.[Medline] [Order article via Infotrieve]
  29. Schwarz MA, Margolis S. Effects of drugs and sterols on cholesterol 7{alpha}-hydroxylase in rat liver microsomes. J Lipid Res. 1983;24:28–33.[Abstract]
  30. Twisk J, Lehmann EM, Princen HMG. Differential feedback regulation of cholesterol 7{alpha}-hydroxylase mRNA and transcriptional activity by bile acids in primary monolayer cultures of rat hepatocytes. Biochem J. 1993;290:685–691.
  31. Princen HMG, Huijsmans CMG, Kuipers F, Vonk RJ, Kempen HJM. Ketoconazole blocks bile acid synthesis in hepatocyte monolayer cultures and in vivo in rat by inhibiting cholesterol 7{alpha}-hydroxylase. J Clin Invest. 1986;78:1064–1071.
  32. Princen HMG, Meijer P, Kwekkeboom J, Kempen HJM. Assay of cholesterol 7{alpha}-hydroxylase activity in rat hepatocytes in primary monolayer culture. Anal Biochem. 1988;171:158–165.[Medline] [Order article via Infotrieve]
  33. Lamprecht W, and Trautschold I. Methoden der enzymatischen Analyse. In: Bergmeijer HU, ed. Berlin: Verlag Chemie. 1970:2024–2033.
  34. De Vries EGE, Meijer C, Timmer-Bosscha H, Berendsen HH, De Leij L, Scheper RJ, Mulder NH. Resistance mechanisms in three human small cell lung cancer cell lines established from one patient during clinical follow up. Cancer Res. 1989;49:4175–4178.[Abstract/Free Full Text]
  35. Su P, Rennert H, Shayiq RM, Yamamoto R, Zheng Y-M, Addya S, Strauss III JF, Avadhani NG. A cDNA encoding rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries. DNA Cell Biol. 1990;9:657–665.[Medline] [Order article via Infotrieve]
  36. Texeira J, Gil G. Cloning, expression, and regulation of lithocholic acid 6ß-hydroxylase. J Biol Chem. 1991;266:21030–21036.[Abstract/Free Full Text]
  37. Chin DW, Gil G, Russell DW, Liscum L, Luskey KL, Basu SK, Okayama H, Berg P, Goldstein JL, Brown MS. Nucleotide sequence of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of endoplasmatic reticulum. Nature. 1984;308:613–617.[Medline] [Order article via Infotrieve]
  38. Gil G, Goldstein JL, Slaughter CA, Brown MS. Cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase from the hamster. I. Isolation and sequencing of a full-length cDNA. J Biol Chem. 1986;261:3710–3716.[Abstract/Free Full Text]
  39. Lee LY, Mohler WA, Schafer BL, Freudenberger JS, Byrne-Connolly N, Eager KB, Mosley ST, Leighton JK, Thrift RN, Davis RA, Tanaka RD. Nucleotide sequence of the rat low density lipoprotein receptor cDNA. Nucleic Acids Res. 1989;17:1249–1260.[Free Full Text]
  40. Twisk J, Hoekman MFM, Lehmann EM, Meijer P, Mager WH, Princen HMG. Insulin suppresses bile acid synthesis in cultured rat hepatocytes by down-regulation of cholesterol 7{alpha}-hydroxylase and sterol 27-hydroxylase gene transcription. Hepatology. 1995;21:501–510.[Medline] [Order article via Infotrieve]
  41. Bligh EG, Dyer WJ. A rapid method for total lipid extraction and purification. Can J Biochem. 1959;193:911–917.
  42. Havekes LM, De Wit ECM, Princen HMG. Cellular free cholesterol in HepG2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity. Biochem J. 1987;247:739–746.[Medline] [Order article via Infotrieve]
  43. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–430.[Medline] [Order article via Infotrieve]
  44. Lange Y, Ramos BV. Analysis of the distribution of cholesterol in the intact cell. J Biol Chem. 1983;258:15130–15134.[Abstract/Free Full Text]
  45. Dietschy JM, Turley SD, Spady DK. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans. J Lipid Res. 1993;34:1637–1659.[Medline] [Order article via Infotrieve]
  46. Wang X, Sato S, Brown MS, Hua X, Goldstein JL. SREBP-1, a membrane-bound transcription factor released by sterol-regulated proteolysis. Cell. 1994;77:53–62.[Medline] [Order article via Infotrieve]
  47. Brown MS, Goldstein, JL. The SREBP Pathway. Regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell. 1997;89:331–340.[Medline] [Order article via Infotrieve]
  48. Horton JD, Cuthbert JA, Spady DK. Regulation of hepatic 7{alpha}-hydroxylase: expression and response to dietary cholesterol in rat and hamster. J Biol Chem. 1995;270:5381–5387.[Abstract/Free Full Text]
  49. Molowa DT, Cimis GM. Co-ordinate regulation of low-density-lipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reductase and synthase gene expression in HepG2 cells. Biochem J. 1989;260:731–736.[Medline] [Order article via Infotrieve]
  50. Cohen LH, Griffioen M. Regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA contents in human hepatoma cell line HepG2 by distinct classes of mevalonate-derived metabolites. Biochem J. 1988;255:61–67.[Medline] [Order article via Infotrieve]



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