493
Biochimica
et Biophysics
Acta,
@ ElsevierlNorth-Holland
488
Biomedical
(1977) 493-501 Press
BBA 57046
PREFERENTIAL UPTAKE AND UTILIZATION OF MEVALONOLACTONE OVER MEVALONATE FOR STEROL BIOSYNTHESIS IN ISOLATED RAT HEPATOCYTES
PETER
A. EDWARDS
*, J. EDMOND,
A.M. FOGELMAN
and G. POPJAK
Department of Biological Chemistry and the Division of Cardiology, Medicine, University of California, School of Medicine, Los Angeles,
(Received
April 12th,
Department of Calif. (U.S.A.)
1977)
Summary Mevalonolactone at micromolar concentrations is taken up by rat hepatocytes and is converted into non-saponifiable lipids much faster than mevalonate. Although the first evidence of decarboxylation of both mevalonolactone and mevalonate (as determined by the release of 14C0 from the 1-14Clabelled substrates) was observed at the same time (2 min), tht subsequent rate of decarboxylation of mevalonolactone was approx. 43-fold of that found with mevalonate. The more rapid utilization of micromolar concentrations of mevalonolactone, compared to mevalonate, can be partly explained by the approx. 2-fold faster entry of the unchanged mevalonolactone into intact cells compared to the anionic mevalonate. This difference in uptake into the cell was observed with both the pure (R)- and the biologically inactive (S)-enantiomers and was independent of temperature. At these micromolar concentrations of the lactone the cell sterol biosynthetic pathway was not saturated and approx. 66% of the (R)-enantiomer was converted within 12 min to either isopentenyl pyrophosphate or non-saponifiable lipids. However, chemical (i.e. non-enzymic) hydrolysis of the lactone is slow, has a half-life of approx. 60 min at 37°C and at pH 7.4 and results in less than 35% of the lactone being converted to the anion mevalonate during a 15 min incubation. Hence the rapid conversion of approx. 66% of the mevalonolactone into sterols can be most easily explained if the cells contain a mevalonolactone hydrolase. _
* To
whom
University
correspondence of California,
should Los
be
Angeles.
sent Calif.
at the 90024.
Division U.S.A.
of
Cardiology,
Department
of Medicine.
Introduction
Mevalonate, the anion present at basic pH, is converted under acidic conditions to the lactone, mevalonoiactone. Mevalonate is converted into cholesterol by a series of reactions [ 1+2]. The first enzyme in this biosynthetic pathway, mevalonate kinase, converts mevalonate to 5qhosphomevalonate 13 ] and is inactive with mevalonolactone [4,5]. A slow apparent reaction of the kinase with mevaIonolactone is presumed to result from the slow hydrolysis of the la&one at the pH (7.4) of the kinase reaction with subsequent rapid phosphorylation of the mevalonate [5]. However, in a previous publication we demonstrated that in isolated rat hepatocytes the incorporation of (KS’)-[Z-‘“C]mevalonolactone into sterols during a 2 h period was approximately eight times greater than the incorporation of mevalonate at concentrations below 6 . 10M4 M [5]. Fumagalli et al. 163 had noted previously that in liver slices, but not brain slices, mevalonolactone was a slightly better substrate for sterol synthesis than mevalonate. In an attempt to elucidate these apparent anomalies we have studied both the uptake of mevalonolactone and mevalonate into isolated rate hepatocytes and the initial rate of entry of these two compounds into the sterol biosynthetic pathway within the intact cell. Methods
Materials. Hyamine hydroxide was obtained from Sigma and Aquasol from New England Nuclear. Sources of all other materials have been described previously [5]. The preparation of (R)-[5-‘4C]mevalonate (11.0 Ci/mol) [5] and (S)-[ 5-‘4C]mevalonate (11.8 Ci/mol) [ 7] has been given in detail elsewhere. ~~n~rnuis. Sprague-Dawley rats (150-250 g) were housed under a 12-h light (7 a.m. to 7 p.m.) and a 12-h dark cycle (7 p.m. to 7 a.m.) with free access to food and water. Animals were killed at around 9 a.m. Rut heputocytes. Rat hepatocytes were isolated from the liver perfused with collagenase as described previously [8,9]. The washed hepatocytes were resuspended in modified Swim’s S-77 medium containing 1.5% bovine serum albumin [a]. The number of cells used in different experiments varied and is given in the legends to the figures. ~evalono~acto~e and meua~onate. Aqueous solutions of (RS)-, (R)- or (S)mevalonate labelled with 14C at C-l, C-2 or C-5 as noted in the legends to the figures, were divided into two halves. One half was converted into mevalonolactone with a small excess of 1 M HCl as described [5]. The solutions of mevalonolactone (pH less than 3.0) and mevalonate (pH greater than 9.0) were incubated at 37°C for 30 min before addition to cell suspensions. Incubation of hepatocytes with mevalonolactone or mevalonate. Suspensions of rat hepatocytes were incubated at 37°C or 4°C with 14C-labelled mevalonate or mevalonolactone. At the times indicate the whole suspension, or an aliquot, was removed and the medium separated from the cells by centrifugation at 75 X g for 3 min. The medium was recentrifuged at 1500 X g for 10 min, an aliquot (0.1 ml) of the supernat~t was added to 0.9 ml ethanol and the resulting precipitate was removed by centrifugation. A sample (0.5 ml) of the
supernatant was removed to det,ermine its lJC content [5]. The elapsed time between the removal of a sample from the incubation and the initial separation of the medium from the cells at 75 X g for 3 min was approx. 4 min. In all experiments in which the uptake into the cells of mevalonolactone and mevalonate was measured, the times given are those from the addition of the lJClabelled compounds to the cell suspension until the medium was separated from the cells after slow speed centrifugation. The relative volumes of packed cells and cell-free medium were obtained in each experiment after centrifugation of aliquots of the cell suspension in hematocrit tubes at 1000 X g for 5 min. These values were used to calculate the “C content of the medium at zero time when the added 14C was presumably totally excluded from the cells. In all uptake experiments a control incubation was made in which the cells were replaced by their volume of medium. The radioactivity present in 0.1 ml of this control incubation was taken as the value for equal distribution of the 14C!label between cells and medium. The “C content of the non-saponifiable lipid fraction was determined after the hepatocyte suspension had been added to an equal volume of 20% trichloroacetic acid, saponification of the precipitate and extraction of the non-saponificable lipids into light petroleum 191. Studies with (ES’)-[ l-‘“Clmevalonolactone or (RS)-[ 1-“Clmevalonate were performed in glass counting vials sealed with a rubber cap. The cell suspension and the substrate were preincubated separately for 10 min at 37°C. The substrate was then injected through the rubber cap and the incubation continued. At the times indicated 0.2 ml 1 M H,SOJ was injected into the suspension and the ‘“CO, collected in 0.3 ml hyamine hydroxide contained in a plastic cup suspended from the rubber cap. After 1 h at 37°C the cup containing the hyamine hydroxide was removed, the external surface wiped clean and the cup placed in a clean counting vial with 10 ml Aquasol. Samples were counted after a 24-h equilibration period and recounted after addition of an internal standard of [ ‘“C] toluene. Results Factors affecting the entry of mevalonolactoue and mevalonate into rat hepatocy tes Incubation of rat hepatocytes at 37°C with 44 PM (R)-[5-‘4C]mevalonolactone or (R)-[5-14C] mevalonate resulted in a rapid decline in the 14C content of the medium (Fig. 1). The decline was significantly faster with mevalonolactone; the 14C content of the medium decreased to 50% of the initial value by 2.25 min with mevalonolactone and in about 9.5 min with mevalonate (Fig. 1). The 14C content of the media from both incubations were similar after 44 min when 85% of the mevalonate and 92% of the lactone had been removed from the media (Fig. 1). Addition of the biologically inactive (S)-[ 5-‘4C]mevalonolactone or (S)[5-‘4C]mevalonate to cell suspensions also resulted in a more rapid decline in the 14C content of the medium with the lactone (Fig. 2). Of particular interest in four studies with mevalonolactone was the consistent finding that the 14Ccontent of the medium fell to a minimum value within 4 min and that during the sub-
496 100
---~T----r
90 5
80
$
70
0
l_ ._.__i_~i_~i__..l._i_.
0
5
..~ i__i.~_i
1015202530354045 lwJBATlON
TIME Imin)
Fig. 1. Uptake of (R)-mevalonolaotone and (R)-mrvalonate into rat heptocytes. (R)-[5-14Clmevalonolactone (0) or (H)-[5-14C]mevalonate (td) (11.0 Cilmol) at a final concentration of 44 !.LM was added to a suspension of rat heptocytes at 3’7’C in a total volume of 2.6 ml. The ratio of packed crlls:cell suspension is given on the was 0.27 (v/v). Total exclusion of the ’ 4 C from thr cells in this and similar experiments ordinate as 100% Aliquots were removed at various times, the medium separated from the eelts, and the 14C content of the medium determined as described in Methods.
sequent incubation it rose slowly, After 20-44 min, the 14C content of the media were similar whether mevalonolactone or mevalonate was used (Fig. 2 and data not shown). We interpret the results with (S)-mevalonolactone to indicate that the cells initially concentrated the la&one against a concentration gradient and that subsequent hydrolysis in the cell of the lactone to mevalonate resulted in the latter’s release from the cell (Fig. 2).
Fig. 2. Uptake of (S)-mevalonolactone tone (0) or (5)-15-l JC]mevalonate ,(o) 2.59 ml suspension of rat hepatocytes 0.40 (v/v). The theoretical 14C content and medium is shown in this and other Methods.
and (S)-mevalonate into rat hepatocytes. (S)_[5-14C]mevalonolac(11.8 Ci/mol) at a final concentration of 18.6 /AMwas added to a incubated at 37’C. The ratio of packed cells:cell suspension was of the medium when the label is equally distributed between cells figures by the horizontal broken line. Other details are given in
491
The results with the (R)- and (S)-enantiomers demonstrated that the uptake by the cells of either form of the compound was not related to their stereochemistry. The differences in uptake of mevalonolactone and mevalonate into rat hepatocytes was also observed at 4°C with both the (R)- and (S)-enantiomers. At this temperature both (R)- and (S)-mevalonolactone were concentrated within the hepatocytes at all times studied (Fig. 3), the 14C content of the medium being lower than expected if the lactones had attained equal distribution between cells and media (Fig. 3; cf. dashed line). The data suggest that mevalonolactone is concentrated in the cells against a concentration gradient. Mevalonate, in contrast to the lactone, was not sequestered in the cells (Fig. 3). At 4” C the conversion of the (R)-isomers into sterol intermediates is presumably slow and would be expected to result in a much slower rate of removal of either substrate from the medium than at 37°C (cf. Figs. 1 and 3). At similar concentrations (RS)-mixtures of mevalonolactone and mevalonate (53 PM) behaved as did the pure (R)-isomers; (RS)-mevalonolactone was initially removed from the medium at a much faster rate than (RS)-mevalonate. After 44 min of incubation the concentration of both mevalonolactone and mevalonate in the medium was approx. 42% of the initial value (Fig. 4A). Calculations from Figs. 1 and 2 with the pure (R)- and (S)-compounds predict a value of 43% after 44 min incubation if the removal of the (R)-isomer from the medium were not affected by the biologically inactive (S)-enantiomer. At high concentrations (7.6 mM) there was no significant difference between the uptake of mevalonolactone and mevalonate at any time tested (Fig. 4B). The enzymic conversion of mevalonolactone and mevalonate into intermediates of the sterol biosynthetic pathway The experiments of Figs. 1, 3 and 4A suggested that mevalonolactone was
8
IskMEVALCNATE
4C_d1I 5
INCUBATION
TIME
10
15
20
Imin)
Fig. 3. Effect of low temperature on the entry of mevalonolactone and mevalonate into isolated rat hepatocytes. A 1.58 ml liver cell suspension (packed cell volume = 0.63 ml) was incubated with (R)-[5-14Clmevalonolactone (0) or (R)-[5-14C]mevalonate (0) (43.2 PM) (A) or (S)-[5-14Clmevalonolactone (0) or (S)-[5-‘4Clmevalonate (0) (40.2 wM) (B). See also legends to Figs. 1 and 2 for experimental details.
40-
0
0
I 10
I 20 INCUBATION
I 30
I 40
50
TIME (mid
Fig. 4. Effect of the concentration of mevalonolactone and mevalonate on their uptake into liver cells. (RS)-[2-14C]mevalonolactone (0) or (RS)-[2-1 4CImevalonate (0) was added to a suspension of rat hepatocytes and at the times indicated 0.6-1111 samples were removed and the 1 4C content of the medium determined. In A the total volume was 2.56 ml. the packed cell volume 0.72 ml and the substrate concentration was 53 PM (5.64 Ci/mol). Corresponding values for B were 2.6 ml, 0.62 ml. 7.6 mM and 0.5 Ci/mol.
not only taken up by the hepatocytes faster than mevalonate, but also that it was apparently utilized enzymically faster than the latter form of the substrate. In order to explore this supposition two types of experiments were carried out. In one set we compared the evolution of 14COZ from (RS)-[ l-14C]mevalonolactone and from mevalonate. This comparison gave a measure of the relative rates of the first enzymic transformations of the two forms of the substrate up to the generation of isopentenyl pyrophosphate. In the second set we examined the conversion of pure (R)- and of (RS)- mixtures of lactone and mevalonate labelled with 14C into non-saponifiable lipids. The evolution of 14C02 from the [ l-14C]lactone and from [ l-14C]mevalonate began with a similar lag period of about 2 min, but even at that time nearly tentimes more 14C02 was generated from the lactone than from the anion. By 4 min 27% of the 14C in the (R)-[1-‘4C]mevalonolactone had been released as 14C02 as compared to only 0.63% from the corresponding mevalonate (Fig. 5). Inclusion of 20 mM bicarbonate buffer in the incubation medium of the cells increased the recovery of 14C02. Under such conditions over 66% of the 14C in (R)-[ l-14C]mevalonolactone, added as the (RS)-mixture, was recovered as i4C0, after a 12 min incubation, as compared to a values of 2.6% for the anion.
499
INCUBATION
INCUBATION
TIME lm,ni
TIME lminl
Fig. 5. Decarboxylation of (RS)-[ l-14Clmevalonolactone and (RS)-[l-14Clmevalonate. l-ml suspensions of rat hepatocytes containing approx. 80 mg wet weight of cells were incubated with (RS)-[l-14Clmevalonolactone (0) or (RS)-[l-14C]mevalonate (0) (0.188 Cilmol) at 51 PM. At the indicated times 0.2 ml 1 M H2S04 was injected through the rubber cap and the incubation continued for 1 h. The “C02 content of the hyamine hydroxide was determined as described in Methods. The percentage of the (R)-1-14Clabelled substrate released as 14C02 is given for mevalonolactone on the left ordinate. and for mevalonate on the right ordinate. Note the lo-fold scale difference. Fig. 6. Incorporation of CR)-mevalonolactone and (R)-mevalonate into non-saponifiable lipids. (R)[5-14Clmevalonolactone (0) or (R)-[5-14Clmevalonate (0) (11.0 Ci/mol) at a final concentration of 45.6 PM was added to a 7 ml suspension of liver cells (approx. 1.4 g wet weight of cells) and incubated at 37’C. Samples (1.0 ml) were removed at the times indicated and the radioactivity in the non-saponifiable lipids determined as described in Methods.
Rates of conversion of mevalonolactone and mevalonate into non-saponifiable lipids in rat hepatocytes The initial rate of synthesis of non-saponifiable lipids was significantly greater with (R)-[5-‘4C]mevalonolactone than with (R)-[5-14C]mevalonate at 46 PM; during the first 5 min the rate was 12.5 times greater with the former
00 INCUBATION
TIME ,mlnl
10 INCUBATION
20
30
40
TIMElminl
Fig. 7. Non-saponifiable lipid synthesis from (RS)-mevalonolactone and (RS)-mevalonate. Incorporation of (RS)-[z-14Clmevalonolactone (0) and (RS)-[2-14Clmevalonate (0) at 53 PM into non-saponifiable lipids. The experimental conditions were the same as described in the legend to Fig. 4A. The 14C content of the non-saponifiable lipids was determined as described in Methods. The percent conversion of the CR)substrate (added as the (RS)-mixture) into non-saponifiables is given on the ordinate. Fig. 8. Effect of high concentrations of mevalonolactone and mevalonate on their incorporation into nonsaponifiable lipids. Incorporation of (RS)-[2-14Clmevalonolactone (0) and (RS)-t2-14Clmevalonate (0) at 7.6 mM into non-saponifiable lipids. The experimental conditions were the same as described in the legend to Fig. 4B. The percent conversion of the (R)-substrate into non-saponifiables is given on the ordinate.
500
than with the latter (Fig. 6}. After 15 min incubation more than 56% of the “C-labelled mevalonolactone was found in non-saponifiable lipids. The corresponding figure for mevalonate was ll%, and whereas the conversion of the lactone into non-saponifiables appeared to have been completed by 15 min, the utilization of mevalonate continued slowly and nearly linearly for at least 40 min (Fig. 6). Essentially similar results were obtained in studies with (RS)[2-14C]mevalonolactone and (RS)-[Z-‘“C]mevalonate at 53 PM (Fig. 7). At high concentrations of the substrates (7.6 m&I), where the entry of the lactone and the anion into the cells was similar (cf. Fig. 4B) there was no significant difference in the rate of synthesis of non-saponifiables from mevalonolactone of mevalonate (Fig. 8). In a previous report we demonstrated that at a concentration of 8 mM sterol synthesis from mevalonolactone and mevalonate during a 2 h period did not differ significantly [ 51. Discussion The previous findings by Fumagalli et al. [63 and Edwards et al. [5] that in liver slices or intact hepatocytes, respectively, mev~onolactone was a better substrate for sterol biosynthesis than mevalonate was at first puzzling because it has been generally accepted that only the latter substrate was involved in the biosynthetic path [ 31. The data presented here confirm and extend our previous findings [5,11]. We demonstrate that at micromolar concentrations the uncharged mevalonolactone is more rapidly taken up by hepatocytes than the anionic mevalonate. Further, this uptake process is not dependent on the chiral nature of the molecule since the difference in uptake between mevalonolactone and mevalonate was observed with both the (R)- and (S)-enantiomers (Figs. l-3). The uptake and concentration of mevalonolactone within the cells is presumed to be independent of an energy process since the phenomenon was observed at both 37 and 4°C (Figs. l-3). No differences in uptake into the cell were observed with mevalonolactone and mevalonate at concentrations of approx. 7.6 mM (Fig. 4B). It is of interest, therefore, that at these relatively high concentrations the rate of sterol synthesis from the two substrates did not differ either (Fig. 8) [ 51 and contrasts with the significant differences in uptake and in sterol synthesis at low substrate concentrations (cf. Figs. 1 and 6; 4A and 7). “C-fabelled mevalonolactone was converted At micromoIar concentrations both into isopentenyl pyrophosphate, with release of “‘CO2 (Fig. 5), and into non-saponifiables (Fig. 6) 43 and 11 times faster than mevalonate. Different cell preparations and incubation conditions result in experimental variations for these figures. The findings that more than 56% of the mevalonolactone was converted into non-saponifiables within 5-15 min (Figs. 6 and 7) and that more than 66% of [ 1-14C]mevalonolactone was decarboxylated during a 12 min incubation indicates that mevalonolactone rapidly entered the sterol biosynthetic pathway. We have previously demonstrated [5] that at 37°C and at pH 7.5 less than 35% of the added mevalonolactone was hydroiysed to mevalohate within 15 min in the presence of excess mevalonate kinase (66% was hydrolysed after
501
60 min).The half-life for the chemical hydrolysis of the lactone at 37°C and a pH 7.5 was shown to be approx. 60 min [5]. However, aqueous solutions of mevalonolactone at pH values below pH 3.0 contain, at equilibrium, approx. 20% of the anion mevalonate as determined with mevalonate kinase [5] or with the use of NMR (Parker, T. and Popjak, G., unpublished). It is therefore necessary, in order to account for the incorporation of 66% of the mevalonolactone into sterol intermediates within the first 12 min of incubation to postulate that hepatocytes contain (a) a mevalonolactone hydrolylase or (b) an area of high pH where hydrolysis of mevalonolactone occurs rapidly. The results presented above do not allow us to differentiate between these alternatives. It will be of interest to determine whether other tissues can also rapidly incorporate mevalonolactone into sterols and to determine whether mevalonate in blood [ 121 is actually in the lactone form. Finally, the results presented above demonstrate that, in rat liver, the rate of incorporation of tracer doses of [14C]mevalonate into sterol intermediates is determined by the transport of the mevalonate across the plasma membrane of the cells and not by the capacity of the cells to synthesize cholesterol. Acknowledgements We are grateful to Dr. T. Parker for the suggestion of the experiment with [l-14C]mevalonolactone and to Drs. T. Parker and J. Bardenheier for helpful information in carrying out the experiment. We are also grateful for the excellent technical assistance of Ms. Donna Lemongello. This work was supported in part by United States Public Health Service Research Grants HL-19063 (P.A.E.), HL-18016 (G.P.) and by grants from the Edna and George Castera Fund at U.C.L.A. P.A.E. is an established investigator of the American Heart Association. References 1
Tavormina,
2
Cornforth.
3
Popjak,
4
Levy,
5
Edwards.
6
Fumagalli,
P.A..
Gibbs,
J.W., G. (1969)
H.R.
M.H.
Cornforth, in Methods
and
Popjak.
P.A..
Grossi,
Huff,
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Gore.
Chem.
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(Clayton.
Biochem.
Fogelman.
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Popjak,
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Popjak,
R.,
and R.H.,
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Academic
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Edmond,
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S. (1962)
Chem.
Arch.
252.
1057-1063
Biochem.
Biophys.
Commun.
68.6449
529-533 7
Fogelman,
8
Edwards.
9
Edwards,
10
Gould,
11
Edwards,
12
Edgren,
A.M., P.A. P.A..
R.G.
Edmond.
(1975)
Fogelman,
and
P.A. B. and
Swyryd.
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A.M. E.A.
Fogelman,
Hellstrom,
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Biochem.
K.
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A.M.
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99,
Biochimica
et Biophysics
Acta,
@ ElsevierlNorth-Holland
488
Biomedical
(1977) 493-501 Press
BBA 57046
PREFERENTIAL UPTAKE AND UTILIZATION OF MEVALONOLACTONE OVER MEVALONATE FOR STEROL BIOSYNTHESIS IN ISOLATED RAT HEPATOCYTES
PETER
A. EDWARDS
*, J. EDMOND,
A.M. FOGELMAN
and G. POPJAK
Department of Biological Chemistry and the Division of Cardiology, Medicine, University of California, School of Medicine, Los Angeles,
(Received
April 12th,
Department of Calif. (U.S.A.)
1977)
Summary Mevalonolactone at micromolar concentrations is taken up by rat hepatocytes and is converted into non-saponifiable lipids much faster than mevalonate. Although the first evidence of decarboxylation of both mevalonolactone and mevalonate (as determined by the release of 14C0 from the 1-14Clabelled substrates) was observed at the same time (2 min), tht subsequent rate of decarboxylation of mevalonolactone was approx. 43-fold of that found with mevalonate. The more rapid utilization of micromolar concentrations of mevalonolactone, compared to mevalonate, can be partly explained by the approx. 2-fold faster entry of the unchanged mevalonolactone into intact cells compared to the anionic mevalonate. This difference in uptake into the cell was observed with both the pure (R)- and the biologically inactive (S)-enantiomers and was independent of temperature. At these micromolar concentrations of the lactone the cell sterol biosynthetic pathway was not saturated and approx. 66% of the (R)-enantiomer was converted within 12 min to either isopentenyl pyrophosphate or non-saponifiable lipids. However, chemical (i.e. non-enzymic) hydrolysis of the lactone is slow, has a half-life of approx. 60 min at 37°C and at pH 7.4 and results in less than 35% of the lactone being converted to the anion mevalonate during a 15 min incubation. Hence the rapid conversion of approx. 66% of the mevalonolactone into sterols can be most easily explained if the cells contain a mevalonolactone hydrolase. _
* To
whom
University
correspondence of California,
should Los
be
Angeles.
sent Calif.
at the 90024.
Division U.S.A.
of
Cardiology,
Department
of Medicine.
Introduction
Mevalonate, the anion present at basic pH, is converted under acidic conditions to the lactone, mevalonoiactone. Mevalonate is converted into cholesterol by a series of reactions [ 1+2]. The first enzyme in this biosynthetic pathway, mevalonate kinase, converts mevalonate to 5qhosphomevalonate 13 ] and is inactive with mevalonolactone [4,5]. A slow apparent reaction of the kinase with mevaIonolactone is presumed to result from the slow hydrolysis of the la&one at the pH (7.4) of the kinase reaction with subsequent rapid phosphorylation of the mevalonate [5]. However, in a previous publication we demonstrated that in isolated rat hepatocytes the incorporation of (KS’)-[Z-‘“C]mevalonolactone into sterols during a 2 h period was approximately eight times greater than the incorporation of mevalonate at concentrations below 6 . 10M4 M [5]. Fumagalli et al. 163 had noted previously that in liver slices, but not brain slices, mevalonolactone was a slightly better substrate for sterol synthesis than mevalonate. In an attempt to elucidate these apparent anomalies we have studied both the uptake of mevalonolactone and mevalonate into isolated rate hepatocytes and the initial rate of entry of these two compounds into the sterol biosynthetic pathway within the intact cell. Methods
Materials. Hyamine hydroxide was obtained from Sigma and Aquasol from New England Nuclear. Sources of all other materials have been described previously [5]. The preparation of (R)-[5-‘4C]mevalonate (11.0 Ci/mol) [5] and (S)-[ 5-‘4C]mevalonate (11.8 Ci/mol) [ 7] has been given in detail elsewhere. ~~n~rnuis. Sprague-Dawley rats (150-250 g) were housed under a 12-h light (7 a.m. to 7 p.m.) and a 12-h dark cycle (7 p.m. to 7 a.m.) with free access to food and water. Animals were killed at around 9 a.m. Rut heputocytes. Rat hepatocytes were isolated from the liver perfused with collagenase as described previously [8,9]. The washed hepatocytes were resuspended in modified Swim’s S-77 medium containing 1.5% bovine serum albumin [a]. The number of cells used in different experiments varied and is given in the legends to the figures. ~evalono~acto~e and meua~onate. Aqueous solutions of (RS)-, (R)- or (S)mevalonate labelled with 14C at C-l, C-2 or C-5 as noted in the legends to the figures, were divided into two halves. One half was converted into mevalonolactone with a small excess of 1 M HCl as described [5]. The solutions of mevalonolactone (pH less than 3.0) and mevalonate (pH greater than 9.0) were incubated at 37°C for 30 min before addition to cell suspensions. Incubation of hepatocytes with mevalonolactone or mevalonate. Suspensions of rat hepatocytes were incubated at 37°C or 4°C with 14C-labelled mevalonate or mevalonolactone. At the times indicate the whole suspension, or an aliquot, was removed and the medium separated from the cells by centrifugation at 75 X g for 3 min. The medium was recentrifuged at 1500 X g for 10 min, an aliquot (0.1 ml) of the supernat~t was added to 0.9 ml ethanol and the resulting precipitate was removed by centrifugation. A sample (0.5 ml) of the
supernatant was removed to det,ermine its lJC content [5]. The elapsed time between the removal of a sample from the incubation and the initial separation of the medium from the cells at 75 X g for 3 min was approx. 4 min. In all experiments in which the uptake into the cells of mevalonolactone and mevalonate was measured, the times given are those from the addition of the lJClabelled compounds to the cell suspension until the medium was separated from the cells after slow speed centrifugation. The relative volumes of packed cells and cell-free medium were obtained in each experiment after centrifugation of aliquots of the cell suspension in hematocrit tubes at 1000 X g for 5 min. These values were used to calculate the “C content of the medium at zero time when the added 14C was presumably totally excluded from the cells. In all uptake experiments a control incubation was made in which the cells were replaced by their volume of medium. The radioactivity present in 0.1 ml of this control incubation was taken as the value for equal distribution of the 14C!label between cells and medium. The “C content of the non-saponifiable lipid fraction was determined after the hepatocyte suspension had been added to an equal volume of 20% trichloroacetic acid, saponification of the precipitate and extraction of the non-saponificable lipids into light petroleum 191. Studies with (ES’)-[ l-‘“Clmevalonolactone or (RS)-[ 1-“Clmevalonate were performed in glass counting vials sealed with a rubber cap. The cell suspension and the substrate were preincubated separately for 10 min at 37°C. The substrate was then injected through the rubber cap and the incubation continued. At the times indicated 0.2 ml 1 M H,SOJ was injected into the suspension and the ‘“CO, collected in 0.3 ml hyamine hydroxide contained in a plastic cup suspended from the rubber cap. After 1 h at 37°C the cup containing the hyamine hydroxide was removed, the external surface wiped clean and the cup placed in a clean counting vial with 10 ml Aquasol. Samples were counted after a 24-h equilibration period and recounted after addition of an internal standard of [ ‘“C] toluene. Results Factors affecting the entry of mevalonolactoue and mevalonate into rat hepatocy tes Incubation of rat hepatocytes at 37°C with 44 PM (R)-[5-‘4C]mevalonolactone or (R)-[5-14C] mevalonate resulted in a rapid decline in the 14C content of the medium (Fig. 1). The decline was significantly faster with mevalonolactone; the 14C content of the medium decreased to 50% of the initial value by 2.25 min with mevalonolactone and in about 9.5 min with mevalonate (Fig. 1). The 14C content of the media from both incubations were similar after 44 min when 85% of the mevalonate and 92% of the lactone had been removed from the media (Fig. 1). Addition of the biologically inactive (S)-[ 5-‘4C]mevalonolactone or (S)[5-‘4C]mevalonate to cell suspensions also resulted in a more rapid decline in the 14C content of the medium with the lactone (Fig. 2). Of particular interest in four studies with mevalonolactone was the consistent finding that the 14Ccontent of the medium fell to a minimum value within 4 min and that during the sub-
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1015202530354045 lwJBATlON
TIME Imin)
Fig. 1. Uptake of (R)-mevalonolaotone and (R)-mrvalonate into rat heptocytes. (R)-[5-14Clmevalonolactone (0) or (H)-[5-14C]mevalonate (td) (11.0 Cilmol) at a final concentration of 44 !.LM was added to a suspension of rat heptocytes at 3’7’C in a total volume of 2.6 ml. The ratio of packed crlls:cell suspension is given on the was 0.27 (v/v). Total exclusion of the ’ 4 C from thr cells in this and similar experiments ordinate as 100% Aliquots were removed at various times, the medium separated from the eelts, and the 14C content of the medium determined as described in Methods.
sequent incubation it rose slowly, After 20-44 min, the 14C content of the media were similar whether mevalonolactone or mevalonate was used (Fig. 2 and data not shown). We interpret the results with (S)-mevalonolactone to indicate that the cells initially concentrated the la&one against a concentration gradient and that subsequent hydrolysis in the cell of the lactone to mevalonate resulted in the latter’s release from the cell (Fig. 2).
Fig. 2. Uptake of (S)-mevalonolactone tone (0) or (5)-15-l JC]mevalonate ,(o) 2.59 ml suspension of rat hepatocytes 0.40 (v/v). The theoretical 14C content and medium is shown in this and other Methods.
and (S)-mevalonate into rat hepatocytes. (S)_[5-14C]mevalonolac(11.8 Ci/mol) at a final concentration of 18.6 /AMwas added to a incubated at 37’C. The ratio of packed cells:cell suspension was of the medium when the label is equally distributed between cells figures by the horizontal broken line. Other details are given in
491
The results with the (R)- and (S)-enantiomers demonstrated that the uptake by the cells of either form of the compound was not related to their stereochemistry. The differences in uptake of mevalonolactone and mevalonate into rat hepatocytes was also observed at 4°C with both the (R)- and (S)-enantiomers. At this temperature both (R)- and (S)-mevalonolactone were concentrated within the hepatocytes at all times studied (Fig. 3), the 14C content of the medium being lower than expected if the lactones had attained equal distribution between cells and media (Fig. 3; cf. dashed line). The data suggest that mevalonolactone is concentrated in the cells against a concentration gradient. Mevalonate, in contrast to the lactone, was not sequestered in the cells (Fig. 3). At 4” C the conversion of the (R)-isomers into sterol intermediates is presumably slow and would be expected to result in a much slower rate of removal of either substrate from the medium than at 37°C (cf. Figs. 1 and 3). At similar concentrations (RS)-mixtures of mevalonolactone and mevalonate (53 PM) behaved as did the pure (R)-isomers; (RS)-mevalonolactone was initially removed from the medium at a much faster rate than (RS)-mevalonate. After 44 min of incubation the concentration of both mevalonolactone and mevalonate in the medium was approx. 42% of the initial value (Fig. 4A). Calculations from Figs. 1 and 2 with the pure (R)- and (S)-compounds predict a value of 43% after 44 min incubation if the removal of the (R)-isomer from the medium were not affected by the biologically inactive (S)-enantiomer. At high concentrations (7.6 mM) there was no significant difference between the uptake of mevalonolactone and mevalonate at any time tested (Fig. 4B). The enzymic conversion of mevalonolactone and mevalonate into intermediates of the sterol biosynthetic pathway The experiments of Figs. 1, 3 and 4A suggested that mevalonolactone was
8
IskMEVALCNATE
4C_d1I 5
INCUBATION
TIME
10
15
20
Imin)
Fig. 3. Effect of low temperature on the entry of mevalonolactone and mevalonate into isolated rat hepatocytes. A 1.58 ml liver cell suspension (packed cell volume = 0.63 ml) was incubated with (R)-[5-14Clmevalonolactone (0) or (R)-[5-14C]mevalonate (0) (43.2 PM) (A) or (S)-[5-14Clmevalonolactone (0) or (S)-[5-‘4Clmevalonate (0) (40.2 wM) (B). See also legends to Figs. 1 and 2 for experimental details.
40-
0
0
I 10
I 20 INCUBATION
I 30
I 40
50
TIME (mid
Fig. 4. Effect of the concentration of mevalonolactone and mevalonate on their uptake into liver cells. (RS)-[2-14C]mevalonolactone (0) or (RS)-[2-1 4CImevalonate (0) was added to a suspension of rat hepatocytes and at the times indicated 0.6-1111 samples were removed and the 1 4C content of the medium determined. In A the total volume was 2.56 ml. the packed cell volume 0.72 ml and the substrate concentration was 53 PM (5.64 Ci/mol). Corresponding values for B were 2.6 ml, 0.62 ml. 7.6 mM and 0.5 Ci/mol.
not only taken up by the hepatocytes faster than mevalonate, but also that it was apparently utilized enzymically faster than the latter form of the substrate. In order to explore this supposition two types of experiments were carried out. In one set we compared the evolution of 14COZ from (RS)-[ l-14C]mevalonolactone and from mevalonate. This comparison gave a measure of the relative rates of the first enzymic transformations of the two forms of the substrate up to the generation of isopentenyl pyrophosphate. In the second set we examined the conversion of pure (R)- and of (RS)- mixtures of lactone and mevalonate labelled with 14C into non-saponifiable lipids. The evolution of 14C02 from the [ l-14C]lactone and from [ l-14C]mevalonate began with a similar lag period of about 2 min, but even at that time nearly tentimes more 14C02 was generated from the lactone than from the anion. By 4 min 27% of the 14C in the (R)-[1-‘4C]mevalonolactone had been released as 14C02 as compared to only 0.63% from the corresponding mevalonate (Fig. 5). Inclusion of 20 mM bicarbonate buffer in the incubation medium of the cells increased the recovery of 14C02. Under such conditions over 66% of the 14C in (R)-[ l-14C]mevalonolactone, added as the (RS)-mixture, was recovered as i4C0, after a 12 min incubation, as compared to a values of 2.6% for the anion.
499
INCUBATION
INCUBATION
TIME lm,ni
TIME lminl
Fig. 5. Decarboxylation of (RS)-[ l-14Clmevalonolactone and (RS)-[l-14Clmevalonate. l-ml suspensions of rat hepatocytes containing approx. 80 mg wet weight of cells were incubated with (RS)-[l-14Clmevalonolactone (0) or (RS)-[l-14C]mevalonate (0) (0.188 Cilmol) at 51 PM. At the indicated times 0.2 ml 1 M H2S04 was injected through the rubber cap and the incubation continued for 1 h. The “C02 content of the hyamine hydroxide was determined as described in Methods. The percentage of the (R)-1-14Clabelled substrate released as 14C02 is given for mevalonolactone on the left ordinate. and for mevalonate on the right ordinate. Note the lo-fold scale difference. Fig. 6. Incorporation of CR)-mevalonolactone and (R)-mevalonate into non-saponifiable lipids. (R)[5-14Clmevalonolactone (0) or (R)-[5-14Clmevalonate (0) (11.0 Ci/mol) at a final concentration of 45.6 PM was added to a 7 ml suspension of liver cells (approx. 1.4 g wet weight of cells) and incubated at 37’C. Samples (1.0 ml) were removed at the times indicated and the radioactivity in the non-saponifiable lipids determined as described in Methods.
Rates of conversion of mevalonolactone and mevalonate into non-saponifiable lipids in rat hepatocytes The initial rate of synthesis of non-saponifiable lipids was significantly greater with (R)-[5-‘4C]mevalonolactone than with (R)-[5-14C]mevalonate at 46 PM; during the first 5 min the rate was 12.5 times greater with the former
00 INCUBATION
TIME ,mlnl
10 INCUBATION
20
30
40
TIMElminl
Fig. 7. Non-saponifiable lipid synthesis from (RS)-mevalonolactone and (RS)-mevalonate. Incorporation of (RS)-[z-14Clmevalonolactone (0) and (RS)-[2-14Clmevalonate (0) at 53 PM into non-saponifiable lipids. The experimental conditions were the same as described in the legend to Fig. 4A. The 14C content of the non-saponifiable lipids was determined as described in Methods. The percent conversion of the CR)substrate (added as the (RS)-mixture) into non-saponifiables is given on the ordinate. Fig. 8. Effect of high concentrations of mevalonolactone and mevalonate on their incorporation into nonsaponifiable lipids. Incorporation of (RS)-[2-14Clmevalonolactone (0) and (RS)-t2-14Clmevalonate (0) at 7.6 mM into non-saponifiable lipids. The experimental conditions were the same as described in the legend to Fig. 4B. The percent conversion of the (R)-substrate into non-saponifiables is given on the ordinate.
500
than with the latter (Fig. 6}. After 15 min incubation more than 56% of the “C-labelled mevalonolactone was found in non-saponifiable lipids. The corresponding figure for mevalonate was ll%, and whereas the conversion of the lactone into non-saponifiables appeared to have been completed by 15 min, the utilization of mevalonate continued slowly and nearly linearly for at least 40 min (Fig. 6). Essentially similar results were obtained in studies with (RS)[2-14C]mevalonolactone and (RS)-[Z-‘“C]mevalonate at 53 PM (Fig. 7). At high concentrations of the substrates (7.6 m&I), where the entry of the lactone and the anion into the cells was similar (cf. Fig. 4B) there was no significant difference in the rate of synthesis of non-saponifiables from mevalonolactone of mevalonate (Fig. 8). In a previous report we demonstrated that at a concentration of 8 mM sterol synthesis from mevalonolactone and mevalonate during a 2 h period did not differ significantly [ 51. Discussion The previous findings by Fumagalli et al. [63 and Edwards et al. [5] that in liver slices or intact hepatocytes, respectively, mev~onolactone was a better substrate for sterol biosynthesis than mevalonate was at first puzzling because it has been generally accepted that only the latter substrate was involved in the biosynthetic path [ 31. The data presented here confirm and extend our previous findings [5,11]. We demonstrate that at micromolar concentrations the uncharged mevalonolactone is more rapidly taken up by hepatocytes than the anionic mevalonate. Further, this uptake process is not dependent on the chiral nature of the molecule since the difference in uptake between mevalonolactone and mevalonate was observed with both the (R)- and (S)-enantiomers (Figs. l-3). The uptake and concentration of mevalonolactone within the cells is presumed to be independent of an energy process since the phenomenon was observed at both 37 and 4°C (Figs. l-3). No differences in uptake into the cell were observed with mevalonolactone and mevalonate at concentrations of approx. 7.6 mM (Fig. 4B). It is of interest, therefore, that at these relatively high concentrations the rate of sterol synthesis from the two substrates did not differ either (Fig. 8) [ 51 and contrasts with the significant differences in uptake and in sterol synthesis at low substrate concentrations (cf. Figs. 1 and 6; 4A and 7). “C-fabelled mevalonolactone was converted At micromoIar concentrations both into isopentenyl pyrophosphate, with release of “‘CO2 (Fig. 5), and into non-saponifiables (Fig. 6) 43 and 11 times faster than mevalonate. Different cell preparations and incubation conditions result in experimental variations for these figures. The findings that more than 56% of the mevalonolactone was converted into non-saponifiables within 5-15 min (Figs. 6 and 7) and that more than 66% of [ 1-14C]mevalonolactone was decarboxylated during a 12 min incubation indicates that mevalonolactone rapidly entered the sterol biosynthetic pathway. We have previously demonstrated [5] that at 37°C and at pH 7.5 less than 35% of the added mevalonolactone was hydroiysed to mevalohate within 15 min in the presence of excess mevalonate kinase (66% was hydrolysed after
501
60 min).The half-life for the chemical hydrolysis of the lactone at 37°C and a pH 7.5 was shown to be approx. 60 min [5]. However, aqueous solutions of mevalonolactone at pH values below pH 3.0 contain, at equilibrium, approx. 20% of the anion mevalonate as determined with mevalonate kinase [5] or with the use of NMR (Parker, T. and Popjak, G., unpublished). It is therefore necessary, in order to account for the incorporation of 66% of the mevalonolactone into sterol intermediates within the first 12 min of incubation to postulate that hepatocytes contain (a) a mevalonolactone hydrolylase or (b) an area of high pH where hydrolysis of mevalonolactone occurs rapidly. The results presented above do not allow us to differentiate between these alternatives. It will be of interest to determine whether other tissues can also rapidly incorporate mevalonolactone into sterols and to determine whether mevalonate in blood [ 121 is actually in the lactone form. Finally, the results presented above demonstrate that, in rat liver, the rate of incorporation of tracer doses of [14C]mevalonate into sterol intermediates is determined by the transport of the mevalonate across the plasma membrane of the cells and not by the capacity of the cells to synthesize cholesterol. Acknowledgements We are grateful to Dr. T. Parker for the suggestion of the experiment with [l-14C]mevalonolactone and to Drs. T. Parker and J. Bardenheier for helpful information in carrying out the experiment. We are also grateful for the excellent technical assistance of Ms. Donna Lemongello. This work was supported in part by United States Public Health Service Research Grants HL-19063 (P.A.E.), HL-18016 (G.P.) and by grants from the Edna and George Castera Fund at U.C.L.A. P.A.E. is an established investigator of the American Heart Association. References 1
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