Effects of peroxisome proliferators on fatty acid-binding protein in rat liver

21

Biochimica et Biophysics Acts, 754 (1983) 21-27

Elsevier

BBA 51508

EFFECTS LIVER YOICHI

OF PEROXISOME

KAWASHIMA,

SAYURI

PROLIFE~TORS

NAKAGAWA.

YUKA

ON FATTY ARID-RINDING

TACHIBANA

and HIROSHI

PRO~IN

IN RAT

KOZUKA

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01 (Japan) (Received

May Znd, 1983)

Key words: Fa/
Peroxisome;

(Rat liver)

The relationship between hepatic fatty acid-binding protein and peroxisomal &oxidation was studied. Rats were fed a diet containing p-chlorophenoxyisobutyric acid (clofibric acid), 2,2’-(decamethylenedithio)-diethanol (tiadenol), di-(2-ethylhexyl)-phthalate (DEHP), di-(2-ethylhexyl)-adipate (DEHA) and acetylsalicylic acid. On the adminis~ation of these peroxisome proliferators, both Il-‘4C]oleic acid-binding capacity and content of fatty acid-binding protein were increased, in association with an increase in peroxisomal /l-oxidation activity. The order of the increase in binding capacity and content of fatty acid-binding protein was tiadenol > DEHP 2 clofibric acid > DEHA = acetylsalicylic acid. The order of the increase in peroxisomal B-oxidation activity in liver was tiadenol > clofibric acid 2 DEHP > DEHA = acetylsalicylic acid. Linear regression analysis between the binding capactiy or content of fatty acid-binding protein and peroxisomal &oxidation activity was highly significant.

Introduction Fatty acid-binding protein has been found in cytosol of various tissues, including liver and intestinal mucosa [1,2]. The protein binds not only long-chain fatty acids, but also their corresponding CoA esters, with high affinity 131. Many attempts have been made to resolve the physiological role of fatty acid-binding protein, and substantial evidence suggesting its physiological significance have been provided. At present, fatty acid-binding protein is considered to participate in uptake and transport of long-chain fatty acid [4-71, esterification of fatty acid [8-111, stimulation of some enzyme actions [12-141 and release of inhibitory action of fatty acyl-CoA to some enzymes 115,161.

Abbreviations: tiadenol. 2,2’-(decamethylenedithio)-diethanol: DEHP. di-(2-ethylhexyl)-phthalate; DEHA, di-(2-ethylhexyl)adipate. ~5-2760/~3/$03.~

a 1983 Elsevier Science Publishers

B.V.

Fleischner et al. [17] found that hypolipidemic drugs, clofibrate and nafenopin, increase the fatty acid-binding protein level in liver of rats. Renaud et al. [18] showed that the increase in the fatty acid-binding protein level by clofibrate treatment resulted in an increase of uptake of fatty acid into liver, but not in esterification of the fatty acid. Clofibrate is known to be a strong peroxisome proliferator [19] and the proliferated peroxisomes are found to contain a high activity of cyanide-insensitive @-oxidation [20,21]. A recent report [22] provided evidence to show the possible participation of fatty acid-binding protein in peroxisomal P-oxidation of fatty acyl-CoA. We [23] have shown perviously that administration of peroxisome proliferators, such as phthalic acid esters which are unrelated structurally to clofibrate, also causes an increase in the amounts of [1-‘4C]oleic acid bound to fatty acid-binding protein in rat liver. To obtain more information about the relationship between fatty acid-binding protein and peroxisomal p-

22

oxidation study.

in rat liver, we carried

out the present

Materials and Methods Chemicals. [ 1- 14C]Oleic acid (57.0 Ci/mol) was obtained from New England Nuclear Co. (Boston, MA. U.S.A.). PalmitoyI-CoA, CoA, clofibric acid, acetylsalicylic acid, bovine serum albumin and Coomassie brilliant blue were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.); DEHP and DEHA were from the Tokyo Chemical Industry Co. (Tokyo, Japan); tiadenol was from the Aldrich Chemical Co. (Milwaukee, WI, U.S.A.); NAD was from the Oriental Yeast Co. (Tokyo, Japan); Sephadex G-50 and aminohexylamino-Sepharose 4B were from Pharmacia Fine Chemicals (Uppsala, Sweden). All other chemicals were of analytical grade. ~~~~a~~. Male rats of the Wistar strain, weighing 160-180 g, were used. The rats were fed ad libitum a commercial diet or a ground diet containing 0.5% (w/w) clofibric acid, 0.5% (w/w) tiadenol, 2% (w/w) DEHP, 2% (w/w) DEHA and 1% (w/w) acetylsalicylic acid for 7 days. The rats were then decapitated. Livers were isolated and perfused with cold 0.9% NaCI to wash out traces of blood. The livers were homogenized with 1.5 vol. of cold 0.25 M sucrose. Cytosol was obtained from the homogenates by differential centrifugation, as previously described 1231. Binding assay of oleic acid to fatty acid-binding protein. Fatty acid-binding protein-associated fatty acid-binding receptors in cytosol were measured by means of binding of [l-‘4C]oleic acid to the fatty acid-binding protien fraction of cytosol, with chromatography on Sephadex G-50 essentially according to the method of Ockner et al. [ll], with some modifications as described previously [23]. Briefly, the incubation mixture contained 120 nmol of [l- “C]oIeic acid (45 nCi), 7.5 mg of cytosolic protein and 3 mg of Triton WR-1339 in 1.5 ml of 0.154 M KCl/O.Ol M potassium phosphate buffer (pH 7.4). The mixture was incubated at 37OC for 30 min. After cooling on ice, I ml of the mixture was subjected to gel filtration on a Sephadex G-50 column (2.2 x 30 cm) equilibrated with 0.154 M KCl/O.Ol M sodium phosphate buffer (pH 7.4) at 4”C, and eluted. Fractions of 2 ml were collected.

The radioactivity in the eluate was measured by a liquid scintillation spectrometer after mixing with a toluene/Triton-based scintillator. A column of Sephadex G-50 was used for not more than 10 samples to obtain good reproducibility of the results. ~e~s~~reF~ejlt of the futty ffcid-b~ndjng ~rote~?? context by amenity chrot~latogra~h~).Oleic acid was coupled to aminohexylamino-Sepharose 4B by the carbodiimide method according to the method of Cuatrecasas 1241. The oleic acid content of the oleoylaminohexylamino-Sepharose was estimated as 0.6 pmol/ml of gel. Affinity chromatography was performed essentially according to the method of Ockner et al. [25], with some modifications, as follows. Fatty acid-binding protein fractions, which were obtained by gel filtration of cytosol and exhibited oleic acid-binding activity, were combined and diluted twice with phosphate-buffered saline. The diluted fatty acid-binding protein solution, containing 300-900 pg of protein, was subjected to a column of oleoylaminohexylamino-Sepharose (0.9 x 2.5 cm). The unabsorbed proteins to the gel were eluted with 10 ml of phosphate-buffered saline. The proteins which bound to the column were eluted with 12 ml of 2.5% ethanol in 0.075 sodium phosphate buffer (pH 6.0). Residual bound proteins were washed out with 5 ml of 50% ethanol in 0.075 M sodium phosphate buffer (pH 2.4) and then 5 ml of 0.05 M NaOH/ethanol (1 : 1, v/v). For reuse, the column was again equilibrated with phosphate-buffered saline. Fatty acid-binding protein was eluted with 25% ethanol in 0.075 M sodium phosphate buffer (pH 6.0). The amounts of protien in this eluate was determined as fatty acid-binding protein. E~ect~o~hores~s.Polyacrylamide disc gel eiectrophoresis was performed according to the method of Davis [26]. 2.5% contracting (pH 6.7) and 7% separating (pH 8.9) gels were buffered with 0.5 M Tris and 0.38 M glycine (pH 8.6) and run at 0.8 mA/gel for 5 h. Proteins were fixed and stained in 0.2% Coomassie brilliant blue in 50% methanol/7% acetic acid for 2 h. The gels were destained in 20% methanoI/7% acetic acid. Enzyme assay. The cyanide-insensitive palmitoyl-CoA oxidation was assayed according to the method of Lazarow and De Duve 1201, with some

23

modifications as previously described [27]. 1 unit of the activity is defined as the activity required to reduce 1 pmol of NAD per min. Orher ~r~ce~~~es. The protein concentration was determined by the method of Lowry et al. [28], with bovine serum albumin as standard. Statistical analysis of the data was performed with regression analysis and significance was determined by Student’s t-test. Results Tabie I shows the effects of various peroxisome proliferators on the amounts of [l-‘4C]oieic acid bound to the fatty acid-binding protein fraction when [1-‘4C]oleic acid was mixed with hepatic cytosol and analyzed with gel filtration. Clofibrate has been known to increase both the binding capacity and the content of fatty acid-binding protein in rat liver 1171.In accordance with earlier reports [17,18] and our previous study [23], treatment with clofibric acid increases approximately 5 times the amount of [1-‘4C]oleic acid which binds to fatty acid-binding protein. Administration of DEHP gave similar effects on the binding capacity of fatty acid-binding protein as those observed with clofibric acid. Of the peroxisome proliferators tested, tiadenol cussed the most pronounced increase in the amounts of [l-‘4C]oleic acid bound to fatty acid-binding protein. The binding capacity increased by tiadenol treatment was 1.4 times, per

TABLE

g

liver, and 1.9 times, per total liver, greater than the increase by clofibric acid treatment. Although acetylsalicylic acid and DEHA also elevated the amounts of [I- “C]oleic acid bound to fatty acidbinding protein, the effects of these compounds were not so marked as those of the other three compounds. Fleischner et al. [17] demonstrated by radioimmunoassay that the increase in the binding capacity of hepatic fatty acid-binding protein is attributable to the increase in the content of fatty acidbinding protein in the iiver. In order to examine whether peroxisome proliferators, regardless of their chemical structure, increase generally the content of hepatic fatty acid-binding protein, we measured the fatty acid-binding protein concentration in cytosol from rats treated by various peroxisome proliferators. Fatty acid-binding protein in cytosol was partially purified by gel filtration. The partially purified fatty acid-binding protein was subjected to affinity chromatography of ol~oylaminohexy~~ino-Sepharose. The proteins unabsorbed to the affinity column were washed out with phosphate-buffered saline and fatty acidbinding protein was eluted with 25% ethanol in 0.075 M sodium phosphate buffer (pH 5.0). Approx. 98% of the fatty acid-binding protein applied to the affinity column was recovered with 25% ethanol/O.075 M sodium phosphate buffer (pH 6.0). This was confirmed using purifed fatty acidbinding protein from hepatic cytosol, with gel

I

EFFECTS OF VARIOUS FATTY ACID-BINDING

PEROXISOME PROTEIN

PROLIFERATORS

ON THE

BINDING

OF [l-‘4C]OLEIC

ACID

TO HEPATIC

Rats were fed a diet containing one of the drugs or chemicals described in the table, for 7 days. Each value represents the mean & S.D. Numbers in parentheses are the number of animals used. Binding of oleic acid to fatty acid-binding protein was assayed as described in the text. Treatment

Control (3) Clofibric acid (3) Tiadenoi (4) DEHP (3) DEHA (4) Acetylsalicylic acid (4)

Oleic acid bound

to fatty acid-binding protein

nmol/mg cytosolic protein

nmoI/g liver

nmol/liver

1.23+0.13 4.35 * 0.21 7.61+ 0.47 5.10*0.50 2.93 & 0.65 2.62k0.17

39.9* 9.1 185.2 -r_10.8 260.0 Lt 50.6 220.0i13.1 116.8k20.8 108.2C 7.3

524+ 46 26841 328 5084kl297 3753+ 418 1632k 397 14511 196

24

TABLE

II

EFFECTS Values

OF VARIOUS

are mean$

determined the text.

PEROXISOME

S.D. Numbers

by affinity

~hrom~tograph~

Treatment

Control (3) Clofibric acid (4) Tiadenol(4) DEHP (4) DEHA (4) Acetyisalicylie acid14)

PROLIFERATORS

in parenthescs

represent

ON FATTY the number

of the fatty amid-binding

Fatty acid-binding

protein

protein

ACID-BINDINC;

of animals fraction

ohtnined

Fatt?

PROTEIN

CONTENT

acid-binding

protcin

hy gel filtrati~~t~ of cytosol

IN LIVER content

its dcscribcd

was in

content

w% cytosolic protein

w/g liver

50.5& 1.5 121.1+24.5 205.0 * 27.8 129.0 ri_ 8.4 li3.Y & x.4 61.31 3.6

l.XO~O.15 4.17 & 1.46 6.X2& 0.38 5.20 i 0.65 4.58 * 0.34 2.53,O.Ih

filtration twice: DEAE-Sephadex A-50 and CMSephadex C-50 ~unpublished data). Moreover, electrophoretic analysis revealed that the phosphate-buffered saline wash did not contain protein bands with migration identical to that of fatty acid-binding protein. Table II shows the effects of peroxisome proliferators on the content of fatty acid-binding protein in liver. In control rats, the concentration of fatty acid-binding protein in cytosol was 50 pg/mg cytosolic protein, indicating that fatty acid-binding protein accounts for 5% of

uacd.

2 I .7 -.- 1.7 74.2 2. 14.2 140.6 + 9.0 X6.3 i 6.7 63.2 i 5.1 34,oj 4.5

cytosolic protein. As anticipated from the results in Table I, both the content of fatty acid-binding protein in livers and the concentration of fatty acid-binding protein in eytosolic protein were increased by the administration of the peroxisome proliferators. In livers of rats treated with tiadenol, the concentration of fatty acid-binding protein increased strikingly and reached approximately 20% of cytosolic protein. Fatty acid-binding protein isolated by affinity chromatography was subjected to disc gel electrophoresis in a nondenaturing buffer. The staining of the isolated fatty acid-binding protein from control liver demonstrates the presence of at least

TABLE

III

EFFECTS OF VARIOUS PEROXISOME PROLIFERATORS ON PEROXISOMAL &OXIDATION IN RAT LIVER Values are mean+S.D. Numbers number of animals used. Treatment

Fig. 1. Disc gel electrophoresis of the hepatic protein isolated by affinity chromatography ciofibric acid-fed rats. Approximately 20 pg acid-binding protein were subjected to each binding protein of control rat liver: b, fatty tein of clofibric acid-fed rat liver.

fatty arid-binding from control and of individual fatty gel. a, Fatty acidacid-binding pro-

Control (3) Clofibric acid (3) Tiadenol(4) DEHP (3) DEHA (4) Acetylsalicylic acid (4)

in parentheses

Palmitovl-CoA

represent

oxidation

units/g liver

units/liver

0.38 IO.05 5.77 + 0.67 8.05 f 0.69 5.39kO.49 2.62 + 0.69 2.48 k 0.72

4.55 0.42 98.1 f 14.0 157.Ok25.3 88.1 i 4.2 39.2 k 13.0 32.7+ 7.6

the

25

two major proteins (Fig. la). Treatment with clofibric acid did not affect the migration of fatty acid-binding protein on polyacrylamide gels (Fig. lb).

All peroxisome prolifecators tested in the present study have been reported to cause an increase in peroxisomal P-oxidation activity in liver [20,21,29-313. In Table III, the increase in the

t

0 Oleic

Oleic acid bound to F&BP

( nmol/g

0

liver

2

)

6

concentration

( mg/g

liver

4

acid

bound

( nmol/liver

4

FABP

I

2

8

-6

FABP

)

I

I

40

80

FABP

)

to

6 x lo3

I

1

120

140

concentration

( mg/liver

)

Fig. 2. The relationship between peroxisomal &oxidation activity vs oleic acid binding capacity to fatty acid-binding protein (FABP) or fatly acid-binding protein content. Regression analyses were performed on six mean data from both Tables I and II1 and Tables II and III. A, Peroxisomal &oxidation activity vs. oleic acid binding capacity to fatty acid-binding protein per g liver, Y = - 1.09 + 0.034 X; r = 0.981; P < 0.001. B, Peroxisomal /3-oxidation activity vs. oleic acid binding capacity to fatty acid-binding protein per total liver. Y = - 10.53+0.032X; r = 0.966; P < 0.01. C, Peroxisomal &oxidation activity vs. fatty acid-binding protein content per g liver. Y = - 1.90 +1.41X; r = 0.926; P < 0.01. D, Peroxisomal P-oxidation activity vs fatty acid-binding protein content per total liver. Y = -1X29+ 1.26X: r = 0.963; P < 0.01. 0. Control; l. clofibric acid; A, tiadenol: A, DEHP; n . DEHA; 0, acelyisaIicyI~c acid,

26

activity of cyanide-insensitive palmitoyl-CoA oxidation following the administration of peroxisome prolifera tors was confirmed. To examine the relationship between the amounts of [114C]01eic acid bound to the fatty acid-binding protein fraction and the activity of peroxisomal ,B-oxidation, a linear reglession analysis was done. As shown in Fig. 2A, correlation between the amounts of [1- 14 C]0Ieic acid bound to fatty acidbinding protein and the activity of peroxisomal ,B-oxidation on the basis of per g liver was seen (r = 0.981, P < 0.001). Similarly, linear regreassion analysis revealed that the amounts of [l_14C]0Ieic acid bound to total hepatic fatty acid-binding protein correlated with total hepatic peroxisomal ,B-oxidation activity (r = 0.966, P < 0.01). In addition, the correlation between fatty acid-binding protein content and peroxisomal ,B-oxidation activity both per g liver and per total liver was also significant, with r = 0.926 (P < 0.01) and r = 0.963 (P < 0.01), respectively (Fig. 2C and D). Discussion Our previous study [23] provided evidence showing that peroxisome prolifera tors, such as DEHP and dibutylphthalate, which are not analogues of clofibrate, are also able to increase the amounts of [1- 14 C]0Ieic acid bound to fatty acidbinding protein in hepatic cytosol. In the present work, three additional peroxisome prolifera tors, tiadenol, DEHA and acetylsalicylic acid, were also shown to cause an increase in the amount of oleic acid bound to fatty acid-binding protein. Moreover, it is evident from the measurement of the fatty acid-binding protein content in cytosol that the increase in oleic acid binding to fatty acid-binding protein in cytosol is responsible for the increase in fatty acid-binding protein content. Based on these findings, it seems to be conclusive that peroxisome prolifera tors, even though their structures differ each other, are able to increase the fatty acid-binding protein content in liver. From the results in Tables 1 and II, the amounts of [1_ 14 C]oleic acid bound to fatty acid-binding protein are calculated to be 25-40 p.mol/mg fatty acid-binding protein, indicating that peroxisome proliferators does not change considerably the binding capacity of the fatty acid-binding protein molecule.

We isolated fatty acid-binding protein by affinity chromatography to study the effects of treatment with peroxisome proliferators on its properties. In accordance with the findings of Ockner et al. [25], the isolated fatty acid-binding protein showed at least two protein bands on polyacrylamide gels. It is known that fatty acid-binding protein exists in several immunologically identical forms which differ in their isoelectric pH and affinity to ligand [25]. Since fatty acid-binding protein isolated by affinity chromatography is considered to be the fatty acid-binding protein with high 'affinity to fatty acid, the determination of fatty acid-binding protein by affinity chromatography may be a more useful procedure than immunological determination, in terms of the determination of functional fatty acid-binding protein in fatty acid binding. Treatment with clofibric acid did not change the electrophoretic pattern of migration of hepatic fatty acid-binding protein, suggesting that clofibric acid may not alter the properties of fatty acid-binding protein. By statistical analysis, a pronounced correlation was found both between the binding capacity of fatty acid-binding protein in cytosol and peroxisomal ,B-oxidation activity and between the fatty acid-binding protein content and peroxisomal ,Boxidation activity. Although the detailed mechanism by which fatty acid-binding protein participates in peroxisomal ,B-oxidation was not clarified by our study, Appelkvist and Dallner [22] suggested from their in vitro experiments that fatty acid-binding protein may be involved in the transport process of fatty acyl-CoA into peroxisomes. Our present results may weIl be in accordance with the conclusion whi.ch they reached. The present study has focused only on the relationship between fatty acid-binding protein and peroxisomal ,B-oxidation. On the one hand, physiological changes caused by the administration of peroxisome proliferators are not limited to the induction of peroxisomal ,B-oxidation activity. Numerous responses to peroxisome proliferators have been found: an increase in synthesis of CoA and fatty acyl-CoA [32] and induction of some enzymes, including stearoyl-CoA desaturase [33], glycerophosphate acyltransferase [34] and acylCoA hydrolase [27,35,36]. The results presented in this report do not exclu de the possibility of in-

27

volvement of fatty acid-binding protein in these biological changes produced by peroxisome proliferators. Nevertheless, it is noteworthy that the change in peroxisomal ~-oxidation activity is the most striking of the biological changes mentioned above. References 1 Ockner. R.K.. Manning J.A., Poppenhausen, R.H. and Ho, W.K.L. (1972) Science 177, 56-5X 2 Mishkin. S.. Stein, L., GaInaitan. Z. and Arias, I.M, (1972) Biochem. Biophys. Res. Commun. 47. 997-1003 3 Mishkin. S. and Turcottc, R. (1974) Biochem. Biohpys. Res. Commun. 37, 91X-926 4 Ockner, R.K. and Manning J.A. (1974) J. Clin. Invest. 54, 326-33X 5 Goresky. C.A., Daly, D.S.. Mishkin. S. and Arias. I.M. (197X) Am. J. Physiol. 234, E 542-E 553 6 Kushlan, M.C., Gollan, J.L., Ma. W.-L. and Ocknrr. R.K. (1981) J. Lipid Res 22.431-436 M.Y., Elson, C. and Shargo. E. (1976) Bio7 Wu-Rideout. them. Biophys. Res. Commun. 71. X09-816 Z. and X Mishkin, S., Stein, L., Fleishner, G., Gatamaitan. Arias. I.R. (1975) Am. J. Physiol. 228, 1634-1640 9 Ockner. R.K. and Manning J.A. (1976) J. Clin. Invest. SK, b32-641 10 Ockner, R.K., Burnett, D.A.. Lysenko. N. J.A. (1979) J. Clin. Invest. 64, 172-181 11 Ockner, R.K., Lysenko, N., Manning~ J.A.. and Brunett, D.A. (1980) J. Clin. Invest, 6.5, 12 Mishkin, S. and Turcotte, R. (1974) B&hem. Commun. 60. 376-381

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