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The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 261, No. 2, March, pp. 291-298,198s
The Effect of Sodium Butyrate on Tyrosine Aminotransferase Induction Primary Cultures of Normal Adult Rat Hepatocytes’ JEFFREY
L. STAECKER
AND
HENRY
in
C. PITOT’
The University of Wisconsin, McArdle Laboratory for Cancer Research, 450 North Randall Avenue, Madison, Wisconsin 5.3i’O6 Received
July
10, 198’7, and in revised
form
November
10.1987
Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et ab, submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT)
manner.
0 1988 Academic
Press, Inc.
Butyrate inhibits histone deacetylation and induces histone hyperacetylation when it is added to cultured cells (1). In addition, butyrate has a variety of other effects on cultured eukaryotic cells, including suppression of the characteristics of transformation in many cell lines, induction of changes in gene expression, and inhibition of cell growth (1). The effects of butyrate listed above have been correlated with butyrate-induced histone acetylation changes in many cultured cells (1). Histone acetylation in HTC cells is represented by at least two kinetic classes: rapid histone acetylation in which acetate turnover occurs with a half-life of approx-
imately 5 min, and slow histone acetylation in which acetate turnover occurs with a half-life of approximately 300 min (2). Similar kinetics for histone acetylation have been reported for cultured human diploid fibroblasts (3). The relationship between histone acetylation and TAT3 induction has been studied in HTC cells. TAT, an enzyme found primarily in the liver, catalyzes the first reaction of a pathway by which tyrosine can ultimately be degraded to fumarate and acetoacetate (4). The induction of TAT in HTC cells is abolished by treatment with 5 mM sodium butyrate (5, 6). Evidence that the inhibition of TAT induction in HTC cells by butyrate is caused by butyrate-induced changes in fast histone acetylation has
i This study was supported in part by Grants CA 07175 and CA 22484 from the National Cancer Institute. J.L.S. was a recipient of a predoctoral traineeship from the National Cance Institute (T32-CA 09135). ‘To whom correspondence should be addressed.
3 Abbreviations used: TAT, tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 291
0003-9861188 Copyright All rights
$3.00
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
292
STAECKER
been provided by Chalkley’s laboratory. Plesko et al. (6) have correlated the effect of butyrate on fast acetylation with TAT induction. Shires and Chalkley (7) isolated numerous HTC variants in which histone deacetylation, as well as cell replication and TAT induction, continued in the presence of butyrate. We have recently demonstrated that butyrate treatment inhibits the loss of some aspects of the differentiated phenotype of primary cultures of normal adult rat hepatocytes and preserves high levels of basal TAT gene expression (Staecker et al., submitted). Previous reports have indicated that butyrate addition to cultured hepatoma cell lines completely inhibits TAT induction without affecting basal TAT gene expression in these cells (5, 6). In continuing our studies of the effects of butyrate on the regulation of gene expression in primary cultures of normal hepatocytes, we have studied the effect of this small-chain fatty acid on TAT gene expression in primary rat hepatocyte culture compared with reports on the HTC hepatoma cell line (5, 6). MATERIALS
AND
METHODS
Hepatocyte isolation and culture. Hepatocytes were isolated by an in situ perfusion technique previously described (method d in Ref. (8)). Viable hepatocytes, as judged by trypan blue exclusion, were separated from nonviable hepatocytes and nonparenchymal cells prior to plating (8), resulting in an initial viability of more than 95%. Cells were then plated onto plastic tissue culture dishes (60 or 100 mm) and cultured in Leibovitz (L-15) medium supplemented with Hepes (15 mM), penicillin (100 pg/ml), streptomycin (100 pg/ml), insulin (1.5 pg/ml), and glucose (1.5 mg/ml). During the initial 2 h of culture (attachment period) the medium was also supplemented with newborn bovine serum (2.5%). Treatment of the cells with butyrate or propionate was begun with the initial medium change unless otherwise noted. Medium containing butyrate or propionate was prepared by addition of an appropriate amount of a filter-sterilized, concentrated, 1 M solution of sodium butyrate or propionate that had been prepared by dissolving the compounds in culture medium. TAT assay. TAT activity was determined according to the method of Diamondstone (9) as modified by Granner and Tomkins (10) and described by Kreamer et al. (8), except that the cells were freezethawed while they were attached to the culture
AND
PITOT
dishes. TAT activity values were averages of duplicate determinations made on three separate plates of cells. Unless otherwise noted, TAT gene expression was induced by the inclusion of dexamethasone (10m6) in the culture medium. RNA analysis. Cells were disrupted in 4 M guanidium thiocyanate, and the RNA was pelleted by centrifugation through a cushion of 5.7 M CsClz-0.1 M EDTA, pH 7.0, as previously described (11). RNA was quantified spectrophotometrically by the relationship: 1.0 unit equivalent of AzW = 32 gg/ml RNA. Total cellular RNA was separated in 1.2% agaroseformaldehyde gels according to the method of Maniatis et al. (12) and transferred to Gene Screen Plus filters (New England Nuclear) for Northern gel analysis. Mammalian and Escherichia coli ribosomal RNAs were used as size markers for Northern gels. Analysis of the Northern transfers was accomplished by probing with nick-translated clone pcTAT-3 for TAT (generously provided by Dr. G. Schiitz, Institute of Cell and Tumor Biology, Heidelberg). Subsequent steps for Northern analysis (prehybridization, hybridization, washings, and autoradiography steps) were identical with those detailed previously (11). Histone isolation and electrophoresis. Histones were isolated from cultured hepatocytes and separated according to the method of Alfageme et al. (13) as described (14). After Coomassie blue staining, the gels were scanned with a Zeineh Model Sl-504-X1 soft laser densitometer (Biomed Corp., Chicago, IL), and the peaks were integrated with a Numonics 1224 electronic digitizer (Numonics Corp., Landsdale, PA). HTC culture. Large quantities of HTC cells were grown in T-flasks (Corning) in modified Swim’s 77 medium with 2.5% calf and 2.5% fetal calf serum, as described by Covault and Chalkley (2). The cells were then plated onto 60- or loo-mm tissue culture plates (Corning) for experimental purposes. Butyrate addition to HTC cells was accomplished in a manner identical with that used for primary hepatocyte cultures, except that the 1 M butyrate stock was made up in Swim’s 77 medium containing 2.5% calf and 2.5 fetal calf serum.
RESULTS
The effects of butyrate on TAT induction by dexamethasone or glucagon were investigated in primary cultures of rat hepatocytes. After numerous preliminary experiments, it became apparent that the effect of butyrate on TAT induction was dependent on the time after induction that TAT activity was assayed. There was little or no TAT induction in butyrate-treated
EFFECT
OF
BUTYRATE
ON
TYROSINE
cultures up to 8 h following the addition of dexamethasone to the cultures (Fig. 1). This delay was followed by an increase in TAT activity observed 16 h after induction was begun (Fig. 1). TAT activity levels 24 h after induction by dexamethasone was initiated were much higher in butyratetreated cells than in cultures without butyrate on both the second and third days of culture (Fig. 1). Similar results were obtained when TAT gene expression was induced by glucagon instead of dexamethasone (Table I). There was variability in TAT induction from experiment to experiment in both control and butyrate-treated cultures, as shown by comparing the results in Figs. 1 and 3-6. However, we always found that after initially inhibiting TAT induction, butyrate treatment resulted in a higher level of TAT induction than in control cultures without butyrate (e.g., Figs. 1 and 3-6). Previous studies (5, 6) have shown that induction of TAT activity in HTC cells is accompanied by increases in TAT mRNA levels and that both TAT activity and mRNA induction are blocked by the presence of 5 mM butyrate (5,6). We measured
”
I 8
’
16 24 6 Hours post-induction
clay 2 of culture
’
16
Day 3 of culture
24 i
FIG. 1. The effect of butyrate on TAT induction in cultured hepatocytes. Cells were cultured as described under Materials and Methods and treated as indicated. Induction was initiated by replacing the medium after 1 day of culture with medium containing 10F6 dexamethasone. Plates were harvested during the second and third days of culture at the indicated times. TAT activity was determined as described under Materials and Methods and expressed as milliunits TAT activity per milligram protein. Similar results were observed in nine other experiments (not shown). TAT induction and the expression of TAT activity were done in an identical manner in Figs. 3-6. Standard deviation of the mean is indicated at data points.
AMINOTRANSFERASE
293
TAT mRNA levels in primary hepatocyte cultures in the presence and absence of butyrate by Northern and dot-blot analysis. The results of a Northern analysis are shown in Fig. 2.; the higher level of TAT activity in butyrate-treated cells was accompanied by a higher level of TAT mRNA than that in control cultures. Similar results (not shown) were observed in different experiments when dot-blot analysis was used to analyze the changes in TAT mRNA levels. Our results suggest that induction of TAT by dexamethasone of normal and butyrate-treated hepatocytes may result from increased transcription of the TAT gene. Previous work has shown that TAT induction in cultured hepatocytes, as well as in other systems, is accomplished primarily by increased transcription of the TAT gene (4, 15). The effects of butyrate on TAT induction by dexamethasone were examined as an extension of a series of experiments in which we determined that butyrate helped to preserve aspects of the differentiated phenotype of cultured hepatocytes (Staecker et ab, submitted). Initially the effects of butyrate on TAT induction in cultures that had been exposed to butyrate prior to and during the induction period were examined. To determine whether the effects of butyrate on TAT induction were due to exposure of the cells prior to or during the induction period, we discontinued butyrate treatment of some cultures while simultaneously beginning butyrate treatment of other cultures. Prior exposure of cultures to butyrate had little effect on the induction of TAT, although a slight increase was observed in cells pretreated with butyrate, commensurate with the increase in the basal TAT activity observed in butyrate-treated cells (Fig. 3). The effects of butyrate on TAT induction were found to be dependent on the concentration of butyrate in the culture medium. A higher level of TAT activity was observed 24 h after induction in cells treated with 1, 2.5, or 5.0 mM butyrate, although inhibition of TAT induction after 8 h of induction was observed only in cultures treated with 2.5 or 5.0 mM butyrate (Fig. 4).
294
STAECKER
AND TABLE
THE EFFECT
OF BUTYRATE
ON TAT
Oh Treatment Control
18.3 28.4
a TAT
activity
INDUCTION
10m6 Dex
f 1.3" +- 1.5
is expressed
42.1 38.4
as microunits
31 3.2 k 3.4
per milligram
We investigated the possibility that TAT induction in butyrate-treated cultures occurred subsequent to the elimination of butyrate by its metabolism, since previous reports indicated that primary hepatocyte cultures readily metabolize butyrate (16, 17). In initial experiments, we found that introducing additional butyrate into butyrate-treated cultures 8 h after induction was initiated inhibited TAT induction (Fig. 5). These results indicate that the metabolism of butyrate by cultured hepatocytes, with concomitant release from inhibition by butyrate, may be responsible for subsequent TAT induction in butyrate-treated cells.
2.3
I BY DEXAMETHASONE
OR GLU~AGON
8 h postinduction
Basal
5 mM butyrate
PITOT
24 h postinduction
10m6 Glucagon 37.4
f
35.7
-t 3.3
protein
10m6 Dex
4.2
92.8
+ standard
+
10m6 Glucagon 6.2
70.8
f
7.9
149.4 i 10.2
89.4
f
3.5
deviation
of the mean.
We were able to obtain direct measurements of the metabolism of butyrate by cultured hepatocytes through analysis of fresh butyrate-supplemented medium and spent butyrate-supplemented medium (spent medium refers to medium removed from butyrate-treated hepatocytes after the second day of culture). Dr. Sharon Fleming (University of California, Berkeley) generously performed the measurement of butyrate in our samples, using an established gas chromatography procedure (18). After 24 h of culture, the metabolism of butyrate by the hepatocyte culture reduced the concentration of butyrate from 5 mM to less than 0.3 mM. The effects of the metabolism of butyrate by primary cultures of hepatocytes are
-
6
Hours
FIG. 2. The effect of butyrate on TAT mRNA levels. RNA was isolated from cells after 2 days of culture, and 20 Kg of RNA was spotted on each lane. RNA was obtained from butyrate-treated (lanes 3 and 4) and control cells (lanes 1 and 2) in which TAT gene expression was induced (lanes 2 and 4) or from uninduced cells (lanes 1 and 3).
16
24
post-induction
FIG. 3. The effect of prior butyrate treatment on TAT induction. After 2 days of culture in the presence or absence of butyrate, the medium was replaced with medium containing dexamethasone with and without 5 nIM butyrate, as indicated. Similar results were observed in two other experiments. Variation at data points is expressed as in Fig. 1.
EFFECT
120
H 0-3 H
100
OF
BUTYRATE
ON
TYROSINE
1 mM butyrate 2.5 mM butyrale 5.0 mM butyrate
.E Z”60 .g h SF 2 3 60 ‘E 40
20
6 16 Hours post-induction
24
FIG. 4. The effect of various concentrations of butyrate on TAT induction. TAT induction by 10m6 dexamethasone was determined on hepatocytes cultured in various concentrations of butyrate, as indicated. Similar results were observed in two other experiments. Variation at data points is expressed as in Fig. 1.
reflected in changes in histone H4 acetylation. We examined the effects of the metabolism of butyrate on histone H4 acetylation levels both in primary cultures of rat hepatocytes and in HTC cells (generously provided by Dr. Roger Chalkley, University of Iowa). After an initial increase in histone acetylation in butyratetreated primary hepatocyte cultures, histone H4 acetylation declined and ap-
-
295
AMINOTRANSFERASE
proached pretreatment levels within 24 h of the addition of butyrate (Table II). Histone H4 acetylation continued to increase in butyrate-treated HTC cells for the entire 24 h period (Table II), indicating that butyrate was metabolized by HTC cells much more slowly than in primary cultures of adult rat hepatocytes. It has previously been shown that HTC cells metabolize butyrate (19). These results, in conjunction with the results shown above, indicate that the TAT induction in butyrate-treated hepatocytes occurs only subsequent to the lowering of butyrate levels by its metabolism. To test the possibility that a metabolite of butyrate was responsible for increased TAT levels at 24 h (Fig. l), we investigated the effects of propionate on TAT induction in primary hepatocyte cultures. Propionate is nearly as effective as butyrate in
TABLE
II
CHANGESINHISTONE H~ACETYLATIONAFTER BUTYRATEADDITION" Hours
after 5
addition
of
mM butyrate
0
1
3
6
24
66
53
45
38
59
-
-
6
6
6
Primary hepatocytes H4-parental
control
6
16
24
Hours post-induction
FIG. 5. The effect on TAT induction
of supplementing butyrate-treated cells with additional butyrate. This experiment is identical with the one represented by Fig. 1, except that an addition of 1 M butyrate (to 5 mM butyrate) was made to some butyrate-treated cultures, as indicated. Similar results were observed in another experiment performed in an identical manner. Variation at data points is expressed as in Fig. 1.
a Numbers reflect percentage of total histone H4 in indicated form at times after the addition of 5 mM butyrate. The most highly acetylated forms were usually present at very low levels that could not be measured (indicated by -). H4Aci refers to monoacetylated H4; H4Aca refers to diacetylated H4, etc. Primary hepatocyte cultures were treated as described for other experiments. HTC cells were treated in a manner identical with that for primary hepatocyte cultures after reaching confluency.
296
STAECKER
inhibiting histone deacetylation (20) and, similar to butyrate, promotes the differentiated phenotype of cultured hepatocytes (Staecker et al., submitted). Propionate is the ionized form of a three-carbon fatty acid and thus is metabolized differently from the even-numbered four-carbon fatty acid butyrate. The different metabolism of butyrate and propionate presumably results in the different effects of these two compounds on glycolysis, lactate formation, and pH changes in primary cultures of adult rat hepatocytes (16). Despite the differences in butyrate and propionate metabolism, we found that both compounds had similar effects on TAT induction by dexamethasone in rat hepatocyte cultures (Fig. 6). DISCUSSION
The studies reported in this paper extend other investigations on the effects of butyrate on gene expression in primary cultures of adult rat hepatocytes. In primary hepatocyte cultures, induction of TAT gene expression did not occur initially in the presence of 5 lllM butyrate, although increased TAT gene expression
120. .
o-2 control H 5 mM butyrate k45mMpmphate
loo-
0 Hours
16 post-induction
24
FIG. 6. The effect of propionate on TAT induction in cultured hepatocytes. TAT activity is shown for cells that were untreated, treated with 5.0 mM propionate, or treated with 5.0 mM butyrate, as indicated. Similar results were observed in three other experiments. Variation at data points is expressed as in Fig. 1.
AND
PITOT
was observed in cultures after butyrate metabolism. Three different experiments indicated that TAT induction occurred subsequent to butyrate depletion: (i) the addition of more butyrate after 8 h of culture inhibited TAT induction; (ii) within 24 h of addition of medium containing 5 mM butyrate to hepatocytes, essentially no butyrate remained; and (iii) while histone acetylation levels initially increased upon the addition of butyrate, the increase was short-lived, with histone acetylation levels returning to near-normal levels by the 24th h of culture. In a previous report it was claimed that butyrate had little effect on TAT induction in primary cultures of rat hepatocytes (14). However, in that study hepatocytes were continuously exposed to dexamethasone, and TAT activity was analyzed only after the daily feeding (14). Previous studies from this laboratory (21) have shown that TAT may be induced within 8 h by addition of cortisone to primary cultures of adult rat hepatocytes. Butyrate is metabolized by primary cultures of hepatocytes (16, 17, and this report) and by HTC cells, a cell line derived from a rat hepatoma (19). Depletion of sodium butyrate from the culture medium of Friend erythroleukemia cells has recently been reported (22). The variable and apparently widespread metabolism of butyrate by cultured cells indicates that any effects of butyrate in vitro should be correlated with butyrate metabolism, particularly when one is working with cultured cells of hepatic origin. We have previously shown that butyrate treatment of primary hepatocyte cultures increased basal TAT gene expression (Staecker et al., submitted). The previously observed increase in basal TAT gene expression as a result of butyrate treatment was constant throughout the culture period and did not fluctuate in relation to the metabolism of butyrate or induction of TAT gene expression shown in this report. We believe that both the stimulation of basal levels and the inhibition of induced levels of TAT gene expression by butyrate may be due to the effects
EFFECT
OF
BUTYRATE
ON
TYROSINE
of butyrate on histone acetylation. Butyrate treatment of cultured cells inhibits histone deacetylation, which may be the mechanism by which butyrate inhibits TAT induction (7). We previously have shown that histone acetylation levels spontaneously decline in primary cultures of adult rat hepatocytes and have suggested that butyrate may preserve aspects of the differentiated phenotype (e.g., TAT gene expression) of these cells by increasing histone acetylation (Staecker et al., submitted). If one assumes that butyrate treatment increased basal TAT gene expression as a result of increased histone acetylation, the same process could be responsible for the higher level of TAT gene expression observed in butyrate-treated, dexamethasone-induced, primary hepatocyte cultures. It is possible that, whereas histone hyperacetylation may block TAT induction (7), minimal histone acetylation levels may be necessary for maximal induction of TAT. It has previously been reported that histone acetylation increases during TAT induction (23). Part of the process involved in TAT induction in HTC cells appears to occur in the presence of butyrate. Simultaneous addition of butyrate and dexamethasone does not result in TAT induction in HTC cells (6). However, in HTC cells previously treated with butyrate and dexamethasone, TAT induction occurs more rapidly after removal of butyrate than is observed when dexamethasone alone is added to untreated cells (6). We tried to duplicate the experiment described above using primary hepatocyte cultures instead of HTC cells. Both times such an experiment was attempted, removal of medium from control and butyrate-treated cells resulted in inhibition of the induction of TAT activity; this suggests that medium replacement may inhibit TAT induction in cultured hepatocytes. The part of the TAT induction process that occurs in the presence of butyrate in HTC cells (see above) may be stimulated by butyrate in primary hepatocyte cultures, resulting in the hyperinduction of TAT gene expression that we observed in butyrate-treated primary he-
297
AMINOTRANSFERASE
patocyte cultures. We have previously suggested that a similar phenomenon (i.e., DNA synthesis stimulation by optimal histone acetylation levels) may be responsible for the stimulation of DNA synthesis in primary rat hepatocyte cultures by 0.5 or 1.0 mM butyrate, despite inhibition of DNA synthesis by 5.0 mM butyrate (Staecker et al, submitted). The results of the present study indicate that butyrate treatment (5.0 mM) of cultured hepatocytes does not result in uniformly high histone acetylation levels, but, because of butyrate metabolism, results in a pulse of increased histone aeetylation. The butyrate-induced fluctuation in histone acetylation levels is obviously not a normal process. However, it has been reported that pulses of increased histone acetylation occur sequentially over chromatin domains, thereby sequentially exposing those domains (24). The artificial, butyrate-induced pulse of increased acetylation in primary hepatocyte cultures that are otherwise deficient in histone acetylation may affect gene expression in
a manner
similar
to the sequential
in-
creases in histone acetylation that normally occur. We have previously determined that treatment with 5 mM butyrate is beneficial to primary cultures of adult rat hepatocytes. However, toxic effects were observed after 3-5 days of culture when primary rat hepatocyte cultures were treated with butyrate if the cells were damaged, were of low viability, or when the exposure of cells to 5 mM butyrate was increased by additional feedings (results not shown). Increasing the concentration of butyrate or increasing the amount of medium while holding the butyrate concentration constant (results not shown) was also toxic to hepatocytes after 3-5 days of culture. We believe that treatment of hepatocyte cultures with one dose of 5 mM butyrate is not toxic because of the subsequent metabolism of butyrate by the cells. Our observations concerning butyrate toxicity are consistent with the toxicity observed when other cultured cells are cultured for a period of time (2 or more days) in the pres-
298
STAECKER
AND
ence of 5 mM butyrate (see (7) for discussion of butyrate toxicity). 11. ACKNOWLEDGMENTS We thank Gerry Sattler and Steve Foltz for their expert technical assistance and Kristen Luick for typing this manuscript. We also thank Dr. Ilse Riegel for her helpful suggestions concerning the preparation of this manuscript. REFERENCES 1. KRUH, J. (1982) Mol. Cell. BioL 42, 65-82. 2. COVAULT, J., AND CHALKLEY, R. (1980) J. Biol. Chem, 255,9110-9116. 3. DUNCAN, M. R., ROBINSON, M. J., AND DELL’ORCO, R. T. (1983) Biochim. Biophys. Acta 762, 221-226. 4. GROENEWALD, J. V., TERBLANCHE, S. E., AND OELOFSEN, W. (1984) Innt. J. Biochem. 16, l-18. 5. TICHONICKY, L., SANTANA-CALDERON, M. A., DEFER, N., GIESEN, E. M., BECK, G., AND KRUH, J. (1981) Eur. J B&hem. 120,427-433. 6. PLESKO, M. M., HARGROVE, J. L., GRANNER, D. K., AND CHALKLEY, R. (1983) J. BioL Chem. 258, 13738-13744. 7. CHALKLEY, R., AND SHIRES, A. (1985) J. BioL Chem. 260,7698-7704. 8. KREAMER, B. L., STAECKER, J. L., SAWADA, N., SATTLER, G. L., HSIA, M. T. S., AND PITOT, H. C. (1986) In Vitro 22, 201-211. 9. DIAMONDSTONE, T. I. (1966) Anal. Biochem. 16, 395-401. 10. GRANNER, D. K., AND TOMKINS, G. M. (1970) in Methods in Enzymology (Tabor, H., and
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14. 15.
16. 17.
18. 19. 20. 21. 22.
23. 24.
PITOT Tabor, C. W., Eds.), Vol. 17, Part A, pp. 633-637, Academic Press, New York. BEER, D. G., SCHWARZ, M., SAWADA, N., AND PITOT, H. C. (1986) Cancer Res. 46,2435-2441. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982) Molecular Cloning, a Laboratory Manual, pp. 202-206, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. ALFAGEME, C. R., ZWEIDLER, A., MAHOWALD, A., AND COHEN, L. H. (1974) J. Biol. Chem. 249, 3729-3736. ENGELMANN, G. L., STAECKER, J. L., AND RICHARDSON, A. G. (1987) In Vitro 23,86-92. NAWA, K., NAKAMURA, T., KUMATORI, A., NODA, C., AND ICHIHARA, A. (1986) J. BioL Chem. 261, 16883-16888. ANDERSON, J. W., AND BRIDGES, S. R. (1984) PTOC. Sot. Exp. BioL Med. 177,372-376. DEMIGNE, C., YACOUB, C., REMESY, C., AND FAFOURNOUX, P. (1986) Biochim. Biophys. Acta 875,535542. FLEMING, S. E., TRAITLER, H., AND KOELLREUTER, B. (1987) Lipids 22,195-200. WRIGHT, P. S., DUDLEY, D. T., AND CHALKLEY, R. (1986) Arch. Biochem. Biophys. 248,243-252. MCKNIGHT, G. S., HAGER, L., AND PALMITER, R. D. (1980) Cell 22,469-477. MICHALOPOULOS, G., SATTLER, G. L., AND PITOT, H. C. (1978) Cancer Res. 38,1550-1555. FRIEND, C., ZAJAC-KAYE, M., HOLLAND, J. G., AND POGO, B. G. T. (1987) Cancer Res. 47, 378-382. GRAAFF, G. D. V., AND VONHOLT, C. (1973) Biochim. Biophys. Acta 299,480-484. PERRY, M., AND CHALKLEY, R. (1982) J. Biol. Chem. 257,7336-7347.
The Effect of Sodium Butyrate on Tyrosine Aminotransferase Induction Primary Cultures of Normal Adult Rat Hepatocytes’ JEFFREY
L. STAECKER
AND
HENRY
in
C. PITOT’
The University of Wisconsin, McArdle Laboratory for Cancer Research, 450 North Randall Avenue, Madison, Wisconsin 5.3i’O6 Received
July
10, 198’7, and in revised
form
November
10.1987
Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et ab, submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT)
manner.
0 1988 Academic
Press, Inc.
Butyrate inhibits histone deacetylation and induces histone hyperacetylation when it is added to cultured cells (1). In addition, butyrate has a variety of other effects on cultured eukaryotic cells, including suppression of the characteristics of transformation in many cell lines, induction of changes in gene expression, and inhibition of cell growth (1). The effects of butyrate listed above have been correlated with butyrate-induced histone acetylation changes in many cultured cells (1). Histone acetylation in HTC cells is represented by at least two kinetic classes: rapid histone acetylation in which acetate turnover occurs with a half-life of approx-
imately 5 min, and slow histone acetylation in which acetate turnover occurs with a half-life of approximately 300 min (2). Similar kinetics for histone acetylation have been reported for cultured human diploid fibroblasts (3). The relationship between histone acetylation and TAT3 induction has been studied in HTC cells. TAT, an enzyme found primarily in the liver, catalyzes the first reaction of a pathway by which tyrosine can ultimately be degraded to fumarate and acetoacetate (4). The induction of TAT in HTC cells is abolished by treatment with 5 mM sodium butyrate (5, 6). Evidence that the inhibition of TAT induction in HTC cells by butyrate is caused by butyrate-induced changes in fast histone acetylation has
i This study was supported in part by Grants CA 07175 and CA 22484 from the National Cancer Institute. J.L.S. was a recipient of a predoctoral traineeship from the National Cance Institute (T32-CA 09135). ‘To whom correspondence should be addressed.
3 Abbreviations used: TAT, tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5); Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. 291
0003-9861188 Copyright All rights
$3.00
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
292
STAECKER
been provided by Chalkley’s laboratory. Plesko et al. (6) have correlated the effect of butyrate on fast acetylation with TAT induction. Shires and Chalkley (7) isolated numerous HTC variants in which histone deacetylation, as well as cell replication and TAT induction, continued in the presence of butyrate. We have recently demonstrated that butyrate treatment inhibits the loss of some aspects of the differentiated phenotype of primary cultures of normal adult rat hepatocytes and preserves high levels of basal TAT gene expression (Staecker et al., submitted). Previous reports have indicated that butyrate addition to cultured hepatoma cell lines completely inhibits TAT induction without affecting basal TAT gene expression in these cells (5, 6). In continuing our studies of the effects of butyrate on the regulation of gene expression in primary cultures of normal hepatocytes, we have studied the effect of this small-chain fatty acid on TAT gene expression in primary rat hepatocyte culture compared with reports on the HTC hepatoma cell line (5, 6). MATERIALS
AND
METHODS
Hepatocyte isolation and culture. Hepatocytes were isolated by an in situ perfusion technique previously described (method d in Ref. (8)). Viable hepatocytes, as judged by trypan blue exclusion, were separated from nonviable hepatocytes and nonparenchymal cells prior to plating (8), resulting in an initial viability of more than 95%. Cells were then plated onto plastic tissue culture dishes (60 or 100 mm) and cultured in Leibovitz (L-15) medium supplemented with Hepes (15 mM), penicillin (100 pg/ml), streptomycin (100 pg/ml), insulin (1.5 pg/ml), and glucose (1.5 mg/ml). During the initial 2 h of culture (attachment period) the medium was also supplemented with newborn bovine serum (2.5%). Treatment of the cells with butyrate or propionate was begun with the initial medium change unless otherwise noted. Medium containing butyrate or propionate was prepared by addition of an appropriate amount of a filter-sterilized, concentrated, 1 M solution of sodium butyrate or propionate that had been prepared by dissolving the compounds in culture medium. TAT assay. TAT activity was determined according to the method of Diamondstone (9) as modified by Granner and Tomkins (10) and described by Kreamer et al. (8), except that the cells were freezethawed while they were attached to the culture
AND
PITOT
dishes. TAT activity values were averages of duplicate determinations made on three separate plates of cells. Unless otherwise noted, TAT gene expression was induced by the inclusion of dexamethasone (10m6) in the culture medium. RNA analysis. Cells were disrupted in 4 M guanidium thiocyanate, and the RNA was pelleted by centrifugation through a cushion of 5.7 M CsClz-0.1 M EDTA, pH 7.0, as previously described (11). RNA was quantified spectrophotometrically by the relationship: 1.0 unit equivalent of AzW = 32 gg/ml RNA. Total cellular RNA was separated in 1.2% agaroseformaldehyde gels according to the method of Maniatis et al. (12) and transferred to Gene Screen Plus filters (New England Nuclear) for Northern gel analysis. Mammalian and Escherichia coli ribosomal RNAs were used as size markers for Northern gels. Analysis of the Northern transfers was accomplished by probing with nick-translated clone pcTAT-3 for TAT (generously provided by Dr. G. Schiitz, Institute of Cell and Tumor Biology, Heidelberg). Subsequent steps for Northern analysis (prehybridization, hybridization, washings, and autoradiography steps) were identical with those detailed previously (11). Histone isolation and electrophoresis. Histones were isolated from cultured hepatocytes and separated according to the method of Alfageme et al. (13) as described (14). After Coomassie blue staining, the gels were scanned with a Zeineh Model Sl-504-X1 soft laser densitometer (Biomed Corp., Chicago, IL), and the peaks were integrated with a Numonics 1224 electronic digitizer (Numonics Corp., Landsdale, PA). HTC culture. Large quantities of HTC cells were grown in T-flasks (Corning) in modified Swim’s 77 medium with 2.5% calf and 2.5% fetal calf serum, as described by Covault and Chalkley (2). The cells were then plated onto 60- or loo-mm tissue culture plates (Corning) for experimental purposes. Butyrate addition to HTC cells was accomplished in a manner identical with that used for primary hepatocyte cultures, except that the 1 M butyrate stock was made up in Swim’s 77 medium containing 2.5% calf and 2.5 fetal calf serum.
RESULTS
The effects of butyrate on TAT induction by dexamethasone or glucagon were investigated in primary cultures of rat hepatocytes. After numerous preliminary experiments, it became apparent that the effect of butyrate on TAT induction was dependent on the time after induction that TAT activity was assayed. There was little or no TAT induction in butyrate-treated
EFFECT
OF
BUTYRATE
ON
TYROSINE
cultures up to 8 h following the addition of dexamethasone to the cultures (Fig. 1). This delay was followed by an increase in TAT activity observed 16 h after induction was begun (Fig. 1). TAT activity levels 24 h after induction by dexamethasone was initiated were much higher in butyratetreated cells than in cultures without butyrate on both the second and third days of culture (Fig. 1). Similar results were obtained when TAT gene expression was induced by glucagon instead of dexamethasone (Table I). There was variability in TAT induction from experiment to experiment in both control and butyrate-treated cultures, as shown by comparing the results in Figs. 1 and 3-6. However, we always found that after initially inhibiting TAT induction, butyrate treatment resulted in a higher level of TAT induction than in control cultures without butyrate (e.g., Figs. 1 and 3-6). Previous studies (5, 6) have shown that induction of TAT activity in HTC cells is accompanied by increases in TAT mRNA levels and that both TAT activity and mRNA induction are blocked by the presence of 5 mM butyrate (5,6). We measured
”
I 8
’
16 24 6 Hours post-induction
clay 2 of culture
’
16
Day 3 of culture
24 i
FIG. 1. The effect of butyrate on TAT induction in cultured hepatocytes. Cells were cultured as described under Materials and Methods and treated as indicated. Induction was initiated by replacing the medium after 1 day of culture with medium containing 10F6 dexamethasone. Plates were harvested during the second and third days of culture at the indicated times. TAT activity was determined as described under Materials and Methods and expressed as milliunits TAT activity per milligram protein. Similar results were observed in nine other experiments (not shown). TAT induction and the expression of TAT activity were done in an identical manner in Figs. 3-6. Standard deviation of the mean is indicated at data points.
AMINOTRANSFERASE
293
TAT mRNA levels in primary hepatocyte cultures in the presence and absence of butyrate by Northern and dot-blot analysis. The results of a Northern analysis are shown in Fig. 2.; the higher level of TAT activity in butyrate-treated cells was accompanied by a higher level of TAT mRNA than that in control cultures. Similar results (not shown) were observed in different experiments when dot-blot analysis was used to analyze the changes in TAT mRNA levels. Our results suggest that induction of TAT by dexamethasone of normal and butyrate-treated hepatocytes may result from increased transcription of the TAT gene. Previous work has shown that TAT induction in cultured hepatocytes, as well as in other systems, is accomplished primarily by increased transcription of the TAT gene (4, 15). The effects of butyrate on TAT induction by dexamethasone were examined as an extension of a series of experiments in which we determined that butyrate helped to preserve aspects of the differentiated phenotype of cultured hepatocytes (Staecker et ab, submitted). Initially the effects of butyrate on TAT induction in cultures that had been exposed to butyrate prior to and during the induction period were examined. To determine whether the effects of butyrate on TAT induction were due to exposure of the cells prior to or during the induction period, we discontinued butyrate treatment of some cultures while simultaneously beginning butyrate treatment of other cultures. Prior exposure of cultures to butyrate had little effect on the induction of TAT, although a slight increase was observed in cells pretreated with butyrate, commensurate with the increase in the basal TAT activity observed in butyrate-treated cells (Fig. 3). The effects of butyrate on TAT induction were found to be dependent on the concentration of butyrate in the culture medium. A higher level of TAT activity was observed 24 h after induction in cells treated with 1, 2.5, or 5.0 mM butyrate, although inhibition of TAT induction after 8 h of induction was observed only in cultures treated with 2.5 or 5.0 mM butyrate (Fig. 4).
294
STAECKER
AND TABLE
THE EFFECT
OF BUTYRATE
ON TAT
Oh Treatment Control
18.3 28.4
a TAT
activity
INDUCTION
10m6 Dex
f 1.3" +- 1.5
is expressed
42.1 38.4
as microunits
31 3.2 k 3.4
per milligram
We investigated the possibility that TAT induction in butyrate-treated cultures occurred subsequent to the elimination of butyrate by its metabolism, since previous reports indicated that primary hepatocyte cultures readily metabolize butyrate (16, 17). In initial experiments, we found that introducing additional butyrate into butyrate-treated cultures 8 h after induction was initiated inhibited TAT induction (Fig. 5). These results indicate that the metabolism of butyrate by cultured hepatocytes, with concomitant release from inhibition by butyrate, may be responsible for subsequent TAT induction in butyrate-treated cells.
2.3
I BY DEXAMETHASONE
OR GLU~AGON
8 h postinduction
Basal
5 mM butyrate
PITOT
24 h postinduction
10m6 Glucagon 37.4
f
35.7
-t 3.3
protein
10m6 Dex
4.2
92.8
+ standard
+
10m6 Glucagon 6.2
70.8
f
7.9
149.4 i 10.2
89.4
f
3.5
deviation
of the mean.
We were able to obtain direct measurements of the metabolism of butyrate by cultured hepatocytes through analysis of fresh butyrate-supplemented medium and spent butyrate-supplemented medium (spent medium refers to medium removed from butyrate-treated hepatocytes after the second day of culture). Dr. Sharon Fleming (University of California, Berkeley) generously performed the measurement of butyrate in our samples, using an established gas chromatography procedure (18). After 24 h of culture, the metabolism of butyrate by the hepatocyte culture reduced the concentration of butyrate from 5 mM to less than 0.3 mM. The effects of the metabolism of butyrate by primary cultures of hepatocytes are
-
6
Hours
FIG. 2. The effect of butyrate on TAT mRNA levels. RNA was isolated from cells after 2 days of culture, and 20 Kg of RNA was spotted on each lane. RNA was obtained from butyrate-treated (lanes 3 and 4) and control cells (lanes 1 and 2) in which TAT gene expression was induced (lanes 2 and 4) or from uninduced cells (lanes 1 and 3).
16
24
post-induction
FIG. 3. The effect of prior butyrate treatment on TAT induction. After 2 days of culture in the presence or absence of butyrate, the medium was replaced with medium containing dexamethasone with and without 5 nIM butyrate, as indicated. Similar results were observed in two other experiments. Variation at data points is expressed as in Fig. 1.
EFFECT
120
H 0-3 H
100
OF
BUTYRATE
ON
TYROSINE
1 mM butyrate 2.5 mM butyrale 5.0 mM butyrate
.E Z”60 .g h SF 2 3 60 ‘E 40
20
6 16 Hours post-induction
24
FIG. 4. The effect of various concentrations of butyrate on TAT induction. TAT induction by 10m6 dexamethasone was determined on hepatocytes cultured in various concentrations of butyrate, as indicated. Similar results were observed in two other experiments. Variation at data points is expressed as in Fig. 1.
reflected in changes in histone H4 acetylation. We examined the effects of the metabolism of butyrate on histone H4 acetylation levels both in primary cultures of rat hepatocytes and in HTC cells (generously provided by Dr. Roger Chalkley, University of Iowa). After an initial increase in histone acetylation in butyratetreated primary hepatocyte cultures, histone H4 acetylation declined and ap-
-
295
AMINOTRANSFERASE
proached pretreatment levels within 24 h of the addition of butyrate (Table II). Histone H4 acetylation continued to increase in butyrate-treated HTC cells for the entire 24 h period (Table II), indicating that butyrate was metabolized by HTC cells much more slowly than in primary cultures of adult rat hepatocytes. It has previously been shown that HTC cells metabolize butyrate (19). These results, in conjunction with the results shown above, indicate that the TAT induction in butyrate-treated hepatocytes occurs only subsequent to the lowering of butyrate levels by its metabolism. To test the possibility that a metabolite of butyrate was responsible for increased TAT levels at 24 h (Fig. l), we investigated the effects of propionate on TAT induction in primary hepatocyte cultures. Propionate is nearly as effective as butyrate in
TABLE
II
CHANGESINHISTONE H~ACETYLATIONAFTER BUTYRATEADDITION" Hours
after 5
addition
of
mM butyrate
0
1
3
6
24
66
53
45
38
59
-
-
6
6
6
Primary hepatocytes H4-parental
control
6
16
24
Hours post-induction
FIG. 5. The effect on TAT induction
of supplementing butyrate-treated cells with additional butyrate. This experiment is identical with the one represented by Fig. 1, except that an addition of 1 M butyrate (to 5 mM butyrate) was made to some butyrate-treated cultures, as indicated. Similar results were observed in another experiment performed in an identical manner. Variation at data points is expressed as in Fig. 1.
a Numbers reflect percentage of total histone H4 in indicated form at times after the addition of 5 mM butyrate. The most highly acetylated forms were usually present at very low levels that could not be measured (indicated by -). H4Aci refers to monoacetylated H4; H4Aca refers to diacetylated H4, etc. Primary hepatocyte cultures were treated as described for other experiments. HTC cells were treated in a manner identical with that for primary hepatocyte cultures after reaching confluency.
296
STAECKER
inhibiting histone deacetylation (20) and, similar to butyrate, promotes the differentiated phenotype of cultured hepatocytes (Staecker et al., submitted). Propionate is the ionized form of a three-carbon fatty acid and thus is metabolized differently from the even-numbered four-carbon fatty acid butyrate. The different metabolism of butyrate and propionate presumably results in the different effects of these two compounds on glycolysis, lactate formation, and pH changes in primary cultures of adult rat hepatocytes (16). Despite the differences in butyrate and propionate metabolism, we found that both compounds had similar effects on TAT induction by dexamethasone in rat hepatocyte cultures (Fig. 6). DISCUSSION
The studies reported in this paper extend other investigations on the effects of butyrate on gene expression in primary cultures of adult rat hepatocytes. In primary hepatocyte cultures, induction of TAT gene expression did not occur initially in the presence of 5 lllM butyrate, although increased TAT gene expression
120. .
o-2 control H 5 mM butyrate k45mMpmphate
loo-
0 Hours
16 post-induction
24
FIG. 6. The effect of propionate on TAT induction in cultured hepatocytes. TAT activity is shown for cells that were untreated, treated with 5.0 mM propionate, or treated with 5.0 mM butyrate, as indicated. Similar results were observed in three other experiments. Variation at data points is expressed as in Fig. 1.
AND
PITOT
was observed in cultures after butyrate metabolism. Three different experiments indicated that TAT induction occurred subsequent to butyrate depletion: (i) the addition of more butyrate after 8 h of culture inhibited TAT induction; (ii) within 24 h of addition of medium containing 5 mM butyrate to hepatocytes, essentially no butyrate remained; and (iii) while histone acetylation levels initially increased upon the addition of butyrate, the increase was short-lived, with histone acetylation levels returning to near-normal levels by the 24th h of culture. In a previous report it was claimed that butyrate had little effect on TAT induction in primary cultures of rat hepatocytes (14). However, in that study hepatocytes were continuously exposed to dexamethasone, and TAT activity was analyzed only after the daily feeding (14). Previous studies from this laboratory (21) have shown that TAT may be induced within 8 h by addition of cortisone to primary cultures of adult rat hepatocytes. Butyrate is metabolized by primary cultures of hepatocytes (16, 17, and this report) and by HTC cells, a cell line derived from a rat hepatoma (19). Depletion of sodium butyrate from the culture medium of Friend erythroleukemia cells has recently been reported (22). The variable and apparently widespread metabolism of butyrate by cultured cells indicates that any effects of butyrate in vitro should be correlated with butyrate metabolism, particularly when one is working with cultured cells of hepatic origin. We have previously shown that butyrate treatment of primary hepatocyte cultures increased basal TAT gene expression (Staecker et al., submitted). The previously observed increase in basal TAT gene expression as a result of butyrate treatment was constant throughout the culture period and did not fluctuate in relation to the metabolism of butyrate or induction of TAT gene expression shown in this report. We believe that both the stimulation of basal levels and the inhibition of induced levels of TAT gene expression by butyrate may be due to the effects
EFFECT
OF
BUTYRATE
ON
TYROSINE
of butyrate on histone acetylation. Butyrate treatment of cultured cells inhibits histone deacetylation, which may be the mechanism by which butyrate inhibits TAT induction (7). We previously have shown that histone acetylation levels spontaneously decline in primary cultures of adult rat hepatocytes and have suggested that butyrate may preserve aspects of the differentiated phenotype (e.g., TAT gene expression) of these cells by increasing histone acetylation (Staecker et al., submitted). If one assumes that butyrate treatment increased basal TAT gene expression as a result of increased histone acetylation, the same process could be responsible for the higher level of TAT gene expression observed in butyrate-treated, dexamethasone-induced, primary hepatocyte cultures. It is possible that, whereas histone hyperacetylation may block TAT induction (7), minimal histone acetylation levels may be necessary for maximal induction of TAT. It has previously been reported that histone acetylation increases during TAT induction (23). Part of the process involved in TAT induction in HTC cells appears to occur in the presence of butyrate. Simultaneous addition of butyrate and dexamethasone does not result in TAT induction in HTC cells (6). However, in HTC cells previously treated with butyrate and dexamethasone, TAT induction occurs more rapidly after removal of butyrate than is observed when dexamethasone alone is added to untreated cells (6). We tried to duplicate the experiment described above using primary hepatocyte cultures instead of HTC cells. Both times such an experiment was attempted, removal of medium from control and butyrate-treated cells resulted in inhibition of the induction of TAT activity; this suggests that medium replacement may inhibit TAT induction in cultured hepatocytes. The part of the TAT induction process that occurs in the presence of butyrate in HTC cells (see above) may be stimulated by butyrate in primary hepatocyte cultures, resulting in the hyperinduction of TAT gene expression that we observed in butyrate-treated primary he-
297
AMINOTRANSFERASE
patocyte cultures. We have previously suggested that a similar phenomenon (i.e., DNA synthesis stimulation by optimal histone acetylation levels) may be responsible for the stimulation of DNA synthesis in primary rat hepatocyte cultures by 0.5 or 1.0 mM butyrate, despite inhibition of DNA synthesis by 5.0 mM butyrate (Staecker et al, submitted). The results of the present study indicate that butyrate treatment (5.0 mM) of cultured hepatocytes does not result in uniformly high histone acetylation levels, but, because of butyrate metabolism, results in a pulse of increased histone aeetylation. The butyrate-induced fluctuation in histone acetylation levels is obviously not a normal process. However, it has been reported that pulses of increased histone acetylation occur sequentially over chromatin domains, thereby sequentially exposing those domains (24). The artificial, butyrate-induced pulse of increased acetylation in primary hepatocyte cultures that are otherwise deficient in histone acetylation may affect gene expression in
a manner
similar
to the sequential
in-
creases in histone acetylation that normally occur. We have previously determined that treatment with 5 mM butyrate is beneficial to primary cultures of adult rat hepatocytes. However, toxic effects were observed after 3-5 days of culture when primary rat hepatocyte cultures were treated with butyrate if the cells were damaged, were of low viability, or when the exposure of cells to 5 mM butyrate was increased by additional feedings (results not shown). Increasing the concentration of butyrate or increasing the amount of medium while holding the butyrate concentration constant (results not shown) was also toxic to hepatocytes after 3-5 days of culture. We believe that treatment of hepatocyte cultures with one dose of 5 mM butyrate is not toxic because of the subsequent metabolism of butyrate by the cells. Our observations concerning butyrate toxicity are consistent with the toxicity observed when other cultured cells are cultured for a period of time (2 or more days) in the pres-
298
STAECKER
AND
ence of 5 mM butyrate (see (7) for discussion of butyrate toxicity). 11. ACKNOWLEDGMENTS We thank Gerry Sattler and Steve Foltz for their expert technical assistance and Kristen Luick for typing this manuscript. We also thank Dr. Ilse Riegel for her helpful suggestions concerning the preparation of this manuscript. REFERENCES 1. KRUH, J. (1982) Mol. Cell. BioL 42, 65-82. 2. COVAULT, J., AND CHALKLEY, R. (1980) J. Biol. Chem, 255,9110-9116. 3. DUNCAN, M. R., ROBINSON, M. J., AND DELL’ORCO, R. T. (1983) Biochim. Biophys. Acta 762, 221-226. 4. GROENEWALD, J. V., TERBLANCHE, S. E., AND OELOFSEN, W. (1984) Innt. J. Biochem. 16, l-18. 5. TICHONICKY, L., SANTANA-CALDERON, M. A., DEFER, N., GIESEN, E. M., BECK, G., AND KRUH, J. (1981) Eur. J B&hem. 120,427-433. 6. PLESKO, M. M., HARGROVE, J. L., GRANNER, D. K., AND CHALKLEY, R. (1983) J. BioL Chem. 258, 13738-13744. 7. CHALKLEY, R., AND SHIRES, A. (1985) J. BioL Chem. 260,7698-7704. 8. KREAMER, B. L., STAECKER, J. L., SAWADA, N., SATTLER, G. L., HSIA, M. T. S., AND PITOT, H. C. (1986) In Vitro 22, 201-211. 9. DIAMONDSTONE, T. I. (1966) Anal. Biochem. 16, 395-401. 10. GRANNER, D. K., AND TOMKINS, G. M. (1970) in Methods in Enzymology (Tabor, H., and
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