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K.J. (1987) J. Hiol. Chem. 262, 13219- 13227 22. Moncecchi, L). M., I’astusyzn, A. & Scallen, T. J. (1991)J. Biol. Chem. 266,9885-9892 23. Haker, M. E., Billheimer, J. T. & Strauss, J. F. (1991) DNA Cell Hiol. 10,695-698 24 He, Z.,Yamamoto, K., Furth, E. E., Schantz, 1,. J., Naylor, S. I,., George, H. & Billheimer, J. T. (1991) DNA Cell Hiol. 10, 559-569 25. Ossendorp, B. C., Van-Heudson, G. I’,, L>eIker, A. I,, Hos, K., Schouten, G. I,. & Wirtz, K. W. (1991) Eur. J. Hiochem. 201,233-239 26. Mori, T., Tsukamoto, T., Mori, H., Tashiro, Y. & Fu.jiki, Y. (1991) Proc. Natl. Acad. Sci. [J.S.A. 88, 4338-4342 27. Seedorf, V. & Assman, H. ( 1 99 1) J. Hiol. Chem. 266, 630-636 28. Hillheimer, J. T., Strehl, I,. I,., Davis, G. 1,. & Strauss, J. F.(1990) DNA Cell Bid. 9, 159- 100 20. Ossendorp, H. C., Van-Heudsen, G. 1’. & Wirtz, ti. W. (1990) Biochem. Hiophys. Kes. Commun. 168, 63 1-636 30. Kesav, S., Moncecchi, L). M. & Scallen, T. J. (1990) FASEH J. 4,732 31. Ossendorp, B. C., Geijtenbeek, H. €1. & Wirtz, K. W. A. (1992) FEBS Lett. 296, 179-183 32. Duffaud, G. L)., March, 1’. E. & Inouvre, M. (1 987) in Methods in Enzymology (Wir, K. & Grossman, I,.. eds.), Academic Press 153,492-507 33. Szabo, 1,. J., Small, G. M. 81 1,azarom. 1’. H. (1989) Gene 75. 1 19-126 34. Huang, C. W., Y a m , K. & Takaji, kl. (100 1) Gene 106, h 1-69 35. Tan. H., Okazaki, K., Kubota, I., Kaniiryo, T. & I Jtiyma,H. (1 990) Eur. J. Biochem. 190, 107- 1 12 3 0 . Gadella, T. W. Jr. & Wirtz, K. W. (1091) Hiochim.

Biophys. Acta 1070,237-245 37. Wirtz, K. W. (1001) Klin. Wochenscher 69, 105-1 1 1 38. Schroeder, I;., Hutko, I’., Nemecz, G. & Scallen, T. J. (1990)J. Biol. Chem. 265, 15 1- 157 39. Butko, P., Hapala, I., Scallen, T. J. & Schroeder, F. (1990) Biochemistry 29,4070-4077 40. Schroeder, F.,Hutko, I’., Hapala, I. & Scallen, T. J. (1990) Lipids 25, 669-677 41. Billheimer, J. T. & Gaylor, J. I,. (1990) Hiochem. Hiophys Acta 1046, 136-1 43 42. Mendis-Handagama, S. M., Watkins, 1’. A., Gelber, S. J., Scallen, T. J., Zirkin, H. K. & Ewing, 1,. 1,. (1 990) Endocrinology 127,2947-2954 43. Thompson, S. I,. & Krisans, S. ti. (1990) J. Hiol. Chem. 265, 5731-5735 44. Kennert, H., Amsterdam. A., Hillheimer, J. T. 81 Strauss. J. F.(1 99 1) Biochemistry 30, 1 1280- 1 1285 45. Trezciak, W. 11.. Simpson. E. K., Scallen, T. Vahouny, G. V. & Waterman, M. K. (1987) J. Hiol. Chem. 262,3713-3717 46. Koff>C. F.,I’astuszyn, A,, Strauss, J. F. & Hillheimer, J. T. (1002)J. Iliol. C’hem..267, 15002- 1SOOX 47. Van Heudson, G. 1’. Il., Hos, K., Kaetz, C. K. H. & Wirtz, K. W. A. (1000) J. Hiol. Chem. 265, 4105-41 10 48. Suzuki, Y., Yamazuchi, S.. Orii, T., Tsuneoka, M. & Tashioro, Y. (1000) Cell Struct. Funct. 15, 301-308 49. Lyons, H. T., Kharroubi. A., Wolins, N., Tenner, S.. Chanderbhan, K. F.?Fiskum, G. & Donaldson, K. 1’. (1991) Arch. Hiochem. Hiophys. 285,238-245 50. Kawata, S.. Imai. Y.. Inada. M., Inui, Y., Kakimoto, H., Fukuda, ti.. Maeda. Y. & Tarui. S. (1991) Clin. Chem. Acta 197, 20 1-208 J.?

Keceived 24 June 1002

A role for fatty acids and liver fatty acid binding protein in peroxisome proliferation? Isabelle Issemann, Rebecca Prince, Jonathan Tugwood and Stephen Green*

IC:I PLC, Central Toxicology Laboratory, Cell and Molecular Biology Section, Alderley Park, Macclesfield, Cheshire SK I0 4TJ,U.K.

Introduction I’eroxisome proliferators are a diverse group of chemicals which when administered to rats and mice produce liver hyperplasia and hypertrophy, the latter being predominantly due to an increase in the size and number of hepatic peroxisomes [l]. Interest in these chemicals stems from the observation that they are rodent liver carcinogens. In parAbbreviations used: I’I’AK. peroxisomc proliferator activated receptor; I’PKE. peroxisonie proliferator response clement; 1;ARI’. fatty acid binding protein. ‘To whom correspondence should be addressed.

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ticular, they are not directly mutagenic and appear to use a novel carcinogenic mechanism. There are therefore two important research questions: how do peroxisome proliferators produce liver tumours in rodents such as rats and mice and what is the relevance of these findings to man? The most potent peroxisome proliferators are hypolipidaemic drugs that were developed for the treatment of coronary heart disease [2]. These drugs lower circulating levels of cholesterol but are more effective at lowering triglycerides. They are therefore used to treat patients with type IV hyperlipidaemia who have high levels of circulating tri-

Lipid-BindingProteins

may be to displace fatty acids from FABP, leading to the activation of PPAR.

glycerides. The administration of peroxisome proliferators to rats and mice increases the level of several hepatic enzymes. These include the peroxisoma1 /?-oxidation enzymes such as acyl CoA oxidase, that is responsible for the metabolism of long chain fatty acids, and the microsomal cytochrome P450 IVA1 that possesses lauric acid hydroxylase activity. Of particular interest is the finding that the induction of these enzymes is regulation, at least in part, at the transcriptional level ~3~41. W e recently identified a member of the steroid hormone receptor superfamily that can be activated by peroxisome proliferators [51. We therefore termed this the peroxisome proliferator activated receptor (PPAR). Such receptors are ligand-activated transcription factors and our more recent results have demonstrated that PPAR can bind to a specific DNA sequence (termed the peroxisome proliferator response element, PPRFC) located upstream of the rat acyl CoA oxidase gene [6]. Therefore PPAR appears to mediate the transcriptional effects of peroxisome proliferators. Some of the questions that remain to be resolved, however, are how do peroxisome proliferators activate PPAR and what is its natural ligand? We present here some preliminary data indicating a role for fatty acids in the regulation of PPAR activity and of PPAR in the regulation of liver fatty acid binding protein (FABP) gene expression. These data suggest that one action of peroxisome proliferators

Results

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Fatty acids activate PPAR

Certain high fat diets induce peroxisome proliferation in rats [7]. We were therefore interested to determine whether fatty acids could activate PPAR. Hepal cells were transfected with the PPAR expression vector and the ACO( - 12731 - 471)G.CAT reporter gene that contains the regulatory sequences of the rat peroxisomal acyl CoA oxidase gene upstream of the rabbit /?-globin promoter and the CAT (chloramphenicol transferase) coding sequence [6]. As shown previously, the peroxisome proliferator Wy- 14,643 strongly activated PPAR at low concentrations producing an increase in CAT enzyme activity from the ACO-G.CAT reporter gene (Fig. 1). A variety of fatty acids were next tested by adding them every 24 h for a total period of 48 h. As shown in Fig. 1 a high concentration of palmitic acid stimulated CAT activity indicating that it could activate PPAR. We next tested a thio-substituted fatty acid (tetrathio decanoic acid [8]) that is structurally similar to palmitic acid except that it has a sulphur atom located between the /? and y carbon atoms and is therefore not a substrate for the /?-oxidation pathway. This compound was far more effective than palmitic acid at activating PPAR suggesting that palmitic acid is a

Fig. I Fatty acids activate PPAR The PPAR expression vector and the acyl CoA oxidase reporter gene were transiently transfected in Hepal cells in the presence of increasing concentrations of palmitic acid, tetrathio decanoic acid (TTA) or the peroxisome proliferator Wy- 14.643. The amount of induced CAT enzyme was measured and expressed as the amoaunt of chloramphenicol acetylated by the cellular extract. Technical details can be found in [S].

50

C

2

30-

-0 5

20

!f

~

V

x z l

10

-

I E-9

I E-8

I E-7

I E-6

I E-5

I E-4

I E-3

Concentration (M)

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poor PPAR activator because it is rapidly metabolized.

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A consensus PPRE in the FABP promoter Both FABP enzyme activity and mRNA levels are upregulated in primary hepatocyte cultures by peroxisome proliferators [9]. We were interested to determine whether FABP was regulated at the transcriptional level and in particular whether the promoter of the FABP gene contained a PPRE. As seen in Fig. 2, inspection of the 5' flanking sequence of the FABP gene [ 101 reveals an imperfect direct repeat element between -68 bp and -56 bp (S'TGACCTA TGGCCT 3') that closely resembles the PPRE (5'TGACC??'TGTCCT 3') identified in the rat acyl CoA oxidase gene [6]. T o determine whether this sequence had any role to play in mediating the effects of peroxisome proliferators we cloned a region of the rat liver FABP promoter ( - 565 to + 21) using the polymerase chain reac-

tion and placed this upstream of the coding sequence of the bacterial enzyme CAT gene. This reporter gene, pFABP( - 56.51 + 21)CAT was transfected into a mouse hepatoma cell line (Hepal) in the absence or presence of a PPAR expression vector (pSG5-PPAR) and the potent peroxisome proliferator Wy- 14,643. Disappointingly, the presence of PPAR and Wy-14,643 had no effect upon CAT enzyme activity indicating that the receptor was unable to stimulate the transcription of the FABP promoter. To investigate whether this was because the PPRE-like sequence was unable to respond to PPAR or whether it was due to the context of this sequence in the FABP promoter, we constructed a second reporter gene pFABP( - 5651 -5O)G.CAT in which the FABP sequences from - 565 bp to - 50 bp were placed upstream of the heterologous rabbit /3-globin promoter (Fig. 2). Using this reporter gene in transfection assays demonstrated that it was responsive to the presence Fig. 2

FABP reporter genes The 5' regulatory sequences of the rat liver fatty acid binding protein gene are shown schematically. The start of transcription is indicated with an arrow at + I and the PPRE-like sequence is underlined between -68bp and -56bp. Two reporter gene constructs were created using the rabbit /3-globin promoter ( - I 10 t o + 20) and t w o using the natural FABP promoter. These were tested in transient transfection assays using Hepal cells in the absence or presence of the PPAR receptor expression vector (R) and the peroxisome proliferator Wy- 14,643 (Wy). The values on the right indicate the percentage of chloramphenicol acetylated by the cell extract. Technical details can be found in [6]. Abbreviation used: TTA, tetrathio decanoic acid.

68 Rat FABP gene and promoter

-565

-565

-56

CAATCACTWCCTATCATATTT

-6 FABP

CAT

Globin

-50

68

J 665

68

1%

1%

1%

1%

+21

e -1%

-

CAATCACTTCTACiATCATATATTT

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10%

+21

pFABP(-565/+21)CAT

pFABP(-565/+21 PPRE)CAT

1%

1%

Lipid-Binding Proteins

of PPAR and Wy-14,643 (Fig. 2). In addition, a third reporter gene, pFABP( - 565/ - 68)G.CAT, which does not contain the PPRE-like sequence was not stimulated by PPAR in the presence of Wy-14,643. These data therefore suggest that PPAR can recognize and activate the PPRE-like sequence present in the FABP promoter but that this sequence is not functional in the context of the natural promoter when transfected into Hepa 1 cells.

Discussion A number of potent peroxisome proliferators are hypolipidaemic drugs and lower the level of circulating triglycerides in man. These chemicals induce enzymes that are important in fatty acid metabolism such as peroxisomal acyl CoA oxidase and cytochrome P450 IVA1. Furthermore, high fat diets can induce peroxisome proliferation in rats and peroxisome proliferators can elevate the level of rat liver FABP. Taken together, therefore, these data suggest an important role of PPAR in regulating fatty acid homeostasis. Examination of the 5’ regulatory sequences of the rat liver FABP indicated the presence of a sequence that is almost identical to the PPRE identified in the 5’ regulatory sequences of the rat acyl CoA oxidase gene. This putative response element appears to be inactive in the context of the natural FABP promoter but is active when placed upstream of the rabbit B-globin promoter (Fig. 2). One reason for the failure of PPAR to activate the FABP-CAT reporter gene may be due to TATA binding proteins that bind to the adjacent TATA box sequence interfering with PPAR binding to the PPRE. Alternatively, it may be that Hepal cells lack a co-factor required for PPAR activity when the PPRE is present in the context of the natural FABP promoter. Clearly, further work is required to determine how peroxisome proliferators regulate FABP expression. Of particular interest was the finding that fatty acids are weak PPAR activators ([ 113, Fig. 1). One explanation for this weak activity is that fatty acids are metabolized rapidly by the /?-oxidation pathway. This is supported by the observation that the thio-substituted fatty acid that is blocked for /?oxidation is a much better PPAR activator (Fig. 1). W e and others have found that a wide range of fatty acids are able to activate PPAR ([ 113, I. Issemann and R. Prince, unpublished results). An important question, therefore, is whether any or all of these fatty acids can bind directly to PPAR or alternatively whether they are metabolized to the ligand or induce the ligand. These data also raise the question

of how synthetic peroxisome proliferators activate PPAR. One possibility is that they bind directly to the receptor in the same way that antioestrogens such as tamoxifen bind to the oestrogen receptor. However, thus far we have been unable to demonstrate any interaction between PPAR and the peroxisome proliferator nafenopin [51. An interesting alternative mechanism for peroxisome proliferator action is that these chemicals bind to FABP displacing fatty acids that activate PPAR. Others [12] have demonstrated that a number of peroxisome proliferators can bind to FABP and displace the oleic acid used to monitor the purification of FABP. It will therefore be important to determine whether peroxisome proliferators act by simply competing with fatty acids for binding to FABP leading to an elevation of fatty acids that activate PPAR, or alternatively whether they and fatty acids can bind directly to PPAR. These experiments and others that determine the true nature of the PPAR ligand will be important in understanding the role of peroxisome proliferators and PPAR in hypolipidaemia and rodent liver cancer. W e thank Jon Bremer for providing the tetrathio decanoic acid used in these studies. 1. Green, S. (1992) Biochem. Pharmacol. 43, 393-401 2. Havel, R. J. & Kane, J. P. (1973) Annu. Rev. Pharmacol. 13,287-308 3. Keddy, J. K., Goel, S. K., Nemali, M. K., Carrino, J. J., Laffler, T. G., Reddy, M. K., Sperbeck, S. J., Osumi, T., Hashimoto, T., Lalwani, N. D. & Kao, M. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1747-1751 4. Hardwick, J. P., Song, B. J., Huberman, E. & Gonzalez, F. J. (1987) J. Biol. Chem. 262,801-810 5. Issemann, I. & Green, S. (1990) Nature 347,645-650 6. Tugwood, J. D. Issemann, I., Anderson, R. G., Bundell, K. R., McPheat, W. L. & Green, S. (1992) EMBO J. 11,433-439 7. Brandes, R., Kaikaus, R. M., Lysenko, N., Ockner, K. K. & Bass, N. M. (1990) Biochem. Biophys. Acta 1034,5341 8. Sweetser, D. A,, Lowe, J. R. & Gordon, J. I. (1986) J. Hiol. Chem. 261,5553-5561 9. Flatmark, T., Nilsson, A,, Kvannes, J. Eikhom, T. S., Fukami, M. H., Kryvi, H. & Christiansen, E. N. (1988) Biochem. Biophys. Acta 962, 122-130 10. Spydevold, 0.& Bremer, J. (1989) Biochem. Biophys. Acta 1003,72-79 11. Gottlicher, M., Widmark, E., Li, Q. & Gustafsson, J.-A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4653-4657 12. Cannon, J. R. & Eacho, P. I. (1991) Biochem. J. 280, 387-391 Received 25 June 1992

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