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The effect of retinoids and clofibric acid on the peroxisomal oxidation of palmitic acid and of 3α,7α,12α-trihydroxy-5β-cholestanoic acid in rat and rabbit hepatocytes
100
ffiochirrucu ef Biophysics Acru, 116X(1993) IOO-107 0 1993 Elsevier Science Publishers B.V. All.rights reserved Ol~S-2760/“).3/$(fh.(K)
BBALIP 54175
The effect of retinoids and clofibric acid on the peroxisomal oxidation of palmitic acid and of 3cq7cYJ2a-trihydroxy-5P-cholestanoic acid in rat and rabbit hepatocytes Ann-Kristin 6stlund Farrants, Astrid Nilsson ‘, Gunhild Trgen, Rune Blomhoff and Jan I. Pedersen Institute for Nutrition Research, School
ofMedicine, University of Oslo, Oslo (Norway)
(Received 7 October 1992)
Key words: Hepatocytes; Morris rat hepatoma cell; Peroxisomal @oxidation; Retinoids; Bile acids; (Rabbit)
The effects of retinoids and the peroxisome proliferator clofibric acid on peroxisomal enzyme pathways were studied in hepatocytes from both rat and rabbit. Retinoic acid and retinal increased the activity of acyl-CoA oxidase in rabbit hepatocytes around 60% and around 30% in rat hepatocytes. Exposure to clofibric acid caused an increase in acyI-CoA oxidase activity of 115% in rat hepatocytes and of 40% in rabbit hepatocytes, indicating that rabbit is less sensitive to peroxisome proliferator than rat. Simultaneous exposure to clofibric acid and retinoids did not act additatively or synergistically. Both rabbit and rat hepatocytes expressed mRNA for the peroxisome proliferator activated receptor, (PPAR), although the transcript in rabbit was slightly smaller compared to that expressed in rat hepatocytes. The effect of retinoic acid in 7800 Cl Morris rat hepatoma cells, a cell line known to have an inducible pero~somal ~-oxidation of fatty acids, was only slight with an increase of the acyl-CaA oxidase activity of 25% compared with control cells. As for clofibric acid, which gave a 2-fold induction of the acyl-CoA oxidase activity, the effect of retinoic acid was potentiated by dexamethasone. These ceils also expressed mRNA for PPAR, with the same size as that found in rat hepatocytes. The oxidation of 3cu,7ar,l2a-trihydroxy-5P-cholestanoic acid (THCA), an intermediate in bile acid formation, in rat hepatocytes increased 110% by clofibric acid and around 80%) by retinoic acid. In rabbit hepatocytes, clofibric acid increased the oxidation rate 75% and retinoic acid 100%. The results presented here show similarities in the effects of retinoids and clofibric acid on the acyl-CoA oxidase activity and the oxidation rate of THCA, since they increase these two peroxisomal activities in hepatocytes in vitro. A decrease in both these enzyme activities occurs during cultivation time in untreated primary hepatocyte cultures. The present data may therefore either be explained by an increased expression or an induced stability of the enzymes involved.
Hypolipidemic drugs, such as clofibrate, and plasticizers cause peroxisomal proliferation and induction of the peroxisomal p-oxidation of fatty acids in rodents (for review see Refs. 1, 2). An increased mRNA level [3,4] with an increased rate of protein synthesis (31 for the induced enzymes is observed after treatment with these agents. The oxidative cleavage of the C,, steroid side chain in bile acid formation has also been found to occur in
Correspondence to: A.K. &tlund Farrants, Institute for Nutrition Research, University of Oslo, Pb 1046, Blindern, N-0316 Oslo, Norway. ’ Present address: MATFORSK, Norwegian Food Research Institute, As, Norway.
the peroxisomes 1.51.It follows a similar reaction mechanism to that of the peroxisomal p-oxidation of fatty acids [6,7], although the first step, the introduction of a double bond, is catalysed by two distinct oxidases, the acyl-CoA oxidase [Bl and the 3ar,7cu,12a-trihydroxy-5@cholestanoic acid (THCAI-CoA oxidase 191. While the activity of acyl-CoA oxidase is increased by administration of ciofibrate in vivo, no change has been observed in the conversion rate of 3q7q12a-trihydroxy-5/?cholestanoic acid (THCA), a C,,-bile acid intermediate, to the primary bile acid cholic acid [lO,ll]. A nuclear receptor which is activated by peroxisome proliferators has been cloned from mouse, the peroxisome proliferator activated receptor (PPAR) [ 121. Three distinct but closely related receptors have also been cloned from Xenopus laevis, xPPARa, p and y [13], and recently a rat PPAR was cloned 1141. These
101 receptors are members of the steroid/ thyroid hormone receptor family of transcription factors and it is likely that the effects of peroxisomal proliferators on gene expression are mediated by PPAR. The mouse PPAR is expressed in liver, kidney and small intestine [12]. This corresponds well with the tissue specificity of peroxisomal proliferation and induction of the peroxisomal p-oxidation of fatty acids [15-171. The endogenous ligand has not yet been identified. The PPAR is closely related to the thyroid hormone receptor (TR) and there are several reports that hyperthyroid rats have an increased peroxisomal P-oxidation compared to euthyroid controls [l&201. However, thyroid hormones given to cultures of rat hepatocytes do not induce the peroxisomal P-oxidation of fatty acids [21]. Other members of the thyroid hormone receptor family are the receptors responding to retinoids, the retinoic acid receptors (RARa, p and y) [22-271 and the retinoid X receptors (RXRa, p and yl [28,291. Both these receptors are activated by all-trans retinoic acid, but 9-cis retinoic acid has been identified as ligand for RXR [30,31]. It has recently been reported that retinoic acid induces the expression of enzymes involved in the peroxisomal P-oxidation of fatty acids in rat hepatocytes [32]. In this study we have tested the effect of clofibric acid, all-trans retinoic acid and all-truns retinol on the peroxisomal P-oxidation of fatty acids and the oxidation of THCA in rat and rabbit hepatocytes. In addition, 7800 Cl Morris rat hepatoma cells have been used to study the effect on the peroxisomal P-oxidation by the same agents in a proliferating system. Materials and Methods Chemicals
Palmitoyl-CoA, clofibric acid, retinoic acid, retinol and dexamethasone (tissue culture grade) were obtained from Sigma (MO, USA). Retinoic acid and retinol were stored as stock solutions in ethanol at - 20°C. Clofibric acid and dexamethasone were stored as stock solutions in ethanol at 4°C for 1 week. 2’,7’Dichlorofluorescin diactetate (LDCF) was obtained from Eastman, Kodak (NY, USA). 3q7a,12cY-Trihydroxy-5@[7& 3H]cholestanoic acid and the unlabelled compound were prepared from alligator bile as previously described [33]. Purification of the substrates was performed using HPLC on a Zorbax ODS column (0.46 x 25 cm) using as eluting solvent 17% 30 mM trifluoroacetic acid (TFA) (pH 2.9 with triethylamine), in methanol. Culture media were purchased from Flow Laboratories (Irvine, Scotland), as well as fetal calf serum, horse serum and gentamicin. Ultroser G was obtained from Gibco (Paisley, Scotland). All solvents were of analytical grade. Other chemicals were of commercial high purity material.
Hepatocyte preparation and culture conditions
Hepatocytes were prepared from perfusion of rat and rabbit liver using the collagenase method described by Blomhoff et al. [341. The hepatocytes were plated on 60 mm Petri dishes in Dulbecco’s Modification of Eagles Medium (DMEM) containing 2% Ultroser G, Gibco, and 2 mM r_-glutamine. During the first 24 h 0.1 mg/ml gentamicin was included in the medium. The treatment period started 24 h after plating and continued for 3 days with a change to fresh medium every day. The cells were harvested in 50 mM potassium phosphate buffer at pH 7.5 and sonicated for 2 x 10 s prior to use. Enzyme activities were measured in these cell homogenates. 7800 Cl Morris rat hepatoma cells were cultured in 1 X Ham’s F-10 medium containing 10% horse serum, 2% fetal calf serum, 0.1 mg/ml gentamicin and 2 mM L-glutamine. The cells were cultivated until confluence, then treated for 3 days and harvested as described for hepatocytes. All cells were kept at 37°C in humidified air/CO, (95 : 5, v : v) atmosphere. Enzyme assays and analytical procedures
Acyl-CoA (palmitoyl-CoA) oxidase was assayed by determination of H,O, production, coupled to the oxidation of LDCF, as described by Small et al. [35] and modified by Leighton (personal communication). The oxidation of LDCF by H,O, to 2’,7’-dichlorofluorescein was followed spectrophotometrically at 502 nm. The reaction mixture contained 0.1 M Tris-HCl (pH 8.51, 0.05 mM LDCF, 50 pg horseradish peroxidase type II (EC 1.11.1.7) 0.015 mM FAD, 0.6 mg/ml bovine serum albumin (BSA), 0.02% Triton X-100 and was started with 60 PM palmitoyl-CoA. All concentrations are given as final values. The reaction mixture contained lo-40 pg of protein in a total volume of 1 ml at 25°C. The LDCF was prepared daily at 5.1 mM in 0.01 M NaOH and stored in a light-tight container under N, gas. The conversion of the C,,-bile acid intermediates to primary bile acids was measured as described by Gstlund Farrants et al. [36]. The standard incubation mixture contained 0.1 M Tris-HCl (pH 8.0), 0.1 mM CoA, 2 mM dithiothreitol, 8.5 mM ATP, 10 mM MgCl,, 75 PM FAD, 2 mg/ml BSA, 0.01% Triton X-100 and 40 PM [3H]THCA, final concentrations. NAD was added after 15 min of incubation to a final concentration of 2 mM. The incubations contained 150-300 pg of protein in a total volume of 0.25 ml. The incubations were stopped with 5 ~1 6 M KOH, and hydrolysed at 60°C for 30 min to remove bound CoA. After acidification and extraction with ethyl acetate, the samples were analysed by reversed phase HPLC on a 5 pm C-18 Nucleosil column (0.5 x 25 cm). The eluting solvent was 24% 30 mM TFA (pH 2.9 with triethylamine)
102 in methanol; 0.8 ml fractions were coilected and assayed for radioactivity. Rates of conversion were calculated from the percentage distribution of radioactivity. Lactate dehydrogenase (LDH) was measured as described by Hohorst [371.
A. /
Acyl-CaA
oxidase
a) Rat b) flabblt hepatocytes
. THCAoxidasa
”
a)Rat
b) Rabbit
Protein determination
Protein was determined
according to Lowry et al.
[381. h!?vA notation
and Northern bIots
mRNA was prepared from total RNA preparations 2391 on Dynabeads (dT,,), Dynal A/S (Norway). Northern blot was transfered onto a Schleicher & Shuell nitrocelloluse filter BA-S 83 (Dassel, Germany) and hybridised using a “‘P-labelled cDNA probe of PPAR at 42°C. Washing of the filter was performed using 2 X saline sodium citrate (SSC>/O.2% sodiumdodecysulphate (SDS) at room temperature twice, and a high stringency wash twice with 1 X SSC/O.2% SDS and then 0.7 x SSC/O.2% SDS at 50°C. Statistical analysis
16
Fig. I. Comparison between (A) the acyl-CoA oxidase activity and (B) the THCA oxidation rate in rat and rabbit hepatocytes harvested 24 h after plating, the initial level. and untreated controls harvested 72 h after the beginning of the treatment period. The activity is related to the initial level. The initial activity of acyl-CoA oxidase in rat hepatocytes was 3.4 (SD:0.9) nmol min -’ mg-‘. and the initial THCA oxidation rate was 2.4 (SD: 1.0) nmol hh’ my ‘. The initial acyl-CoA oxidase activity in rabbit hepatocytes was 3.8 (SD:O.Y) nmol min- ’ mg-‘, and 7.4 (SD : 1.3) nmol h ’ mg I. Bars indicate standard deviation, n, = number of observations.
Tukey’s analysis of variance for multiple comparisons was used to evaluate the significance of differences between populations. Results Characterisation of the cell system
The activity of LDH in cell extracts from rat and rabbit hepatocytes did not decline during the time-span of the experiment, 130% and 93% in 3 day untreated control cultures compared to the initial level, respectively. The activity also remained approximately at the same level independent of exposure (Table I>. This indicates that the stability of the cells was acceptable and that the increase in acyl-CoA oxidase was specific.
TABLE I The LDH activity in rat and rabbit hepatocyte cultures exposed to clofibric acid (Clo) retinoic acid (MI, retinol (ROH), in addition to clofbric acid in combination with either retinoid The activity is related to untreated controls (lag), standard deviation in brackets, and n = number of observations, number of experiments. Rat hepatocytes Y0 3-Day control 0.5 mM Clo 10 PM RA 30gMROH 10 FM RA + 0.5 mM Clo 10 PM ROH+OS mM Clo
100 (5) n = 6, 2 101 (13) n = 7,2 95 (1 I) n = 6,2 99 (12) n = 5, 2 116 (10) n = 6, 2 121 (13) n = 6. 2
Rabbit hepatocytes o/c IO0 (13) n = 4,2 96(ll)n=S,2 113(16)n=5.2 109 (6)n=4,2 98 (15) n = 4,2 106 (13) n = 5, 2
The acyl-CoA oxidase activity in untreated rat and rabbit primary hepatocyte cultures decreased to about 70% of the initial activity after 3 days in culture (Fig. 1A). The oxidation of THCA, measured as cholic acid formation, decreased more in rat hepatocytes than in rabbit hepatocytes with time (Fig. 1B). After 3 days in culture only 25% of the initial activity was found in rat hepatocyte cultures, whereas about 55% of the activity remained in rabbit hepatocytes. The effect of clofibric acid and retinoids on the peroxisoma1 p-oxidation of fatty acids in rat and rabbit hepatocyte ~uItures, and ~8~ Cl worry rat hepatoma cells
The exposure of rat hepatocyte cultures to 0.5 mM clofibric acid for 3 days resulted in an increase of 115% in the acyl-CoA oxidase activity compared to untreated controls (Fig. 2a). Increasing the concentration to 1.0 mM did not result in a further increase. Clofibric acid had less of an effect in rabbit hepatocyte cultures than in rat hepatocyte cultures (Fig. 2bf. Exposure to 0.5 mM clofibric acid for 3 days increased the acyl-CoA oxidase activity 40%. 1.0 mM and 5.0 mM clofibric acid had the same effect as 0.5 mM clofibric acid indicating that the concentration was sufficient for maximal effect. The lower increase with 0.25 mM clofibric acid, indicates that this effect is dose-dependent. 7800 Cl Morris hepatoma cells, which have an inducible peroxisomal p-oxidation of fatty acids 1401,
103 300
a)Ralhepatocytes
n, =21 "2 =7
7
2
24 7
9 3
a)fIal hepafcqtes
b)Rabbithepalocylesc)78OOCl MoIriscells
24 6
3 17 16
9 3
14 6
7 3
n, =21 "2'72
Fig. 2. The effect of different concentrations of clofibric acid on the acyl-CoA oxidase activity in (a) rat hepatocytes, (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. All cells were exposed for 3 days and the activity is expressed as percentage of untreated controls harvested at the same time as exposed cultures. Bars indicate standard deviation, n, = number of observations, n2 = number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
exposed to 0.5 mM clofibric acid for 3 days had a 2-fold increased acyl-CoA oxidase activity above the level found in control cells harvested at the same time (Fig. 2~). Thi s e ff ec t was potentiated by 250 nM dexamethasone and an increase of the activity of 500% to 600% was obtained. In this study dexamethasone had a 30% increase on its own (not shown). Exposure of rat hepatocytes to retinoids also increased the acyl-CoA oxidase activity. Both retinoic acid (10 PM) (Fig. 3a) and retinol (30 PM) gave increases of about 30% (Fig. 4a). The effect of retinoids on rabbit hepatocytes was stronger. The acyl-CoA oxidase activity in cultures exposed to 10 PM retinoic acid or 30 PM retinol for 3 days was increased 65% and 54%, respectively, compared with controls (Fig. 3b and 4b, respectively). Compared with the initial level an increase of 20% was obtained. This effect was clearly dose-dependent with less of an increase obtained with
6 2
6
6 6
b)RabMh@atocyies
24 6
10 12 17 4 4 6
c)78OOCl MOWS cd!s
14 6
12 6
Fig. 3. The effect of retinoic acid on the acyl-CoA oxidase activity in (a) rat hepatocytes, (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. The activity is related to untreated control. Bars indicate standard deviation, n, = number of observations, nz = number of experiments. ** Different from control, P < 0.001; * different from control, P < 0.05.
lower concentrations of retinoids (Fig. 3b and 4b, respectively). Retinoic acid given to 7800 Cl Morris rat hepatoma cells for 4 days increased the acyl-CoA oxidase activity slightly, 25% (Fig. 3c), whereas exposure to retinol produced a weaker response, only 10% (Fig. 4~1. Consistent with the findings with clofibric acid, 250 nM dexamethasone increased the response to both retinoic acid and retinol, but less dramatically, 95% and 70%, respectively. The cells were exposed to clofibric acid in combination with either retinoic acid or retinol to see whether these agents acted additively or synergistically. No additative or potentiating effects were observed in rat or rabbit hepatocytes, nor in 7800 Cl rat hepatoma cells (Table 111. The effect of clofibric acid and retinoids on THCA oxidation in rat and rabbit hepatocytes
Rat hepatocytes exposed to clofibric acid for 3 days
TABLE II The effect of retinoic acid (RA) or retinol (ROH) with combination clofibric acid (Clo) on the acyl-CoA oxidase in rat hepatocytes, rabbit hepatocytes and 7800 Cl Morris cells
The activity is related to untreated controls (lOO%), standard deviation in brackets, and n = number of observations, number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
3-Day control 0.5 mM Clo 10 PM RA 30 PM ROH 10 yM RA+O.5 mM Clo 30 p M ROH + 0.5 mM Clo
Rat hepatocytes %
Rabbit hepatocytes %
Morris cells %
100 (8) n = 21, 7 214(39)n=24,7 132 (14) n = 18,6 129 (22) n = 19, 6 234(52)n=22,6 217 (43) n = 21,6
100 (9) n = 24,8 138 (25) n = 17,6 164(44)n=17,6 154 (28) n = 17, 6 158(28)n=18,6** 144 (41) n = 17,6
100 (15) n = 191(27) n = 125 (11) n = 107 (20) n = 156 (31) n = 162 (25) n =
** * * ** *
* ** ** *
14, 6 7,3 * * 12, 6 * 12, 6 6,2 * * 6,2 * *
104 3oo r
a)Ralhepatocytes
b)RabM hepalocytes c)78ooCl Morris cells
t
n, =21 n2.7
9 3
6 2
19 6
24 6
6 3
8 3
17 6
14 6
300
a)
Rat
F
hepatocytes
b)RsbM hepatocyles
12 6
Fig. 4. The effect of retinol on the acyl-CoA oxidase activity in (a) rat hepatocytes; (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. The activity is related to the untreated control. Bars indicate standard deviation, n, = number of observations, n2 = number of experiments. ** Different from control, P < 0.001; * different from control. P < 0.05.
Fig. 6. The effect of 10 WM retinoic acid and 30 PM retinol on the THCA oxidation rate in (a) rat hepatocytes and (b) rabbit hepatocytes. The activity is related to untreated control. Bars indicate standard deviation, n, = number of observations, 12~= number of experiments. ** Different from control. P < 0.001: * different from control, P < 0.05.
had a higher THCA oxidation rate than untreated cells, an increase of 100% with 1 mM clofibric acid (Fig. 5a). The same trend was found in rabbit hepatocyte cultures after 3 days, where the THCA oxidation rate in cultures exposed to clofibric acid was 75% higher than in control cells (Fig. 5b). This shows that clofjbrie acid also has effect on the peroxisomal oxidation of THCA. The effect of retinoids on the oxidation rate of THCA in rat and rabbit hepatocytes followed the trends
as found in acyl-CoA oxidase (Fig. 6a and b, respectively). Retinoic acid had a small effect in rat hepatocytes causing an increase of 40%. In rabbit hepatocytes, however, both retinoic acid and retinoids in-
300
200
E .z 8 .? 0
a)Rat hepatocyles
b)Rabtst hepatocytes
4.4-
1 1
100
9.57.5-
241:4-
1
c n1 n2
22 7
9 3
6 2
Fig. 5. The effect of clofibric acid on the oxidation rate of THCA, measured as formation of cholic acid, in (a) rat hepatocytes and (b) rabbit hepatocytes. The activity is related to the untreated control. Bars indicate standard deviation, nr = number of observations, n2 = number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
l24Fig. 7. Northern blot analysis of PPAR mRNA in (I) rabbit hepatocytes; (2) rat hepatocytes; and (3) 7800 Cl Morris cells. The RNA ladder size markers in kb (BRL, Gaithersburg, MD, USA) are aligned to the left of the autoradiograph.
105 creased the formation rate more than clofibric acid, 82% for retinoic acid and 75% for retinol. No further additional or potentiating effect on the oxidation rate of THCA was obtained by simultaneous exposure to clofibric acid and either retinoid (not shown). Expression of the peroxisomal proliferator activated receptor, PPAR, in rat and rabbit hepatocytes, and 7800 Cl Morris hepatoma cells
Cross-hybridisation with the cloned PPAR isolated from mouse to mRNA preparations from rat and rabbit hepatocytes showed that PPAR was expressed in both cells. The mRNA isolated from rabbit hepatocytes was smaller than that found in rat hepatocytes (Fig. 7). The 7800 Cl M orris rat hepatoma cells also expressed mRNA for PPAR, at the same size as rat hepatocytes. Discussion The results presented here show that retinoids and clofibric acid both increase the acyl-CoA oxidase activity in rat and rabbit hepatocytes, as well as in 7800 Cl Morris rat hepatoma cells, indicating that retinoids have actions similar to peroxisome proliferators. Both agents also increase the oxidation rate of THCA. The increase in acyl-CoA oxidase activity caused by clofibric acid in rat hepatocytes in this study is low compared to what others have found [41,42]. This may be due to the absence of glucocorticoids in the culture media. These hormones are known to potentiate the effect of peroxisome proliferators [431. The decision not to add glucocorticoids to the medium in the present study was based on the fact that they work through nuclear receptors related to those mediating the effect of retinoids and possibly also clofibric acid. Another experimental factor which must be considered is the use of 2% Ultroser G in the medium for both rat and rabbit hepatocytes, a serum less commonly used in studies of peroxisomal enzyme activities in hepatocytes. Ultroser G was chosen since it has been shown to be a suitable serum for rabbit hepatocytes and has been used in several studies both with rat and rabbit hepatocytes (Trend Berg, personal communication). The fact that the specific activity of LDH did not drop throughout the duration of the experiments indicates that the hepatocytes were stable (Table I). We also performed random viability tests with trypan blue at day 3, which showed that the viability was still high after 3 days in culture (estimated to be over 90%, not shown). The response to peroxisome proliferators is different in different species, some species such as rat and mouse respond strongly, while others, such as man and monkey, hardly at all [15-17,44,45]. Similarly, the effect of clofibric acid and retinoids on the peroxisomal P-oxidation is different in rat and rabbit hepatocytes.
Rabbit hepatocytes exposed to clofibric acid had an increased acyl-CoA oxidase activity, although lower than in rat hepatocytes. That indicates that rabbit is less sensitive to peroxisome proliferators. Peroxisome proliferators are known to increase the mRNA level for the enzymes involved in the peroxisomal p-oxidation of fatty acids [3,4] and it is likely that this effect is mediated by the nuclear receptor PPAR 1121. This receptor is expressed both in rat and rabbit hepatocytes. It is also expressed in 7800 Cl Morris rat hepatoma cells, a cell line where clofibric acid had a similar effect on the peroxisomal P-oxidation as it had in rat hepatocytes. In the present study, retinoic acid and retinol had similar effects as did clofibric acid on the peroxisomal P-oxidation of fatty acid. These similarities in the response to retinoic acid and peroxisome proliferators have also been found by other workers. It has been reported that rat hepatocytes exposed to retinoic acid for 2 days have a 1.5-fold increased acyl-CoA oxidase activity and 3-fold increased mRNA level of the enzymes in the peroxisomal P-oxidation compared with controls [32]. In addition, we also found that the effect of clofibric acid and the effect of retinoids in 7800 Cl Morris cells are potentiated by dexamethasone. This is consistent with the report that dexamethasone potentiates the induction caused by sulphur-substituted fatty acid analogues in these cells [54]. These cells are, however, defective in important enzymes involved in the oxidation of THCA (A.-K. Gstlund Farrants, A. Nilsson and J.I. Pedersen, unpublished work). The effects of retinoids are mediated by nuclear receptors, RARs and RXRs. These receptors are closely related to PPAR and both types are expressed in liver [26,28]. The response element for PPAR found in the acyl-CoA oxidase gene and that found for RXR are similar, whereas that for RAR is slightly different [46,471. In addition, RXR has been shown to form heterodimers with PPAR [481, a heterodimerisation necessary for proper binding to DNA. Heterodimerisation to RXR is also essential for the action of the thyroid hormone receptors, the RARs, and the vitamin D, receptor [49-531. The effect of retinoids on the acyl-CoA oxidase activity could be explained by RXR either recognising the response element for PPAR by itself or by forming a heterodimer with PPAR. Furthermore, it has been shown that clofibric acid and 9-cis retinoic acid, the ligand of RXR, given in combination result in a synergistic induction of gene expression in a transactivation assay [48]. In the present study, however, the combination of clofibric acid and retinoids did not have an additative or synergistic effect on the acyl-CoA oxidase activity, neither in hepatocytes nor in rat hepatoma cells. This could mean that the effect caused by retinoids and clofibric acid is mediated by the same mechanism, such as regulating
106 transcription through similar response elements. Another possible explanation is sequestration of RXR in hepatocytes since it is involved in the response of many nuclear receptors [49-531. These mechanisms in cells are complex, however, and many factors are likely to be involved. In vivo studies in rat have shown that clofibrate has no effect on the oxidation rate of THCA, whereas it induces the peroxisomal P-oxidation of fatty acids [lO,lll. In the present study with hepatocytes we find an increase in THCA oxidation in both rat and rabbit hepatocytes exposed to clofibric acid compared to control. However, the effect of clofibric acid in rat hepatocytes on these two peroxisomal enzyme pathways is different when compared to the initial level at the beginning of the treatment period. Clofibric acid increased the acyl-CoA oxidase activty above the initial level, an increase not obtained for the oxidation rate of THCA. Instead, a drop to 65% of the initial activity was found, which is less than in control cells (about 25%, Fig. 1). It has been shown that two distinct oxidases are involved in the peroxisomal @oxidation of fatty acids and in the oxidation of THCA, acyl-CoA oxidase [8] and THCA-CoA oxidase [9], respectively. These findings, together with previous reports, indicate that these two enzymes are differently regulated. However, since the results presented here show that clofibric acid (and retinoids in rabbit hepatocytes) increase the THCA oxidation compared to control cells, it not excluded that the THCA-CoA oxidase gene contain a response element for PPAR. The results presented here demonstrate that both retinoids and clofibric acid increase the activity of peroxisomal &oxidation and THCA oxidation in rabbit and rat hepatocytes. In rat hepatoma cells the effects of retinoids and clofibric acid could be potentiated by dexamethasone. Whether this is a result of an increased expression, mediated by nuclear receptors, or an increased stability of the enzymes involved needs to be further examined. Acknowledgements
The skilful technical assistance of Eva Torma Grabner is much appreciated. We thank Dr. S. Green for kindly providing the cDNA clone for PPAR, Prof. Ingemar BjGrkhem for THCA and Dr. A.H. Tasjuan for the 7800 Cl Morris rat hepatoma cells. We also thank Per Lea, MATFORSK, for performing the statistics. This work was supported by the Norwegian Council on Cardiovascular Diseases, The Norwegian Cancer Society and Anders Jahre’s Foundation. References 1 Lock, E.A., Mitchell, A.M. and Elcombe, CR. (1989) Annu. Rev. Pharmacol. Toxicol. 29, 145-163. 2 Vamecq, J. and Draye, J.P. (1989) Essays Biochem. 24, 115-225.
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107 37 Hohorst, H.J. (1963) in Methods of enzymatic analysis (Bergmeyer, H.U., ed.), pp. 226-227, Academic press, London and New York. 38 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-2’75. 39 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156. 40 Spydevold, 8. and Bremer, J. (1989) Biochim. Biophys. Acta 1003, 72-79. 41 Ozasa, H., Miyazawa, S., Furuta, S. Osumi, T. and Hashimoto, T. (1985) J. Biochem. 97, 1273-1278. 42 Foxworthy, P.S. and Eacho, PI. (1986) Toxicol. L&t. 30, 189-196. 43 Mitchell, A.M., Bridges, J.W. and Elcombe, CR. (1984) Arch. Toxicol. 55, 239-246. 44 Elcombe, CR. (1985) Arch. Toxicol. Suppl. 8, 6-17. 45 Foxworthy, P.S., White, S.L., Hoover, D.M. and Eacho, PI. (1990) Toxicol. Appt. Pharmacol. 104, 386-394. 46 Mangelsdorf, D.J., Umesono, K., Khewer, S.A., Borgmeyer, U., Ong, ES. and Evans, R.M. (1991) Cell 66, 555-561.
47 Tugwood, J.D., Issemann, I., Anderson, R.G., Bundeli, K.R., McPheat, W.L. and Green, S. (1992) EMBO J. 11,433-439. 48 Kliewer, S.A., Umesono, K., Noonan, D.J., Heyman, R.A. and Evans, R.M. (1992) Nature 358, 771-773. 49 Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J-Y, Staub, A., Gamier, J.M., Mader, S. and Chambon, P. (1992) Cell 68377-395. 50 Zhang, X, Hoffmann, B., Tran, P.B-V., Graupner, G. and Pfahl, M. (1992) Nature 355, 441-446. 51 Kliewer, S.A., Umesono, K., Mangelsdorf, D.J. and Evans, R.M. (1992) Nature 355, 446-449. 52 Bugge, T.H., Pohl, J., Lonnoy, 0. and Stunnenberg, H.G. (1992) EMBO J. 11, 1409-1418. 53 Marks, M.S., Hallenbeck, P.L.. Nagata, T., Segars, J.H., Appella, E., Nikodem, V.M. and Ozato, K. (1992) EMBO J. Ii, 1419-1435. 54 Norrheim, L., Sorensen, H., Gautvik, K., Bremer, J. and Spydevold, 0. (1990) Biochim. Biophys. Acta 1051, 319-323.
ffiochirrucu ef Biophysics Acru, 116X(1993) IOO-107 0 1993 Elsevier Science Publishers B.V. All.rights reserved Ol~S-2760/“).3/$(fh.(K)
BBALIP 54175
The effect of retinoids and clofibric acid on the peroxisomal oxidation of palmitic acid and of 3cq7cYJ2a-trihydroxy-5P-cholestanoic acid in rat and rabbit hepatocytes Ann-Kristin 6stlund Farrants, Astrid Nilsson ‘, Gunhild Trgen, Rune Blomhoff and Jan I. Pedersen Institute for Nutrition Research, School
ofMedicine, University of Oslo, Oslo (Norway)
(Received 7 October 1992)
Key words: Hepatocytes; Morris rat hepatoma cell; Peroxisomal @oxidation; Retinoids; Bile acids; (Rabbit)
The effects of retinoids and the peroxisome proliferator clofibric acid on peroxisomal enzyme pathways were studied in hepatocytes from both rat and rabbit. Retinoic acid and retinal increased the activity of acyl-CoA oxidase in rabbit hepatocytes around 60% and around 30% in rat hepatocytes. Exposure to clofibric acid caused an increase in acyI-CoA oxidase activity of 115% in rat hepatocytes and of 40% in rabbit hepatocytes, indicating that rabbit is less sensitive to peroxisome proliferator than rat. Simultaneous exposure to clofibric acid and retinoids did not act additatively or synergistically. Both rabbit and rat hepatocytes expressed mRNA for the peroxisome proliferator activated receptor, (PPAR), although the transcript in rabbit was slightly smaller compared to that expressed in rat hepatocytes. The effect of retinoic acid in 7800 Cl Morris rat hepatoma cells, a cell line known to have an inducible pero~somal ~-oxidation of fatty acids, was only slight with an increase of the acyl-CaA oxidase activity of 25% compared with control cells. As for clofibric acid, which gave a 2-fold induction of the acyl-CoA oxidase activity, the effect of retinoic acid was potentiated by dexamethasone. These ceils also expressed mRNA for PPAR, with the same size as that found in rat hepatocytes. The oxidation of 3cu,7ar,l2a-trihydroxy-5P-cholestanoic acid (THCA), an intermediate in bile acid formation, in rat hepatocytes increased 110% by clofibric acid and around 80%) by retinoic acid. In rabbit hepatocytes, clofibric acid increased the oxidation rate 75% and retinoic acid 100%. The results presented here show similarities in the effects of retinoids and clofibric acid on the acyl-CoA oxidase activity and the oxidation rate of THCA, since they increase these two peroxisomal activities in hepatocytes in vitro. A decrease in both these enzyme activities occurs during cultivation time in untreated primary hepatocyte cultures. The present data may therefore either be explained by an increased expression or an induced stability of the enzymes involved.
Hypolipidemic drugs, such as clofibrate, and plasticizers cause peroxisomal proliferation and induction of the peroxisomal p-oxidation of fatty acids in rodents (for review see Refs. 1, 2). An increased mRNA level [3,4] with an increased rate of protein synthesis (31 for the induced enzymes is observed after treatment with these agents. The oxidative cleavage of the C,, steroid side chain in bile acid formation has also been found to occur in
Correspondence to: A.K. &tlund Farrants, Institute for Nutrition Research, University of Oslo, Pb 1046, Blindern, N-0316 Oslo, Norway. ’ Present address: MATFORSK, Norwegian Food Research Institute, As, Norway.
the peroxisomes 1.51.It follows a similar reaction mechanism to that of the peroxisomal p-oxidation of fatty acids [6,7], although the first step, the introduction of a double bond, is catalysed by two distinct oxidases, the acyl-CoA oxidase [Bl and the 3ar,7cu,12a-trihydroxy-5@cholestanoic acid (THCAI-CoA oxidase 191. While the activity of acyl-CoA oxidase is increased by administration of ciofibrate in vivo, no change has been observed in the conversion rate of 3q7q12a-trihydroxy-5/?cholestanoic acid (THCA), a C,,-bile acid intermediate, to the primary bile acid cholic acid [lO,ll]. A nuclear receptor which is activated by peroxisome proliferators has been cloned from mouse, the peroxisome proliferator activated receptor (PPAR) [ 121. Three distinct but closely related receptors have also been cloned from Xenopus laevis, xPPARa, p and y [13], and recently a rat PPAR was cloned 1141. These
101 receptors are members of the steroid/ thyroid hormone receptor family of transcription factors and it is likely that the effects of peroxisomal proliferators on gene expression are mediated by PPAR. The mouse PPAR is expressed in liver, kidney and small intestine [12]. This corresponds well with the tissue specificity of peroxisomal proliferation and induction of the peroxisomal p-oxidation of fatty acids [15-171. The endogenous ligand has not yet been identified. The PPAR is closely related to the thyroid hormone receptor (TR) and there are several reports that hyperthyroid rats have an increased peroxisomal P-oxidation compared to euthyroid controls [l&201. However, thyroid hormones given to cultures of rat hepatocytes do not induce the peroxisomal P-oxidation of fatty acids [21]. Other members of the thyroid hormone receptor family are the receptors responding to retinoids, the retinoic acid receptors (RARa, p and y) [22-271 and the retinoid X receptors (RXRa, p and yl [28,291. Both these receptors are activated by all-trans retinoic acid, but 9-cis retinoic acid has been identified as ligand for RXR [30,31]. It has recently been reported that retinoic acid induces the expression of enzymes involved in the peroxisomal P-oxidation of fatty acids in rat hepatocytes [32]. In this study we have tested the effect of clofibric acid, all-trans retinoic acid and all-truns retinol on the peroxisomal P-oxidation of fatty acids and the oxidation of THCA in rat and rabbit hepatocytes. In addition, 7800 Cl Morris rat hepatoma cells have been used to study the effect on the peroxisomal P-oxidation by the same agents in a proliferating system. Materials and Methods Chemicals
Palmitoyl-CoA, clofibric acid, retinoic acid, retinol and dexamethasone (tissue culture grade) were obtained from Sigma (MO, USA). Retinoic acid and retinol were stored as stock solutions in ethanol at - 20°C. Clofibric acid and dexamethasone were stored as stock solutions in ethanol at 4°C for 1 week. 2’,7’Dichlorofluorescin diactetate (LDCF) was obtained from Eastman, Kodak (NY, USA). 3q7a,12cY-Trihydroxy-5@[7& 3H]cholestanoic acid and the unlabelled compound were prepared from alligator bile as previously described [33]. Purification of the substrates was performed using HPLC on a Zorbax ODS column (0.46 x 25 cm) using as eluting solvent 17% 30 mM trifluoroacetic acid (TFA) (pH 2.9 with triethylamine), in methanol. Culture media were purchased from Flow Laboratories (Irvine, Scotland), as well as fetal calf serum, horse serum and gentamicin. Ultroser G was obtained from Gibco (Paisley, Scotland). All solvents were of analytical grade. Other chemicals were of commercial high purity material.
Hepatocyte preparation and culture conditions
Hepatocytes were prepared from perfusion of rat and rabbit liver using the collagenase method described by Blomhoff et al. [341. The hepatocytes were plated on 60 mm Petri dishes in Dulbecco’s Modification of Eagles Medium (DMEM) containing 2% Ultroser G, Gibco, and 2 mM r_-glutamine. During the first 24 h 0.1 mg/ml gentamicin was included in the medium. The treatment period started 24 h after plating and continued for 3 days with a change to fresh medium every day. The cells were harvested in 50 mM potassium phosphate buffer at pH 7.5 and sonicated for 2 x 10 s prior to use. Enzyme activities were measured in these cell homogenates. 7800 Cl Morris rat hepatoma cells were cultured in 1 X Ham’s F-10 medium containing 10% horse serum, 2% fetal calf serum, 0.1 mg/ml gentamicin and 2 mM L-glutamine. The cells were cultivated until confluence, then treated for 3 days and harvested as described for hepatocytes. All cells were kept at 37°C in humidified air/CO, (95 : 5, v : v) atmosphere. Enzyme assays and analytical procedures
Acyl-CoA (palmitoyl-CoA) oxidase was assayed by determination of H,O, production, coupled to the oxidation of LDCF, as described by Small et al. [35] and modified by Leighton (personal communication). The oxidation of LDCF by H,O, to 2’,7’-dichlorofluorescein was followed spectrophotometrically at 502 nm. The reaction mixture contained 0.1 M Tris-HCl (pH 8.51, 0.05 mM LDCF, 50 pg horseradish peroxidase type II (EC 1.11.1.7) 0.015 mM FAD, 0.6 mg/ml bovine serum albumin (BSA), 0.02% Triton X-100 and was started with 60 PM palmitoyl-CoA. All concentrations are given as final values. The reaction mixture contained lo-40 pg of protein in a total volume of 1 ml at 25°C. The LDCF was prepared daily at 5.1 mM in 0.01 M NaOH and stored in a light-tight container under N, gas. The conversion of the C,,-bile acid intermediates to primary bile acids was measured as described by Gstlund Farrants et al. [36]. The standard incubation mixture contained 0.1 M Tris-HCl (pH 8.0), 0.1 mM CoA, 2 mM dithiothreitol, 8.5 mM ATP, 10 mM MgCl,, 75 PM FAD, 2 mg/ml BSA, 0.01% Triton X-100 and 40 PM [3H]THCA, final concentrations. NAD was added after 15 min of incubation to a final concentration of 2 mM. The incubations contained 150-300 pg of protein in a total volume of 0.25 ml. The incubations were stopped with 5 ~1 6 M KOH, and hydrolysed at 60°C for 30 min to remove bound CoA. After acidification and extraction with ethyl acetate, the samples were analysed by reversed phase HPLC on a 5 pm C-18 Nucleosil column (0.5 x 25 cm). The eluting solvent was 24% 30 mM TFA (pH 2.9 with triethylamine)
102 in methanol; 0.8 ml fractions were coilected and assayed for radioactivity. Rates of conversion were calculated from the percentage distribution of radioactivity. Lactate dehydrogenase (LDH) was measured as described by Hohorst [371.
A. /
Acyl-CaA
oxidase
a) Rat b) flabblt hepatocytes
. THCAoxidasa
”
a)Rat
b) Rabbit
Protein determination
Protein was determined
according to Lowry et al.
[381. h!?vA notation
and Northern bIots
mRNA was prepared from total RNA preparations 2391 on Dynabeads (dT,,), Dynal A/S (Norway). Northern blot was transfered onto a Schleicher & Shuell nitrocelloluse filter BA-S 83 (Dassel, Germany) and hybridised using a “‘P-labelled cDNA probe of PPAR at 42°C. Washing of the filter was performed using 2 X saline sodium citrate (SSC>/O.2% sodiumdodecysulphate (SDS) at room temperature twice, and a high stringency wash twice with 1 X SSC/O.2% SDS and then 0.7 x SSC/O.2% SDS at 50°C. Statistical analysis
16
Fig. I. Comparison between (A) the acyl-CoA oxidase activity and (B) the THCA oxidation rate in rat and rabbit hepatocytes harvested 24 h after plating, the initial level. and untreated controls harvested 72 h after the beginning of the treatment period. The activity is related to the initial level. The initial activity of acyl-CoA oxidase in rat hepatocytes was 3.4 (SD:0.9) nmol min -’ mg-‘. and the initial THCA oxidation rate was 2.4 (SD: 1.0) nmol hh’ my ‘. The initial acyl-CoA oxidase activity in rabbit hepatocytes was 3.8 (SD:O.Y) nmol min- ’ mg-‘, and 7.4 (SD : 1.3) nmol h ’ mg I. Bars indicate standard deviation, n, = number of observations.
Tukey’s analysis of variance for multiple comparisons was used to evaluate the significance of differences between populations. Results Characterisation of the cell system
The activity of LDH in cell extracts from rat and rabbit hepatocytes did not decline during the time-span of the experiment, 130% and 93% in 3 day untreated control cultures compared to the initial level, respectively. The activity also remained approximately at the same level independent of exposure (Table I>. This indicates that the stability of the cells was acceptable and that the increase in acyl-CoA oxidase was specific.
TABLE I The LDH activity in rat and rabbit hepatocyte cultures exposed to clofibric acid (Clo) retinoic acid (MI, retinol (ROH), in addition to clofbric acid in combination with either retinoid The activity is related to untreated controls (lag), standard deviation in brackets, and n = number of observations, number of experiments. Rat hepatocytes Y0 3-Day control 0.5 mM Clo 10 PM RA 30gMROH 10 FM RA + 0.5 mM Clo 10 PM ROH+OS mM Clo
100 (5) n = 6, 2 101 (13) n = 7,2 95 (1 I) n = 6,2 99 (12) n = 5, 2 116 (10) n = 6, 2 121 (13) n = 6. 2
Rabbit hepatocytes o/c IO0 (13) n = 4,2 96(ll)n=S,2 113(16)n=5.2 109 (6)n=4,2 98 (15) n = 4,2 106 (13) n = 5, 2
The acyl-CoA oxidase activity in untreated rat and rabbit primary hepatocyte cultures decreased to about 70% of the initial activity after 3 days in culture (Fig. 1A). The oxidation of THCA, measured as cholic acid formation, decreased more in rat hepatocytes than in rabbit hepatocytes with time (Fig. 1B). After 3 days in culture only 25% of the initial activity was found in rat hepatocyte cultures, whereas about 55% of the activity remained in rabbit hepatocytes. The effect of clofibric acid and retinoids on the peroxisoma1 p-oxidation of fatty acids in rat and rabbit hepatocyte ~uItures, and ~8~ Cl worry rat hepatoma cells
The exposure of rat hepatocyte cultures to 0.5 mM clofibric acid for 3 days resulted in an increase of 115% in the acyl-CoA oxidase activity compared to untreated controls (Fig. 2a). Increasing the concentration to 1.0 mM did not result in a further increase. Clofibric acid had less of an effect in rabbit hepatocyte cultures than in rat hepatocyte cultures (Fig. 2bf. Exposure to 0.5 mM clofibric acid for 3 days increased the acyl-CoA oxidase activity 40%. 1.0 mM and 5.0 mM clofibric acid had the same effect as 0.5 mM clofibric acid indicating that the concentration was sufficient for maximal effect. The lower increase with 0.25 mM clofibric acid, indicates that this effect is dose-dependent. 7800 Cl Morris hepatoma cells, which have an inducible peroxisomal p-oxidation of fatty acids 1401,
103 300
a)Ralhepatocytes
n, =21 "2 =7
7
2
24 7
9 3
a)fIal hepafcqtes
b)Rabbithepalocylesc)78OOCl MoIriscells
24 6
3 17 16
9 3
14 6
7 3
n, =21 "2'72
Fig. 2. The effect of different concentrations of clofibric acid on the acyl-CoA oxidase activity in (a) rat hepatocytes, (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. All cells were exposed for 3 days and the activity is expressed as percentage of untreated controls harvested at the same time as exposed cultures. Bars indicate standard deviation, n, = number of observations, n2 = number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
exposed to 0.5 mM clofibric acid for 3 days had a 2-fold increased acyl-CoA oxidase activity above the level found in control cells harvested at the same time (Fig. 2~). Thi s e ff ec t was potentiated by 250 nM dexamethasone and an increase of the activity of 500% to 600% was obtained. In this study dexamethasone had a 30% increase on its own (not shown). Exposure of rat hepatocytes to retinoids also increased the acyl-CoA oxidase activity. Both retinoic acid (10 PM) (Fig. 3a) and retinol (30 PM) gave increases of about 30% (Fig. 4a). The effect of retinoids on rabbit hepatocytes was stronger. The acyl-CoA oxidase activity in cultures exposed to 10 PM retinoic acid or 30 PM retinol for 3 days was increased 65% and 54%, respectively, compared with controls (Fig. 3b and 4b, respectively). Compared with the initial level an increase of 20% was obtained. This effect was clearly dose-dependent with less of an increase obtained with
6 2
6
6 6
b)RabMh@atocyies
24 6
10 12 17 4 4 6
c)78OOCl MOWS cd!s
14 6
12 6
Fig. 3. The effect of retinoic acid on the acyl-CoA oxidase activity in (a) rat hepatocytes, (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. The activity is related to untreated control. Bars indicate standard deviation, n, = number of observations, nz = number of experiments. ** Different from control, P < 0.001; * different from control, P < 0.05.
lower concentrations of retinoids (Fig. 3b and 4b, respectively). Retinoic acid given to 7800 Cl Morris rat hepatoma cells for 4 days increased the acyl-CoA oxidase activity slightly, 25% (Fig. 3c), whereas exposure to retinol produced a weaker response, only 10% (Fig. 4~1. Consistent with the findings with clofibric acid, 250 nM dexamethasone increased the response to both retinoic acid and retinol, but less dramatically, 95% and 70%, respectively. The cells were exposed to clofibric acid in combination with either retinoic acid or retinol to see whether these agents acted additively or synergistically. No additative or potentiating effects were observed in rat or rabbit hepatocytes, nor in 7800 Cl rat hepatoma cells (Table 111. The effect of clofibric acid and retinoids on THCA oxidation in rat and rabbit hepatocytes
Rat hepatocytes exposed to clofibric acid for 3 days
TABLE II The effect of retinoic acid (RA) or retinol (ROH) with combination clofibric acid (Clo) on the acyl-CoA oxidase in rat hepatocytes, rabbit hepatocytes and 7800 Cl Morris cells
The activity is related to untreated controls (lOO%), standard deviation in brackets, and n = number of observations, number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
3-Day control 0.5 mM Clo 10 PM RA 30 PM ROH 10 yM RA+O.5 mM Clo 30 p M ROH + 0.5 mM Clo
Rat hepatocytes %
Rabbit hepatocytes %
Morris cells %
100 (8) n = 21, 7 214(39)n=24,7 132 (14) n = 18,6 129 (22) n = 19, 6 234(52)n=22,6 217 (43) n = 21,6
100 (9) n = 24,8 138 (25) n = 17,6 164(44)n=17,6 154 (28) n = 17, 6 158(28)n=18,6** 144 (41) n = 17,6
100 (15) n = 191(27) n = 125 (11) n = 107 (20) n = 156 (31) n = 162 (25) n =
** * * ** *
* ** ** *
14, 6 7,3 * * 12, 6 * 12, 6 6,2 * * 6,2 * *
104 3oo r
a)Ralhepatocytes
b)RabM hepalocytes c)78ooCl Morris cells
t
n, =21 n2.7
9 3
6 2
19 6
24 6
6 3
8 3
17 6
14 6
300
a)
Rat
F
hepatocytes
b)RsbM hepatocyles
12 6
Fig. 4. The effect of retinol on the acyl-CoA oxidase activity in (a) rat hepatocytes; (b) rabbit hepatocytes; and (c) 7800 Cl Morris cells. The activity is related to the untreated control. Bars indicate standard deviation, n, = number of observations, n2 = number of experiments. ** Different from control, P < 0.001; * different from control. P < 0.05.
Fig. 6. The effect of 10 WM retinoic acid and 30 PM retinol on the THCA oxidation rate in (a) rat hepatocytes and (b) rabbit hepatocytes. The activity is related to untreated control. Bars indicate standard deviation, n, = number of observations, 12~= number of experiments. ** Different from control. P < 0.001: * different from control, P < 0.05.
had a higher THCA oxidation rate than untreated cells, an increase of 100% with 1 mM clofibric acid (Fig. 5a). The same trend was found in rabbit hepatocyte cultures after 3 days, where the THCA oxidation rate in cultures exposed to clofibric acid was 75% higher than in control cells (Fig. 5b). This shows that clofjbrie acid also has effect on the peroxisomal oxidation of THCA. The effect of retinoids on the oxidation rate of THCA in rat and rabbit hepatocytes followed the trends
as found in acyl-CoA oxidase (Fig. 6a and b, respectively). Retinoic acid had a small effect in rat hepatocytes causing an increase of 40%. In rabbit hepatocytes, however, both retinoic acid and retinoids in-
300
200
E .z 8 .? 0
a)Rat hepatocyles
b)Rabtst hepatocytes
4.4-
1 1
100
9.57.5-
241:4-
1
c n1 n2
22 7
9 3
6 2
Fig. 5. The effect of clofibric acid on the oxidation rate of THCA, measured as formation of cholic acid, in (a) rat hepatocytes and (b) rabbit hepatocytes. The activity is related to the untreated control. Bars indicate standard deviation, nr = number of observations, n2 = number of experiments. * * Different from control, P < 0.001; * different from control, P < 0.05.
l24Fig. 7. Northern blot analysis of PPAR mRNA in (I) rabbit hepatocytes; (2) rat hepatocytes; and (3) 7800 Cl Morris cells. The RNA ladder size markers in kb (BRL, Gaithersburg, MD, USA) are aligned to the left of the autoradiograph.
105 creased the formation rate more than clofibric acid, 82% for retinoic acid and 75% for retinol. No further additional or potentiating effect on the oxidation rate of THCA was obtained by simultaneous exposure to clofibric acid and either retinoid (not shown). Expression of the peroxisomal proliferator activated receptor, PPAR, in rat and rabbit hepatocytes, and 7800 Cl Morris hepatoma cells
Cross-hybridisation with the cloned PPAR isolated from mouse to mRNA preparations from rat and rabbit hepatocytes showed that PPAR was expressed in both cells. The mRNA isolated from rabbit hepatocytes was smaller than that found in rat hepatocytes (Fig. 7). The 7800 Cl M orris rat hepatoma cells also expressed mRNA for PPAR, at the same size as rat hepatocytes. Discussion The results presented here show that retinoids and clofibric acid both increase the acyl-CoA oxidase activity in rat and rabbit hepatocytes, as well as in 7800 Cl Morris rat hepatoma cells, indicating that retinoids have actions similar to peroxisome proliferators. Both agents also increase the oxidation rate of THCA. The increase in acyl-CoA oxidase activity caused by clofibric acid in rat hepatocytes in this study is low compared to what others have found [41,42]. This may be due to the absence of glucocorticoids in the culture media. These hormones are known to potentiate the effect of peroxisome proliferators [431. The decision not to add glucocorticoids to the medium in the present study was based on the fact that they work through nuclear receptors related to those mediating the effect of retinoids and possibly also clofibric acid. Another experimental factor which must be considered is the use of 2% Ultroser G in the medium for both rat and rabbit hepatocytes, a serum less commonly used in studies of peroxisomal enzyme activities in hepatocytes. Ultroser G was chosen since it has been shown to be a suitable serum for rabbit hepatocytes and has been used in several studies both with rat and rabbit hepatocytes (Trend Berg, personal communication). The fact that the specific activity of LDH did not drop throughout the duration of the experiments indicates that the hepatocytes were stable (Table I). We also performed random viability tests with trypan blue at day 3, which showed that the viability was still high after 3 days in culture (estimated to be over 90%, not shown). The response to peroxisome proliferators is different in different species, some species such as rat and mouse respond strongly, while others, such as man and monkey, hardly at all [15-17,44,45]. Similarly, the effect of clofibric acid and retinoids on the peroxisomal P-oxidation is different in rat and rabbit hepatocytes.
Rabbit hepatocytes exposed to clofibric acid had an increased acyl-CoA oxidase activity, although lower than in rat hepatocytes. That indicates that rabbit is less sensitive to peroxisome proliferators. Peroxisome proliferators are known to increase the mRNA level for the enzymes involved in the peroxisomal p-oxidation of fatty acids [3,4] and it is likely that this effect is mediated by the nuclear receptor PPAR 1121. This receptor is expressed both in rat and rabbit hepatocytes. It is also expressed in 7800 Cl Morris rat hepatoma cells, a cell line where clofibric acid had a similar effect on the peroxisomal P-oxidation as it had in rat hepatocytes. In the present study, retinoic acid and retinol had similar effects as did clofibric acid on the peroxisomal P-oxidation of fatty acid. These similarities in the response to retinoic acid and peroxisome proliferators have also been found by other workers. It has been reported that rat hepatocytes exposed to retinoic acid for 2 days have a 1.5-fold increased acyl-CoA oxidase activity and 3-fold increased mRNA level of the enzymes in the peroxisomal P-oxidation compared with controls [32]. In addition, we also found that the effect of clofibric acid and the effect of retinoids in 7800 Cl Morris cells are potentiated by dexamethasone. This is consistent with the report that dexamethasone potentiates the induction caused by sulphur-substituted fatty acid analogues in these cells [54]. These cells are, however, defective in important enzymes involved in the oxidation of THCA (A.-K. Gstlund Farrants, A. Nilsson and J.I. Pedersen, unpublished work). The effects of retinoids are mediated by nuclear receptors, RARs and RXRs. These receptors are closely related to PPAR and both types are expressed in liver [26,28]. The response element for PPAR found in the acyl-CoA oxidase gene and that found for RXR are similar, whereas that for RAR is slightly different [46,471. In addition, RXR has been shown to form heterodimers with PPAR [481, a heterodimerisation necessary for proper binding to DNA. Heterodimerisation to RXR is also essential for the action of the thyroid hormone receptors, the RARs, and the vitamin D, receptor [49-531. The effect of retinoids on the acyl-CoA oxidase activity could be explained by RXR either recognising the response element for PPAR by itself or by forming a heterodimer with PPAR. Furthermore, it has been shown that clofibric acid and 9-cis retinoic acid, the ligand of RXR, given in combination result in a synergistic induction of gene expression in a transactivation assay [48]. In the present study, however, the combination of clofibric acid and retinoids did not have an additative or synergistic effect on the acyl-CoA oxidase activity, neither in hepatocytes nor in rat hepatoma cells. This could mean that the effect caused by retinoids and clofibric acid is mediated by the same mechanism, such as regulating
106 transcription through similar response elements. Another possible explanation is sequestration of RXR in hepatocytes since it is involved in the response of many nuclear receptors [49-531. These mechanisms in cells are complex, however, and many factors are likely to be involved. In vivo studies in rat have shown that clofibrate has no effect on the oxidation rate of THCA, whereas it induces the peroxisomal P-oxidation of fatty acids [lO,lll. In the present study with hepatocytes we find an increase in THCA oxidation in both rat and rabbit hepatocytes exposed to clofibric acid compared to control. However, the effect of clofibric acid in rat hepatocytes on these two peroxisomal enzyme pathways is different when compared to the initial level at the beginning of the treatment period. Clofibric acid increased the acyl-CoA oxidase activty above the initial level, an increase not obtained for the oxidation rate of THCA. Instead, a drop to 65% of the initial activity was found, which is less than in control cells (about 25%, Fig. 1). It has been shown that two distinct oxidases are involved in the peroxisomal @oxidation of fatty acids and in the oxidation of THCA, acyl-CoA oxidase [8] and THCA-CoA oxidase [9], respectively. These findings, together with previous reports, indicate that these two enzymes are differently regulated. However, since the results presented here show that clofibric acid (and retinoids in rabbit hepatocytes) increase the THCA oxidation compared to control cells, it not excluded that the THCA-CoA oxidase gene contain a response element for PPAR. The results presented here demonstrate that both retinoids and clofibric acid increase the activity of peroxisomal &oxidation and THCA oxidation in rabbit and rat hepatocytes. In rat hepatoma cells the effects of retinoids and clofibric acid could be potentiated by dexamethasone. Whether this is a result of an increased expression, mediated by nuclear receptors, or an increased stability of the enzymes involved needs to be further examined. Acknowledgements
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