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IN VITRO MECHANISTIC STUDIES OF PEROXISOME PROLIFERATION BY
CHIRAL CLOFIBRATE ANALOGS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Shamina M. Rangwala, B. Pharm.
*****
The Ohio State University
1997
Dissertation Committee:
Dr. Dennis R. Feller, Adviser Approved by
Dr. Norman J. Uretsky, Co-Adviser
Dr. Lane J. Wallace Adviser ' Dr. Bethany J Holycross College of Pharmacy ÜMI Number: 9801766
UMI Microform 9801766 Copyright 1997, by UMI Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code.
UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT
The mechanism by which clofibrate causes peroxisome proliferation is not known.
A series of chiral clofibrate-related 2-(4-chlorophenoxy)acetic acid (CPAA) analogs were
selected to examine the effects of stereoselectivity on activation of peroxisome
proiiferator-activated receptor (PPAR) and stimulation of peroxisomal activity. The
abilities of these compounds to activate the receptor were examined using transactivation
assays using exogenously added PPARa in CV-1 and endogenous PPAR in H4IIEC3
cells. In both systems, the 2-n-propyl and 2-phenyl-substituted CPAA analogs were highly
stereoselective activators of PPARa, suggesting that these compounds act via a receptor.
The effects of these analogs on peroxisomal acyl-CoA oxidase aciidty was determined.
There exists a strong correlation between the ability of a compound to activate PPAR and
stimulate fatty acyl-CoA oxidase (AGO) activity, implicating a causal relationship between
these events. Small modifications in structure around the 2 position of CPAA can result in
dramatic alterations in stereoselectivity and potency, e.g., changing the n-propyl
substituent to the isopropyl substituent results in the loss of stereoselectivity, while the
substitution of the n-hexyl group with the phenyl group at the 2 -position results in a
decrease in activity, however, the stereoselectivity increases strikingly. Enlargement of the chlorophenoxy group to the chlorobenzyloxy group leads to an increase in activity of the compound; however, addition of one more phenyl ring to this side-chain decreased activity, but increased stereoselectivity. We showed that the rank order o f stereoselectivity observed for activating PPAR and stimulating ACO activity is not conserved for displacing tritiated oleic acid from L-FABP. This indicates that binding to liver fatty acid binding protein (L-FABP) is not predictive of the peroxisome proliferative effects of these compounds. However, for non-metabolizable fatty acids tested, such as 2- bromopalmitic acid and perfluorinated octanoic acid, L-FABP may play an important role in the signaling pathways for these molecules as compared to xenobiotic peroxisome proliferators. Ciprofibrate and 2-n-propyl- and 2-phenyl-substituted CPAA analogs bind to the Xenopus PPARa ligand binding domain, displacing leukotriene B$ from this binding site. Thus, the peroxisome proliferative effects of the fibrates are probably mediated by the direct binding of these compounds to the PPARa.
in To my parents, Mohsin and Sehra Rangwala, without your encouragement, faith and support, I would have never come this far.
With my love and thanks.
IV ACKNOWLEDGMENTS
I would like to thank;
Dr. Dennis Feller, for his advice, support and constant enthusiasm throughout my
graduate career.
Dr. Norman Uretsky, my co-adviser at OSU, for his advice and help, especially in
the past two years. Other members of my dissertation committee, Drs. Wallace and
Holycross, for their assistance during the preparation of this dissertation.
Dr. Dan Noonan, Michelle O’Brien and George Yuan, at the University of
Kentucky, for their friendship, help and support throughout this research.
Dr. David Pasco, Chuan-Li Xu, Vincent Siu and all the other members of the
Pasco lab at Ole Miss for their assistance and friendship and for tolerating my constant questions while setting up some of the experimental methods!
Dr. JefiF Lawrence and Dr. Patrick Eacho, Eli Lilly and Company, for their assistance.
Rose Smith and Kathy Brooks at OSU for their cheerful, eflBcient personalities, which helped making the move to Ole Miss less stressful. Becky Drewery for her friendship and help, especially while this dissertation was being written. My friends, both at OSU and Ole Miss; Suzette, Joya, Marina, Subbu, Ratna,
Anish, Edwin, David, Reshma, Grace and Vivek, for their companionship which always made me feel like I belonged; thank you for making my graduate school experience an entirely enjoyable one.
My family, whose love, encouragement and support has sustained me throughout my graduate study.
VI VITA
June 13, 1971 ...... Born-Bombay, India 1992 ...... Bachelor of Pharmaceutical Sciences, University of Bombay. 1992 - 1993 ...... University Fellow, The Ohio State University. 1993 - present ...... Graduate Teaching and Research Associate The Ohio State University
PUBLICATIONS
O'Brien ML, Rangwala SM, Henry KW, Weinberger C, Crick DC, Waechter CJ, Feller DR and Noonan DJ. Convergence of three steroid receptor pathways in the mediation of nongenotoxic hepatocarcinogenesis. Carcinogenesis 17: 185-190, 1996.
Feller DR, O'Brien M, Rangwala SM, Tortorella V, Loiodice F and Noonan DJ. Structural requirements of chiral clofibric acid analogs for activation of the rat peroxisome proiiferator-activated receptora (rPPARa). Ann. N. Y. Acad. Sci. 804: 713-715, 1996.
Rangwala SM, O'Brien ML, Loiodice F, Longo A, Tortorella V, Noonan DJ and Feller DR. Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proiiferator- activated receptor (PPARa) activation and peroxisomal fatty acid B-oxidation. Chirality 9: 37-47, 1997.
vu Rangwala SM, O'Brien ML, Noonan DJ and Feller DR. Differential effects of statins on peroxisomal acyl CoA activity and the rat peroxisome proliferator activated receptor (rPPAR). FASEBJ. 9: A690, 1995.
Rangwala SM, O'Brien M, Loiodice F, Tortorella V, Noonan DJ and Feller DR. Specificity of the effects of isomeric clofibric acid analogs on hepatic peroxisome proliferation. Toxicologist 3d'. 1\0, 1996
Rangwala SM, O’Brien M, Lawrence J, Eacho P, Tortorella V, Loiodice F, Longo A and Feller DR. Studies on the mechanism of peroxisome proliferation by clofibric acid analogs. Toxicologist 252, 1997.
FIELD OF STUDY
Major Field: Pharmacy Pharmacology
vui TABLE OF CONTENTS
Page Abstract...... ii
Dedication ...... iv
Acknowledgments ...... v
Vita...... vii
List of Figures ...... xiii
List of Tables ...... xvi
Chapters:
1. Introduction ...... 1 1.1 Peroxisomes ...... 1 1.1.1 Morphology ...... 1 1.1.2 Biogenesis ...... 2 1.1.3 Biochemistry...... 2 1.2 Peroxisome proliferation ...... 5 1.2.1 Definition ...... 5 1.2.2 Hepatic peroxisome proliferation ...... 5
1.2.2.1 Hepatomegaly ...... 6
1.2.2.2 Peroxisomal and microsomalenzyme induction 6 1.2.2.3 Hepatic carcinogenesis ...... 7 1.2.3 Species differences and relevance to humans ...... 9 1.3 Peroxisome proliferators ...... 10 1.4 Mechanism of peroxisome proliferation ...... 13 1.4.1 Fatty acid overload theory ...... 13 1.4.2 Receptor hypothesis ...... 15 1.4.3 Peroxisome proiiferator-activated receptor (PPAR) ...... 16 1.4.3.1 PPAR isoforms ...... 17
1 .4.3.2 Differential expression of PPARs ...... 17
IX 1.4.3.3 Structure of PPARs ...... 19 1.4.3.4 DNA binding of PPARs ...... 20 1.4.4 Peroxisome proliferator response elements (PPREs) ...... 22 1.5 PPARa and lipid metabolism ...... 24 1.5.1 Role of PPARa as a physiological regulator ...... 24 1.5.2 Hypolipidemic effects of fibrates ...... 26 1.5.2.1 Effects on HDL constituents ...... 26 1.5.2.2 Effects on metabolism of triglyceride-rich lipoproteins ...... 26 1.5.3 Ligands for PPARa ...... 27
1 . 6 PPARy and adipogenesis ...... 28 1.7 Differential activation of PPARs ...... 30 1.8 Statement of the problem ...... 32 1. 8. 1 Objective and rationale ...... 32 1.8.2 Significance ...... 37
2. Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proiiferator-activated receptor a (rPPARa) activation and peroxisomal fatty acid P-oxidation ...... 48 2.1 Introduction ...... 48 2.2 Methods ...... 52 2.2.1 Materials...... 52 2.2.2 Cell culture...... 53 2.2.3 Co-transfection assay for CV-1 cells using calcium phosphate coprecipitation ...... 53 2.2.4 Transfection of H4HEC3 cells ...... 55 2.2.5 Cell treatment and assay for peroxisomal P-oxidation ...... 56 2.2.6 Data analysis...... 57 2.3 Results...... 58 2.3.1 Effects of 2-substituted analogs of 2-(4-chlorophenoxy)acetic acid (CPAA) on rPPARa activation...... 58 2.3.2 Effect of the chemical modification of the 4-chlorophenoxy group of the CPAA analogs on activation of rPPARa 59 2.3.3 Characterization of the PPAR in H4IIEC3 cells ...... 60 2.3.4 Activation of rPPARa in H4IIEC3 cells by 2-substituted analogs of CPAA...... 61 2.3.5 Effects of modification of 4-chlorophenoxy group on acivation of PPARa in H4IIEC3 cells ...... 61 2.3.6 Effects of isomeric CPAA analogs on peroxisomal ACO activity in H4IIEC3 cells...... 62 2.3.7 Studies of the effects of inactive isomers of analogs HI and VI on PPAR activation in H4IIEC3 cells ...... 63 2.4 Discussion ...... 64 3. Role of liver fatty acid binding protein (L-FABP) in peroxisome proliferation ...... 97 3.1 Introduction ...... 97 3.2 Methods ...... 101 3.2.1 Materials...... 101 3.2.2 Isolation and purification of L-FABP ...... 102 3.2.3 L-FABP binding assay ...... 102 3.2.4 Transfection assay ...... 103 3.2.5 Data analysis...... 104 3.3 Results...... 105 3.3.1 Evaluation of the aflSnities of clofibric acid (CFA) and ciprofibrate (CIPRO) for binding liver fatty acid binding protein (L-FABP) ...... 105 3.3.2 Evaluation of the aflBnities of 2-bromopalmitic acid (BP A), 12-hydroxydodecanoic acid (12-OH) and dodecanoic acid (DA) for binding to L-FABP ...... 106 3.3.3 Determination of the potencies of the stereoisomeric analogs of 2-(4-chlorophenoxy)acetic acid (CPAA) for binding to L-FABP ...... 106 3.3.4 Evaluation of the afRnities of perfluorinated fatty acids to bind to L-FABP ...... 107 3.3.5 Activation of PPARa in H4UEC3 cells by perfluorinated fatty acids ...... 108 3.4 Discussion ...... 108
4. Evaluation of the ability of fibric acid analogs to bind to the ligand binding domain of xPPARa ...... 128 4.1 Introduction ...... 128 4.2 Methods ...... 131 4.2.1 Materials...... 131 4.2.2 Expression of xPPARa(LBD)-GST fusion protein ...... 132 4.2.3 Purification of xPPARa(LBD)-GST fusion protein ...... 133 4.2.4 Identification of purified protein using SDS-PAGE ...... 134 4.2.5 Ligand binding assay ...... 135 4.2.6 Data analysis...... 136 4.3 Results...... 136 4.3 .1 Isolation and purification of GST-xPP ARa(LBD) ...... 136 4.3.2 Ability of clofibric acid (CFA), ciprofibrate (CIPRO)
and LTB4 to displace [^HJ-LTB^ ...... 137 4.3.3 Binding of n-propyl and phenyl 2-(4-chlorophenoxy)acetic acid analogs to xPPARa(LBD)-GST ...... 139
XI 4.4 Discussion ...... 139
5. Summary and conclusions ...... 150
Appendix ...... 157 List of references ...... 159
XU LIST OF FIGURES
Figure Page
1.1. Pathway of peroxisomal cholesterol biosynthesis ...... 39
1.2 Chemical structures of fibric acid and non-fibric acid hypolipidemic agents ...... 40
1.3 Chemical structures of plasticizers and phenoxy herbicides ...... 41
1.4 Chemical structures of miscellaneous drugs and fatty acids that cause peroxisome proliferation ...... 42
1.5 Schematic representing the organization of PPARs into functional domains and the binding of the PPAR/RXR heterodimer to a PPRE ...... 43
1. 6 Structures of compounds that can bind PPARa ...... 44
1.7 Prostaglandin J, metabolites and thiazolidinediones that are PPARy ligands 45
1. 8 Structures of 2-(4-chlorophenoxy)acetic acid (CPAA) analogs...... 47
2.1 Concentration-response curves for the activation of rPPARa in CV-1 cells by 2-substituted analogs of 2-(chlorophenoxy)acetic acid [CPAA] and clofibric acid ...... 75
2.2 Concentration-response curves for the activation of rPPARa in C V-1 cells by 2-substituted analogs of 2-(chlorophenoxy)acetic acid [CPAA] ...... 77
2.3 Concentration-response curves for the activation of rPPARa in CV-1 cells by 2-substituted analogs of 2-(chlorophenoxy)acetic acid [CPAA] ...... 79
2.4 Concentration-response curves for the activation of rPPARa in CV-1 cells by isomers of 2-(4-chlorobenzyloxy)-propanoic acid and 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid ...... 81
xm 2.5 Concentration-response curves for the activation of rPPARa in H4IIEC3 cells by clofibric acid (CFA), ciprofibrate (CIPRO) and BRL 49653 ...... 83
2.6 Concentration-response curves for the activation of rPPARa in H4IIEC3 cells by 2-substituted analogs of 2-(chlorophenoxy)acetic acid [CPAA] and clofibric acid (CFA) ...... 84
2.7 Concentration-response curves for the activation of rPPARa in H4IIEC3
cells by 2 -substituted analogs of 2-(chIorophenoxy)acetic acid [CPAA] ...... 8 6
2.8 Concentration-response curves for the activation of rPPARa in H4IIEC3 cells by isomers of 2-(4-chlorobenzyloxy)-propanoic acid and
2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid ...... 8 8
2.9 Concentration-response curves for the stimulation of acyl-CoA oxidase activity in H4IIEC3 cells by 2-substituted analogs of 2-(chlorophenoxy)acetic acid [CPAA]...... 90
2. 1 0 Concentration-response curves for the stimulation of acyl-CoA oxidase activity in H4IIEC3 cells by 2-substituted analogs of 2-(4-chlorophenoxy)acetic acid [CPAA]...... 92
2.11 Concentration-response curves for the stimulation of acyl-CoA oxidase activity in H4IIEC3 cells by isomers of 2-(4-chlorobenzyloxy)-propanoic acid and 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid ...... 94
2.12 Efiects of increasing concentrations of (+)-(R)-in, the inactive isomer of the 2-substituted n-propyl analog of CPAA on the ciprofibrate-induced rPPARa activation ...... 96
3.1 Concentration-response curves of clofibric acid (CFA) and ciprofibrate (CIPRO) for the displacement of [^H]-oleic acid fi’om liver fatty acid binding protein ...(L-FABP)...... 118
3.2 Concentration-response curves of 12-hydroxydodecanoic acid ( 12-OH), 2-bromopalmitic acid (BPA), dodecanoic acid (DA) and clofibric acid (CFA) for the displacement of [^H]-oleic acid fi"om liver fatty acid binding protein (L-FABP)...... 119
3.3 Concentration-response curves of 2-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs I and EQ for the displacement of [^H]-oleic acid fi-om liver fatty acid binding protein (L-FABP) ...... 120
XIV 3.4 Concentration-response curves of 2-substituted 2-(4-chiorophenoxy)acetic acid (CPAA) analogs IV and VT for the displacement of -oleic acid from liver fatty acid binding protein (L-FABP) ...... 122
3.5 Concentration-response curves of 2-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs Vn and VUI for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP) ...... 124
3.6 Concentration-response curves of perfluorooctanoic acid (PFOA), perhuorobutanoic acid (PFBA), perfluorodecanoic acid (PFDA), perfluorooctanol (PFOL) and clofibric acid (CFA) for the displacement of -oleic acid from liver fatty acid binding protein (L-FABP)...... 126
3.7 Concentration-response curves of perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA), perfluorooctanol (PFOL), clofibric acid (CFA) and ciprofibrate (CIPRO) for the activation of rPPARa in H4IIEC3 ceUs...... 127
4.1 SDS-Polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the fusion protein, xPPARa(LBD)-GST ...... 145
4.2 A Effect of clofibric acid (CFA) and ciprofibrate (CIPRO) on the binding
of [^H]-LTB 4 to xPPARa(LBD)-GST. B. Effects of unlabeled LTB^ on the
binding of [^H]-LTB 4 to xPPARa(LBD)-GST. C. Representative experiment
demonstrating the concentration-dependent displacement of [^H]-LTB 4 by CIPRO...... 146
4.3 Displacement of [^H]-LTB 4 by the stereoisomers of the 2-n-propyl-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs (analog HI) ...... 148
4.4 Displacement of [^H]-LTB 4 by the stereoisomers of the 2-rt-propyl-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs (analog IE) ...... 149
XV LIST OF TABLES
Table Page
2.1 Representative experiment showing the eflfects of varying voltages on the transfection of a luciferase plasmid into H4IIEC3 cells ...... 70
2.2 Effects of ciprofibrate on luciferase activity when the PPRE-less reporter plasmid was transfected into H4IIEC3 cells ...... 71
2.3 Effects of (+)-(R)-isomer of analog m (n-propyl derivative of CPAA) on ciprofibrate-induced PPARa activation in H4HEC3 cells ...... 72
2.4 Effects of (-)-(S)-isomer of analog VI (2-phenyl derivative of CPAA) on ciprofibrate-induced PPARa activation in H4IIEC3 cells ...... 73
2.5 Effects of (+)-(R)-isomer of analog EQ («-propyl derivative of CPAA) on 2-bromopalmitic acid-induced PPARa activation in H4IIEC3 cells ...... 74
3. 1 Chemical structures o f fatty acid analogs ...... 115
3.2 IC5 0 and Kj values for clofibric acid (CFA), ciprofibrate (CIPRO), 2-bromopalmitic acid (BPA), 12-hydroxydodecanoic acid( 12-OH) and dodecanoic acid (DA) for the displacementof [^H]-oleic acid fi’om liver fatty acid binding protein (L-FABP) ...... 116
3.3 ICjo and Kj values for 2-(4-chlorophenoxy)acetic acid (CPAA) for the displacement of [^H]-oleic acid fiom liver fatty acid binding protein (L-FABP)...... 117
4.1 Kj values for ciprofibrate (CIPRO) and 2-«-propyl and 2-phenyl-substituted 2-(4-chlorophenoxy)acetic acid (CPAA)
for displacing LTB 4 fiom the xPPARa (LBD)-GST fusion protein ...... 144
XVI CHAPTER 1
INTRODUCTION
1.1 Peroxisomes
Peroxisomes are small, cytosolic organelles that are present in most eukaryotic cells. They were first discovered by Rhodin., in 1954 in the mouse renal proximal convoluted tubular epithelium. Due to their small appearance, he termed these organelles
"microbodies". De Duve et al. (1965) and others (DeDuve and Bandhuin, 1966; Reddy and Lalwani, 1983; Krisans, 1996) biochemically characterized these organelles, and subsequently, discovered enzymes involved in gluconeogenesis, fatty acid metabolism, thermogenesis, cholesterol and bile acid synthesis and purine metabolism . DeDuve used the term “peroxisome” since hydrogen peroxide was generated by enzymes contained in these bodies.
1.1.1 Morphology
Morphologically, under electron micrograph, peroxisomes appear to be round, approximately 0.5 - 1.0 pm in diameter, consisting of a single membrane surrounding a homogenous granular matrix (Reddy and Lalwani, 1983; Goldfischer and Reddy, 1984). The shape and size of peroxisomes may vary with the type of cell under investigation. In
most species, peroxisomes contain an electron-dense crystalloid core which contains urate
oxidase (Hruban and Swift, 1964). Human hepatic peroxisomes lack this urate oxidase
core (Goldfischer and Reddy, 1984).
1.1.2 Biogenesis
Biogenesis of peroxisomes occurs by the division of existing ones. Peroxisomes do not contain DNA, therefore, genes encoding the peroxisomal proteins are located in the nucleus (Lazarow and Fujuki, 1985). The newly synthesized proteins are targeted to the peroxisomes by means of peroxisomal targeting sequence (PTS), which consists of a C- terminal peptide, which is typically SKL (Gould et al, 1987). Recently, it has been demonstrated that a N-terminal oligopeptide (11-16 amino acids) and internal sequences may also act as PTSs (Lazarow et al., 1996). Peroxisomes have a half-life of 1.5 days; they are destroyed by autophagy, which is the fusion of the entire peroxisome with lysosomes (Lazarow et ai, 1996).
1.1.3 Biochemistry
Peroxisomes contain a large variety of enzymes, a majority of which are involved in lipid metabolism. Like the mitochondria, peroxisomes are capable of degrading fatty acids via P-oxidation (Lazarow and deDuve, 1976). The peroxisomal B-oxidation pathway is different from that of the mitochondria in many aspects, including the following: 1) The enzymes in each system are different proteins (Tolbert, 1981; Hashimoto, 1982; Hashimoto, 1996; Mannaerts and Debeer, 1982; Moser 1987; Schulz,
1991). 2) Peroxisomal P-oxidation system is carnitine-independent. 3) Peroxisomal fatty
acid P-oxidation is a cyanide-insensitive palmitoyl-CoA-dependent NAD’’ reduction. 4)
The first enzyme of the peroxisomal pathway, fatty acyl-CoA oxidase, is a flavin- containing oxidase which produces H^O;. 5) The second and third steps in peroxisomes are catalyzed by a bifunctional enzyme, which exhibits both the enoyl CoA hydratase and
3-hydroxy fatty acyl-CoA dehydrogenase activities, whereas the mitochondria contains two separate enzymes. The peroxisomal bifunctional enzyme protein also possesses enoyl-CoA isomerase activity, making it a trifUnctional protein, but it is yet referred to as
the bifunctional protein. 6 ) Mitochondrial fatty acid B-oxidation is coupled to the electron transport chain and oxidative phosphorylation, therefore, all the energy produced during this process is conserved as high-energy phosphate. In the peroxisomal fatty acid B- oxidation pathway, the energy is either lost as heat or is conserved as high energy level electrons of NADH*. 7) Mitochondria oxidize short-, medium- and long-chain fatty acids, but cannot act on very-long-chain fatty acids which are degraded exclusively in the peroxisomes. This is because mitochondria lack the enzyme very-long-chain acyl CoA synthetase, which activates these fatty acids for p-oxidation (Singh et al., 1987).
Peroxisomes, in contrast, can process medium-, long- and very-long-chain fatty acids, but cannot act on short-chain fatty acids, as the first enzyme in the peroxisomal P-oxidation
pathway, fatty acyl-CoA oxidase requires a substrate to have a minimum chain length of 8 carbon atoms (Hashimoto, 1996). Isolated mitochondria and peroxisomes can degrade 2- methyl-branched fatty acids and certain eicosanoids (Vanhove et ai, 1991; Diczfalusy and Alexson, 1990). However, peroxisomes play an important role in the B-oxidation of bile
acid intermediates and dicarboxylic acids (Mannaerts and Van Veldhoven, 1990; Leighton
et ai, 1989). Thus, the major role of the peroxisomes is chain-shortening o f fatty acids, to
produce fatty acids that are good substrates for mitochondrial B-oxidation (Hashimoto,
1982; Mannaerts and Van Veldhoven, 1990).
Besides fatty acyl-CoA oxidase, peroxisomes contain other flavin oxidases (urate
oxidase, D-amino acid oxidase, glycolate oxidase) which generate H^O;. Catalase present
in these organelles is responsible for the breakdown of this peroxide. Catalase is the main
marker enzyme for peroxisomes and is used for the diaminobenzidine staining for
morphologic visualization of these organelles (Novikoflf and Goldfischer, 1969).
Recently, it has been found that certain enzymes involved in cholesterol biosynthesis are located in the peroxisomes (Krisans, 1996) (Fig. 1.1). Two such enzymes, mevaionate kinase and famesyl diphosphate kinase, are present exclusively in the peroxisomes. The enzymes involved in the initial stages of cholesterol biosynthesis, in the conversion of acetyl CoA to HMG CoA reductase, are present both in the peroxisomes and the cytosol. The next stage, i.e., the conversion of HfvtG CoA to mevaionate occurs both in the endoplasmic reticulum and In the peroxisomes, while the further conversion of mevaionate to famesyl diphosphate takes place predominantly in the peroxisome.
Conversion of famesyl diphosphate to squalene occurs solely in the endoplasmic reticulum, and lastly, the metabolism of lanosterol to cholesterol occurs both in the ER and the peroxisomes. Thus, the peroxisomal compartment is vital for another aspect of lipid metabolism, that of cholesterol synthesis. Famesyl diphosphate, which is synthesized
in the peroxisomes, is an important precursor for heme a, ubiqumone and dolichol
synthesis, as well as for the isoprenylation of proteins (Krisans, 1996).
In humans, peroxisomes also contain enzymes involved in the a oxidation (also
known as the oxidative decarboxylation) of branched-chain fatty acids, such as phytanic
acid, which cannot undergo direct P-oxidation, due to the presence of a 3-methyl
substitution (Singh et ai, 1993). Peroxisomes are also responsible for the synthesis of
ether lipids (Tolbert, 1981). Children suflfering from Zellweger's syndrome, a disease
characterized by a lack of peroxisomes, lack plasmalogens, a class of unsaturated ether
phospholipids that contribute a substantial part of the phospholipids of the heart and brain.
Other enzymes present in the mammalian peroxisomes include enzymes present in purine
and amino acid catabolism and in glyoxylate metabolism.
1.2. Peroxisome proliferation
1.2.1 Definition
Paget first reported the changes in the hepatic ultrastructure of rats on administration of clofibrate (1963). In 1965, Hess et al. and, later, Svoboda and Azamoff
(1966), described the remarkable increase in the number of peroxisomes in the liver of rodents which were administered clofibrate. Reddy et al. (1975) coined the term
“peroxisome proliferator” for drugs that caused the proliferation of peroxisomes, and the phenomenon was termed peroxisome proliferation. 1.2.2 Hepatic peroxisome proliferation
The most prominent effects of peroxisome proliferators were seen on the liver.
The kidney, though aSected, did not show the same extent of changes. The increase in the number and volume density of the peroxisomes in the hepatic parenchymal cells is accompanied by hepatomegaly and induction of certain peroxisomal and microsomal enzyme levels. The hepatic responses to peroxisome proliferators are discussed below.
1.2.2.1 Hepatomegaly
The hepatic effects observed in rats, mice and hamsters involve hyperplasia (an increase in cell number) as well as hypertrophy (an Increase in cell size) (Reddy and
Lalwani, 1983). Liver enlargement occurs within a few days of the administration of the compound, and reaches a steady state within 10 to 14 days. These effects are discernable as long as the compound is administered. The hepatomegalic effect is dose dependent, and is observed in both sexes. After cessation of drug treatment, the rat liver returns to normal after a period of 1-2 weeks (Reddy and Lalwani, 1983; Marsman et ai, 1988).
1.2.2.2 Peroxisomal and microsomal enzyme induction
Several enzymes are induced on treatment with peroxisome proliferators. The enzymes of the peroxisomal P-oxidative pathway, namely, fatty acyl-CoA oxidase
(Lazarow and De Duve, 1976; Reddy and Krishnakantha, 1975; Osumi and Hashimoto,
1979), enoyl-CoA hydratase (Lazarow, 1978; Osumi and Hashimoto, 1979; Reddy and
Lalwani, 1983), 3-hydroxyacyl-CoA dehydrogenase (Lazarow and DeDuve, 1976; Osumi and Hashimoto, 1979) and 3-ketoacyI-CoA thiolase (Lazarow, 1978; Osumi and
Hashimoto, 1979) are induced 10-30 fold. In comparison, catalase, the enzyme which
degrades the HjOj produced during P-oxidation, is induced only about 2 fold (Reddy and
Lalwani, 1983). Of the peroxisomal non-fatty acid P-oxidative enzymes, carnitine
acetyltransferase (Moody and Reddy, 1974), carnitine octanoyl transferase (Chatteijee et
al, 1988) and acyl-CoA dihydroxyacetone phosphate acyltransferase (Reddy and Lalwani,
1983) are also induced in vivo. Besides these peroxisomal proteins, the CZP4A subfamily,
which catalyze the w-hydroxylation of fatty acids (Gibson et al, 1982; Gibson, 1996) and
prostaglandins Ej and (Van den Bosch et ai, 1992), are induced by peroxisome
proliferators.
1.2.2.3 Hepatic carcinogenesis
A long-term exposure to hypolipidemic drugs and industrial plasticizers results in development of hepatic tumors in rodents (Reddy et a l, 1980; Reddy and Lalwani, 1983).
However, the mechanism of carcinogenesis is unknown. These compounds are non- genotoxic in in vitro and in vivo assays (Warren et al, 1980; Reddy et a l, 1980; Kluwe et al, 1982) and they do not bind or damage DNA, nor do they cause an increase in unscheduled DNA synthesis (Gupta et al 1985; Glauert et al, 1984; Rao and Reddy,
1996). The oxidative stress hypothesis proposes that the hydrogen peroxide produced as a consequence of the increased peroxisomal B-oxidation during peroxisome proliferation, leads to a state of oxidative stress, and, consequently, DNA damage and tumor initiation
(Reddy and Lalwani, 1983). This is supported by reports of increases in 8 -hydroxy- deoxyguanosine lesions, and, after prolonged administration, an accumulation of
lipofuscin, a measure of oxidative damage, in livers of rats fed with peroxisome
proliferators (Kasai et ai, 1989, Conway et al., 1989). Chu et al. (1995) recently found
that overexpressing fatty acyl CoA oxidase caused CV-1 cells to be transformed into
anchorage-independent cells that develop into tumors when transplanted into nude mice.
An increased activity of the transcription factor NF-kB is seen in livers of rats fed 0.01 %
ciprofibrate in their diets. This could indicate increased levels of reactive oxygen
intermediates in the liver, as NF-kB activity is linked to the levels of active oxygen species
(Lie/a/., 1996).
It has been suggested that cell proliferation may be the principal mechanism by
which peroxisome proliferators cause tumors (Butterworth et ai, 1987). Ames and Gold
(1990) have proposed that any agent that causes persistent cell proliferation may be
mutagenic, as it increases the chance of converting endogenous DNA damage to
mutations. Cellular proliferation in response to peroxisome proliferators peaks after 3-4
days and then declines (Butterworth et al., 1987; Rao and Reddy, 1996). For most
peroxisome proliferators, there is a secondary wave of proliferation over the next 1-2
weeks (Yeldandi et al., 1989). The relative importance of the role of proliferation of peroxisomes versus hyperplasia in the subsequent hepatocarcinogenesis is under debate.
But even if peroxisome proliferation itself is the factor responsible for tumor initiation, cellular proliferation may be an important factor for promoting the lesions initiated by peroxisome proliferators. The hypolipidemic agent nafenopin has been shown to suppress apoptosis, which
could indicate that the hepatocarcinogenic potential of peroxisome proliferators is due to
their ability to prevent the removal of DNA-damaged, potentially initiated cells from the
liver (Bayly et ai, 1994). These cells, protected from apoptosis, then undergo clonal
expansion, and may develop into adenocarcinomas. Upon withdrawal of the drug, only
about 10 % of newly synthesized cells undergo apoptosis; a vast majority of the cells (90
%) that apoptose were not present before treatment with the drug (Roberts, 1996).
However, no other workers have confirmed that peroxisome proliferators inhibit
apoptosis.
1.2.3 Species differences and relevance to humans
Rats and mice are most sensitive to peroxisome proliferators (Reddy and Lalwani,
1983; Elcombe et ai, 1985; Lock et ai, 1989). Reddy and coworkers (1984) have reported that ciprofibrate causes peroxisome proliferation in monkeys, but other investigators have not been able to demonstrate peroxisome proliferation in dogs. Rhesus monkeys, marmosets and guinea pigs (Lake et ai, 1989; Eacho et ai, 1986; Watanabe et ai, 1989). In humans, the available data suggests that peroxisome proliferators have no effects on the hepatic peroxisome number or on peroxisomal enzyme activity. Livers from patients on long-term fibrate therapy have been morphologically examined, and show no significant increase in peroxisomal number or volume (De la Iglesia et ai, 1982; Blümcke et ai, 1983; Hanefeld et ai, 1983). When tested in primary cultures of human hépatocytes, clofibric acid and mono(2-ethyihexyI)phthalate did not cause any peroxisome
proliferation (Bichet et ai, 1990). Therefore, the response to peroxisome proliferators in
rodents may not be predictive of the response in humans.
1.3 Peroxisome proliferators
A large variety of chemicals possessing diverse chemical structures cause
peroxisome proliferation. If a clear set of structure-activity relationships could be defined
for peroxisome proliferators, it would help in the elucidation of the mechanism of this
phenomenon. Comprehensive structure-activity analyses for these compounds are not
available; however, some investigators have examined this issue (Eacho et al., 1989; Feller
and Intrasuksri, 1993; Esbenshade e/a/., 1990; Lakea/., 1986, 1987 and 1988; Lewis
etal, 1987; Feller era/., 1996; Rangwala era/., 1997). Peroxisome proliferators exhibit two characteristic structural properties: they possess an acidic or a potential acidic group, and they have a lipophilic, unmetabolizable structure. Moody et al. (1991) classified peroxisome proliferators into seven groups. Figures 1.2-1.4 show structures of a few chosen peroxisome proliferators.
Group 1 : Fibric acids
The hypolipidemic agent, clofibrate (ethyl-a-p-chlorophenoxyisobutyrate), was the first compound shown to cause changes in the peroxisomes (Paget, 1963). The active form of the compound is the fi-ee acid; it is hydrolyzed in vivo to form clofibric acid, which possesses lipid-lowering properties. Other clinically used hypotriglyceridemic fibric acids
10 include ciprofibrate, bezafibrate, fenofibrate, beclobrate and gemfibrozil. Some of these
drugs are more potent than clofibric acid at lipid-lowering and inducing peroxisomal
enzymes and hepatomegalic effects (Witiak et ai, 1977; Reddy and Lalwani, 1983).
Group 2: Non-fibric acid hypolipidemic drugs
Wy-14,643 (pirinixic acid), tibric acid, tiadenol, BM-15,766 and DL-40 are
hypolipidemic agents that are peroxisome proliferators in rodents (Reddy and
Krishnakantha, 1975; Reddy e/a/., 1979; Reddy and Lalwani, 1983; Hawkins e/a/., 1987;
Moody e/a/., 1991).
Group 3; Industrial plasticizers
Di-(2-ethylhexyl)phthalate (DEHP) and di-(2-ethylhexyl)adipate (DEHA) cause peroxisome proliferation and hepatocarcinogenesis in rodents (Reddy and Lalwani, 1983).
The active compound is formed by the in vivo transformation of DEHP into mono-(2- ethylhexyOphthalate (MEHP) (Mitchell et ai, 1985). These compounds are widely used as industrial plasticizers in polyvinyl chloride (PVC) plastics. These substances could leach out of the plastics and present a possible health hazard to humans (Lake et ai,
1975).
Group 4; Phenoxy acid herbicides
Phenoxy acid herbicides resemble fibric acids structurally. These compounds produce peroxisome proliferation in rats in vitro and in vivo (Lundgren et ai, 1987a).
11 Group 5: Miscellaneous agents
Several compounds such as an antipyretic drug, acetylsalicylic acid (Ishii and Suga,
1979), anticonvulsant valproic acid (Horie and Suga, 1985) and the steroid dehydroepiandrosterone (DHEA)(Wu et ai, 1989) cause peroxisome proliferation.
Although most peroxisome proliferators possess a carboxylate function, LY-171,883, a
leukotriene D 4 antagonist does not. It is a peroxisome proliferator in rodents (Eacho et ai, 1986, 1989). The presence of an acidic tetrazole group may chemically substitute for a carboxylate moiety. Recently, it has been shown that non-steroidal anti-inflammatory drugs, such as ibuprofen and inodomethacin may be peroxisome proliferators (Lehmann et ai, 1997).
Group 6 : Fatty acids
Among the fatty acids that cause peroxisome proliferation in rodents are the perfluorinated fatty acids (Ikeda et ai, 1985; Intrasuksri and Feller, 1991), p,(3'-methyl substituted dicarboxylic acid analogs (MEDICA) (Hertz et ai, 1988; Bar-Tana et ai,
1989) and sulfur-substituted fatty acid analogs (Aarsland et ai, 1989; Berge et ai, 1989,
1989a).
Perfluorinated fatty acids are widely used commercially for their anti-wetting and surfactant properties and chemical and thermal stability (Guenthner and Victor, 1962).
Intrasuksri and Feller (1991) demonstrated that in primary hepatocyte cultures perfluorinated fatty acids can cause an increase in the peroxisomal enzyme activities, and that these effects are dependent on the chain length of the fatty acid, that is,
12 perfluorodecanoic acid (PFDA) is more active than perfluorooctancic acid (PFOA), which
in turn is more active than perfluorobutanoic acid (PFBA). Perfluorooctanol (PFOL),
which lacks the carboxylic group, was active in vivo (Ikeda et ai, 1985) but was inactive
in primary culture (Intrasuksri and Feller, 1991). This implies that PFOL is metabolized in
vivo to produce an active compound.
Group 7: Physiological and dietary effects
Several physiological and dietary effects cause peroxisome proliferation. These
include conditions such as high fat diet (Ishii et ai, 1980; Neat et ai, 1980), cold
adaptation (Nedergaard et ai, 1980), starvation (Ishii et ai, 1980), vitamin E deficiency
(Reddy e/a/., 1981) and diabetes (Horie e/û/., 1981). These conditions produce morphological and enzymatic effects in the peroxisomes that are far less in magnitude than xenobiotics (Reddy and Lalwani, 1983).
1.4 Mechanism of peroxisome proliferation
To explain the mechanism by which peroxisome proliferation occurred, there existed two theories. These were the receptor hypothesis and the fatty acid overload mechanism.
1.4.1 Fatty acid overload theory
The basis of the latter theory arose fi'om the fact that structurally dissimilar chemicals could cause peroxisome proliferation (Lock et ai, 1989). Besides the presence
13 of an acidic function and a lipophilic, non-metabolizable sidechain, peroxisome
proliferators have not been shown to demonstrate any apparent structural similarities. It
was believed that these compounds could not undergo B-oxidation due to the substitutions
on the a carbon atom, therefore, the resulting perturbation in lipid metabolism initiates the
process. This belief was corroborated by the observations that : I) Peroxisome
proliferative compounds exhibited hypolipidemic activity (Reddy and Lalwani, 1983). 2)
Administration of peroxisome proliferators caused the transient accumulation of lipid
droplets in the hepatocytes. Elcombe and Mitchell (1986) proposed that the peroxisome
proliferator DEHP, which caused the accumulation of lipid droplets in the liver, caused a
decrease in the palmitic acid oxidation in isolated rat hepatocytes, and selectively inhibited
medium-chain fatty acid oxidation in the mitochondria. This inhibition may be due to
depletion of CoA or due to sequestration of medium-chain fatty acids (Lock et al., 1989).
3) The peroxisomal fatty acid 6 -oxidation enzymes were induced but other peroxisomal
enzymes such as urate oxidase and catalase were not significantly affected. The levels of
CoA and carnitine are increased. Thus, it was proposed that this alteration in lipid
metabolism was responsible for peroxisome proliferation (Elcombe and NCtchell, 1986).
4) The microsomal CEP4A1, a fatty acid w hydroxylase was induced by peroxisome proliferators. This enzyme could convert fatty acids to dicarboxylic fatty acids, which might be the proximal stimuli for peroxisome proliferation (Lock eta/., 1989). 5)
Inhibitors of mitochondrial fatty acid oxidation, e.g., valproic acid (Horie and Suga,
1985), induced peroxisomal B-oxidation. 6 ) High fat diets (Ishii et al, 1980; Neat et al,
1980) and certain physiological conditions such as cold adaptation (Pollera et al, 1983)
14 and vitamin E deficiency (Reddy et ai, 1981) can stimulate peroxisomal proliferation, and
7) Reddy and Lalwani (1983) suggested that peroxisome proliferators or their metabolites
may serve as substrates for peroxisomal 6 -oxidation, thereby inducing peroxisomal
enzyme induction.
This evidence suggests that there is a correlation between the perturbation of lipid
metabolism and peroxisome proliferation. However, no single event can be identified as being the key to the induction process.
1.4.2 Receptor hypothesis
The receptor hypothesis was proposed by Lalwani et al. (1987) who first reported the existence of a pool of saturable binding sites for nafenopin in the rat liver. This led to the isolation of a 70 kD protein fi’om the rat liver cytosol that could bind [^H]-nafenopin.
When clofibric acid and ciprofibrate were used as aflBnity ligands instead of nafenopin, the same protein was isolated. This study could not be reproduced by other workers (Milton et ai, 1988), casting doubts on the results obtained by Lalwani et al. (1987). One of the proteins isolated by Lalwani et al. (1987) was identified as HSP72, a member of the heat shock protein family (Alvares et ai, 1990). These findings led to other observations which supported the hypothesis of the existence of a receptor protein for peroxisome proliferators. They are: 1) The response to peroxisome proliferators was found to be highly tissue specific (Reddy and Lalwani, 1983). The phenomenon is limited to the liver and the proximal tubular epithelium of the kidney. Hepatocytes transplanted into the anterior chamber of the eye or into fatty pads of syngeneic or xenogeneic hosts respond to
15 peroxisome proliferators (Reddy et a i, 1984). 2) In response to peroxisome proliferators,
the mRNAs for peroxisomal B-oxidation and P450IVAI enzymes were rapidly increased
(Hardwick et ai, 1987; Reddy et al., 1986). 3) Using computer-aided molecular
modeling, some investigators have developed structure-activity relationships that indicate
a receptor-mediated event (Eacho et al., 1989; Feller and Intrasuksri, 1993; Esbenshade et
ai, 1990; Lake et ai, 1986, 1987 and 1988; Lewis et al., 1987), and, 4) The most
convincing evidence for the receptor theory was the discovery of the peroxisome
proliferator-activated receptor (PPAR), a member of the family of nuclear hormone
receptors by Issemann and Green in 1990. Targeted disruption of the PPARa gene in
mice led to an abolition of the typical hepatic response to peroxisome proliferators (Lee et al., 1995).
1.4.3 Peroxisome proliferator-activated receptor (PPAR)
The fact that the phenomenon of peroxisome proliferation involved the rapid transcriptional upregulation of several genes (Hardwick et ai, 1987; Reddy et ai, 1986) gave rise to the idea that peroxisome proliferators influenced the activity of a transcription factor (Reddy and Rao, 1986). The nuclear hormone receptors are a class of receptors that are ligand-dependent transcription factors (Evans, 1988). Prompted by this, in 1990,
Issemarm and Green probed a mouse liver cDNA library using a probe derived from the combined nucleotide sequence of several nuclear receptors, including the estrogen, retinoid and the thyroid hormone receptors. Of the four clones isolated, it was found that one clone could be activated by peroxisome proliferators. This was done by constructing
16 chimeric receptors consisting of the ligand binding portion of the isolated clones and the
DNA binding portion of the estrogen receptor. The second plasmid consisted of the estrogen response element attached to a chloramphenicol acetyl transferase reporter construct. When this chimeric receptor was tested for its ability to activate gene transcription using several diverse peroxisome proliferators, there was a good correlation between their ability to activate the receptor and their potency to induce peroxisome proliferation. This receptor was termed the peroxisome proliferator-activated receptor
(PPAR). Although Issemann and Green (1990) showed the transcriptional activation of
PPAR in response to peroxisome proliferators, they could not demonstrate the binding of
[^H]-nafenopin to ER-PPAR.
1.4.3.1 PPAR isoforms
In the next few years, many related PPARs were discovered in vertebrate species.
The PPAR family is found to consist of three distinct subtypes: PPARa, PPARy and
PPARÔ (also known as PPARP, NUC-1 or F AAR). These receptors have been found in a variety of species, e.g., Xenopus contains a, P and y(Dreyer et ai, 1992), a, y and ô cloned from mouse (Issemann and Green, 1990; Chen e/a/., 1993; Zhu era/., 1993;
Kiiewer et ai, 1994; Amri et ai, 1995), a and ô isoforms from rat (Gottlicher et ai,
1992; Xing et ai, 1995), y from hamster (Aperlo et ai, 1995) and a, y and ô(or NUC-1) isoforms from humans (Schmidt et ai, 1992; Sher et ai, 1993; Greene et ai, 1995).
17 1.4.3.2 Differential expression of PPARs
The expression patterns of these receptors have been extensively studied (Kiiewer
et al., 1994; Braissant et al., 1996). PPARs show a distinct pattern of expression, which
is mostly conserved across species and between the sexes.
PPARa shows high levels of expression in the liver, kidney, heart, mucosa of the
stomach and duodenum and brown adipose tissue. Other organs that express significant
amounts of this isoform include retina, adrenal gland and skeletal muscle. Hepatic
expression of PPARa is positively and negatively regulated by glucocorticoids and insulin
respectively (Lemberger e/a/., 1994; Steineger eta/., 1994). Therefore, PPARa
expression in liver is increased by stress or fasting, and its level cycles according to the
circadian rythmn of circulating glucocorticoids (Lemberger et al., 1996b).
PPARy occurs as two isoforms, y, and y;, which are splice variants of each other.
PPARy% is expressed at very high levels in brown and white adipose tissue (Tontonoz et al., 1994), whereas PPARy i is present more abundantly in non-adipose tissues (Reddy and
Chu, 1996). It was found that the forced expression of PPARy 2 can cause fibroblasts to accumulate lipid and convert to adipocytes (Tontonoz et al., 1995). PPARy is also expressed at lower levels in the spleen, mucosa of the duodenum, retina and in other lymphoid nodes such as the Peyer’s patches, the lymphoid nodes of the digestive tract
(Braissant et al., 1996). In humans, as compared to rodents, the pattern of expression seems to be less restrictive, with mRNA being detected in spleen, bone marrow, liver, testis, skeletal muscle and brain (Elbrecht et al., 1996); although some workers have failed to detect any PPARy in the spleen (Greene et al., 1995). Humans also possess y^ and yj
18 isoforms, both are expressed in adipose tissue (Mukheijee et ai, 1997; Elbrecht et al.,
1996), while the y, isoform is predominant in skeletal muscle (Mukheijee et al., 1997).
PPARÔ is widely expressed in adult and embryo tissue, although its levels are
lower in tissues where other isoforms of the receptor are expressed (Kiiewer et al., 1994).
This implies that the ratio of the PPAR isoforms may play an important role in establishing
the degree of responsiveness of tissues to peroxisome proliferators (Kiiewer et ai, 1994).
PPARÔ is particularly abundant in the central nervous system and epidermis (Lemberger et
al., 1996a). It is also expressed in the mucosa o f the entire digestive tract, liver, pancreas,
nephron, lymphocyte proliferation centers of the immune system and in the genital cells,
where it is abundant in the Sertoli cells of the testis. Due to the widespread expression of
PPARÔ, it is often referred to as the housekeeping receptor (Kiiewer et al., 1994).
Thus, fi'om the expression patterns of the receptors it is clear that PPARa is abundant in tissues that have a high utilization of fatty acids and therefore, this receptor is important for fatty acid catabolism. In contrast, PPARy is important for lipid accumulation and storage.
1.4.3.3 Structure of PPARs
PPARs, like other members of the nuclear hormone receptor family, display a modular structure consisting of four functional domains; 1) the AB domain which is poorly conserved 2) the DNA binding C domain (DBD) 3) the D domain, consisting of the
DBD carboxy-terminal extension and 4) the E domain which is responsible for ligand
19 binding (LBD), mediates hormone-dependent transactivation and provides the
dimerization surface (Lemberger et ai, 1996c). Figure 1.5 shows the organization of
PPAR into functional domains.
The DNA binding domain is the most conserved domain of the nuclear receptors.
It contains two zinc fingers. The amino acids between the first and second cysteines of the
first zinc finger, or the P box, are responsible for specific recognition of the DNA binding
site and the D box, in the second zinc finger, which in PPARs contains only three amino
acids, is involved in dimerization.
A distinct feature of the PPAR family is that the average divergence in the LBD between the different isoforms is greater than among the isoforms of the thyroid hormone receptor (TR) or the retinoic acid receptor (RAR) families (Lemberger et ai, 1996c;
Schoojans et ai, 1996) and, further, the divergence between species is greater than the interspecies differences seen among other nuclear receptors. This implies that the three subtypes of PPAR have different ligand affinities and, therefore different biological roles.
Nuclear receptors contain transactivation domains which are believed to be responsible for the capability of these receptors to turn on or turn off* the expression of genes. Activation function 1 (AF-1) is located in the A/B domain; AF-1 is ligand- independent, that is, it is constitutively active. AF-2, the ligand-dependent activating domain is present in the C terminal part of the molecule in the E domain. The PPAR is also believed to possess these two domains, implying the possibility of ligand-independent activation under the influence of certain promoters.
2 0 1.4.3.4 DNA binding of PPARs
Nuclear hormone receptors can be broadly divided into two classes depending on
the localization of the receptor in the unbound state (Schoojans et aL, 1996). The first
class, consisting of the classical steroid hormone receptors like the androgen, estrogen and
the glucocorticoid receptors, in their unbound state are localized in the cytosol, associated
with the heat shock proteins. The second class of receptors is localized in the nucleus
independent of its liganded state. The PPARs, thyroid hormone receptor (TR), retinoic
acid receptors (RARs and RXRs) and vitamin D receptors (VDR) are all included in this
class.
The basic motif on the DNA recognized by this second class of receptors is the hexamer of AGGTCA. Nuclear hormone receptors in general bind as dimers, the receptors of the second class bind as heterodimers with RXRs (Kiiewer et aL, 1992). The first PPAR response element (PPRE) was characterized fi'om the promoter of the acyl-
CoA oxidase gene and was defined as a DR-1 type of sequence, that is, a direct repeat of the core recognition sequence, separated by one nucleotide (Dreyer et ai, 1992;
Tugwood et ai, 1992). Using in vitro mobility shift assays, it was found that PFAR-RXR dimers preferentially bind DR-1 sequences (Kiiewer et aL, 1992) over other DR sequences
(e g, DR-3, DR-5, etc.). Other receptor dimers such as RAR-RXR and RXR-RXR can also bind to the same DR-1 sequence bound by PFAR-RXR This implies that a possible mechanism of cross-talk between the various receptor signaling pathways could involve competition for the DR-1 sequences. Figure 1.5 illustrates the binding of PPAR/RXR to a
DR-1 sequence.
21 Since all members of the RXR/PPAR/TR class of nuclear receptors heterodimerize with the RXR, competition for RXR provides for potential interactions between the receptors. It has been demonstrated that TR and PPAR compete for RXR causing the dominant receptor to sequester the available RXR (Chu et aL, 1995). The relative amount of the two receptors available and their afiSnities for RXR decides which pathway is inhibited and the extent of inhibition. Similar competition occurs between PPAR subtypes
(Jow and Mukheijee, 1995); in any tissue containing more than one subtype of PPAR given limited amounts of RXR and PPREs, only one subtype of the receptor will be functionally active.
1.4.4 Peroxisome proliferator response elements (PPREs)
Response elements for the PPAR known as PPREs, have been found in several genes involved in lipid metabolism. The rate-limiting enzyme in the peroxisomal fatty acid
P-oxidation pathway, acyl-CoA oxidase was the first gene which was shown to have two
PPREs in its promoter region (Osumi et ai, 1991; Tugwood et a i, 1992). Subsequently, all the other enzymes of the peroxisomal pathway, enoyl-CoA hydratase/dehydrogenase bifunctional enzyme and keto-acyl-thiolase, were shown to be transcriptionally regulated through the PPAR (Zhang et ai, 1992; Bardot et ai, 1993; Lee et ai, 1995). Besides these peroxisomal enzymes, the acyl-CoA synthetase enzyme contains a PPRE in one of its promoter regions (Schoojans et ai, 1995). The CYP4A6 gene, encoding the microsomal laurate w-hydroxylase contains two PPREs. A cryptic PPRE in this gene can be activated by PPAR-RXR through removal of transcriptional repression by
2 2 apolipoprotein regulatory protein (ARP-1). HMG-CoA synthase contains a PPRE in the
proximal promoter region (Rodriguez et ai, 1994). The medium acyl-CoA
dehydrogenase is one of the chain-length mitochondrial enzymes that catalyzes the first
step of mitochondrial B-oxidation. The gene for this enzyme contains a nuclear receptor
regulatory element (NRRE) to which the PPAR can bind (Gulick et aL, 1994).
Besides mitochondrial and peroxisomal proteins, the transcription of several
cytosolic proteins is controlled by PPAR. The fatty acid binding proteins (FABP), liver
FABP (Issemann e/a/., 1992; Kaikaus e/a/., 1993) and the adipose-specific aP2
(adipocyte fatty acid binding protein) (Tontonoz et aL, 1994) contain PPREs in the promoter regions of their genes. The gene encoding the malic enzyme contains a PPRE
(Castelein et aL, 1994), as does the gene for PEPCK (phosphoenol pyruvate carboxy kinase), an enzyme involved in gluconeo- and glyceroneogenesis. The PEPCK gene contains two elements PCKl and PCK2, that can bind PPAR/RXR (Tontonoz et aL,
1995); however, only PCK2 is functional in adipose tissue.
PPAR plays a vital role in the regulation of the expression of extracellular proteins involved in lipid transport. Fenofibrate represses the human apoA-I gene by a PPAR- independent phenomenon. There exists a PPRE in the promoter of this gene, through which PPAR can counter effect the repression of this gene by fenofibrate (Vu-Dac et aL,
1994). PPAR controls the expression of the human apoA-II gene expression (Vu-Dac et aL, 1995). Transcription of the apoC-IH gene is inhibited by peroxisome proliferators and fatty acids due to competition of the PPRE on this gene between PPAR/RXR heterodimers and HNF-4 (hepatocyte nuclear factor 4), a liver-enriched transcriptional
23 factor (Staels et ai, 1995; Hertz et ai, 1995). Lipoprotein lipase (LPL) activity in hepatic
cells and adipocytes is controlled by PPAR via a PPRE present in the LPL promoter
(Schoojans et aL, 1996). Peroxisome proliferators also modify the expression of mouse
liver stearoyl-CoA desaturase and mouse adipocyte GLLrT4 expression. The activity of
stearoyl-CoA desaturase is induced by clofibrate, whereas polyunsaturated fatty acids
(PUFAs) decrease its expression (Miller and Ntambi, 1996). This is the first case where such opposing eflfects of PUFAs and clofibrate have been observed. Arachidonic acid and eicosatetraenoic acid and clofibric acid have shown to decrease the expression of GLUT4 gene, which encodes for a subtype of a membrane glucose promoter (Long and Pekala,
1996). This inhibition was shown, /« vitro, to be due to the loss of binding of PPAR to a
PPRE; however, the presence of a PPRE in the GLUT4 gene is yet to be demonstrated
(Long and Pekala, 1996).
1.5 PPARa and lipid metabolism
1.5.1 Role of PPARa as a phyiological regulator
Based on the PPAR target genes identified, there is growing evidence that PPARa plays an important role in the regulation of the fatty acid metabolism in the liver. Fatty acids are taken up by the liver mainly as non-esterified fatty acids carried by serum albumin and fi’om the hydrolysis of triglycerides carried in chylomicrons. Free fatty acids are also carried by the cytosolic fatty acid binding proteins (FABPs) in various tissues, the regulation of which is controlled by PPAR (Issemarm et ai, 1992; Kaikaus et ai, 1993;
24 Tontonoz et ai, 1994). Triglycerides are hydrolyzed extracellularly into glycerol and fatty
acids by lipoprotein lipase, a gene which is stimulated by PPARs (Schoojans et aL, 1996).
The cellular uptake of fatty acids is facilitated by fatty acid transporter protein, the
expression of which is stimulated by fibrates or fatty acid treatment (Schoojans et ai,
1996). Once the fatty acids enter the hepatocytes, they are activated into acyl-CoA
thioesters by various acyl-CoA synthetases. One acyl-CoA synthetase has been shown to
be regulated by PPARa at the transcriptional level (Schoojans et ai, 1995). This acyl-
CoA could then undergo P-oxidation. The very-long-chain fatty acyl-CoAs (C > 20)
undergo oxidation in the hepatic peroxisomes. All the enzymes of this pathway are
regulated by the PPARa (Osumi e/a/., 1991; Tugwood e/a/., 1992; Zhang e/a/., 1992;
Bardot e/a/., 1993; Lee e/a/., 1995).
The long- and medium-chain fatty acids can be oxidized by the mitochondria or the
peroxisomes; the entry of the fatty acids into the mitochondria is via a carnitine-dependent
system involving the enzyme carnitine palmitoyl transferase I (CPT I) which is responsive
to peroxisome proliferators (Brady et ai, 1989; Foxworthy et ai, 1990), and may be a
potential PPAR target. The mitochondrial medium-chain acyl-CoA dehydrogenase,
which catalyzes the first step of fatty acid p-oxidation in the mitochondria, is directly
regulated by PPARa (Gulick et ai, 1994). The acetyl-CoA formed during P-oxidation is
converted into ketone bodies, through a pathway involving HMG-CoA synthetase which
is controlled by PPARa (Rodriguez et aL, 1994). Further, peroxisome proliferators have
been known to induce the rate of hepatic mitochondrial P-oxidation and ketogenesis
(Mannaerts et aL, 1979; Foxworthy et al, 1990); in mice lacking PPARa, there is an
25 accumulation of lipid droplets in the liver, which represents an imbalance between estérification and oxidation, due to a lack of regulation of P-oxidation and ketogenesis
(Lemberger et ai, 1996c).
PPARa is stimulated during fasting and stress by elevated glucocorticoid levels
(Lemberger et ai, 1994). In the case of fasting, insulin levels also decrease which would also stimulate PPARa (Steineger et aL, 1994). Fasting and stress situations also stimulate lipolysis, which would result in high levels of fatty acids in the liver; which, in turn, would enhance the fatty acid uptake, fatty acid P-oxidation and ketogenesis, all of which are under PPARa control. Fasting also results in an increased rate of gluconeogenesis; the expression of a key enzyme in the gluconeogenetic pathway, viz., phosphoenol pyruvate carboxy kinase, is also regulated by PPARa (Tontonoz et ai, 1995). Thus, PPARa controls fatty acid metabolism at multiple sites in the oxidative pathway.
1.5.2 Hypolipidemic effects of fibrates
1.5.2.1 Effects on HDL constituents
Fibrates have a negative effect on apoA-I transcription via the PPAR, while the apoA-U plasma concentrations increase after fibrate treatment. An increase in apoA-U production would result in a shift of apoA-I from Lp A-1 to Lp A-I;A-U, resulting in lower Lp A-I and higher Lp A-I: A-U levels. It is through these mechanisms that fibrates favor the occurrence of the less protective HDL profile consisting of increased Lp A-LA- n and decreased Lp A-I particles (Lussier-Cacan at al., 1989; Schoojans et ai, 1996).
2 6 1.5.2.2 Effects on metabolism of triglyceride-rich lipoproteins
Lipoprotein lipase (LPL) is important for the clearance of plasma triglycerides
(Auwerx e/a/., 1992). LPL hydrolyzes the triglyceride of chylomicrons and VLDL
particles, resulting in the release of free fatty acids. ApoC-ED inhibits the clearance of
plasma trigycerides, by interfering with the apoE-mediated uptake of these particles by
cellular receptors. Fibrates and fatty acids stimulate the transcription of the LPL gene in
hepatocytes and adipocytes via a PPRE (Schoojans et ai, 1996), which explains the
increased LPL activity seen on administration of these compounds. Concomitantly, the
decreased apoC-EQ secretion enhances the LPL-mediated lipoprotein metabolism, leading to an increased clearance of VLDL particles. These effects result in a less atherogenic lipoprotein profile (Schoojans et a i, 1996).
1.5.3 Ligands for PPARa
Until recently, there were no studies demonstrating the direct binding of any molecule to the receptor. Lalwani et al. (1987) demonstrated the binding of^H-nafenopin to a protein isolated from rat liver, but this was later identified to be a heat shock protein
(Alvares et ai, 1990). Issemann and Green (1990) attempted to demonstrate the binding of [^EE]-nafenopin to the PPARa, but failed to do so. This was due to a low afiBnity of nafenopin for the receptor and a large amount of endogenous binding activity in the cell lines tested (Essemann and Green, 1990). Some investigators proposed that peroxisome proliferators may bind to an auxiliary protein, such as liver fatty acid binding protein (L-
FABP), resulting in the displacement of fatty acid(s) that may actually be the proximate
27 PPARa ligand (Issemann et ai, 1992). This theory has been supported by evidence that
1) peroxisome proliferators bind purified rat liver FABP (Carmon and Eacho, 1991), 2)
induce the levels of L-FABP in rat hepatocytes (Kaikaus et ai, 1993), and, 3) there exists
a PPRE in the promoter region of the L-FABP gene (Issemarm et ai, 1992).
Recent reports showed that peroxisome proliferators produced a conformational
change in PPARa (Dowell et ai, 1997). These workers deleted various portions of the
receptor to show that fatty acids like eicosatetraenoic acid (ET Y A) and Wy-14,643 bind
to the PPAR through distinct regions. Devchand et ai (1996) demonstrated the binding
of [^H]-leukotriene B^ ([^H]-LTBJ to bacterially expressed PPARa. This was the first
ligand to be shown to bind to PPARa. Using competition binding studies, these
investigators demonstrated that Wy-14,643 can bind PPARa. In another set of studies,
using a similar binding assay, Kiiewer et a i (1997) reported the binding of a novel fibrate compound, GW2331, to xPPARa and xPPARy with almost similar afBnities. Kiiewer et ai also tested a number of fatty acids for binding to the xPPARa and xPPARy. Oleic, petroselenic, linoleic, linolenic and arachidonic acids bound xPPARs a and y, as was evidenced by the displacement of [^H]-GW2331 fi’om these receptors. Also, it was found
that 8 (S)-hydroxyeicosatetraenoic acid selectively bound to PPARa. Figure 1.6 illustrates the structures of compounds that have been shown to bind PPARa. However, neither of these studies tested the traditional fibrate peroxisome proliferators for binding to PPAR.
Forman et ai (1997) have demonstrated that the presence of Wy-14,643, clofibric acid
2 8 and ciprofibrate causes the PPAR to bind to a PPRE; in this assay, the rank order with
which the fibrates promoted the binding of PPAR to PPRE accurately reflected their
potencies as peroxisome proliferators.
1 . 6 PPARy and adipogenesis
Adipocytes arise fi'om mesodermal pluripotent ceils. These cells can diflerentiate
into adipocytes, chondrocytes or myocytes, depending on the external stimulus
(Lemberger et ai, 1996). The advent of adipogénie conversion of fibroblasts is marked by
the expression of several genes, including the family of CCAAT enhancer binding proteins
(C/EBPs a, P and Ô) and PPARs y and p. The sequence of induction is as follows:
PPARP, C/EBPÔ, C/EBPP, PPARy and C/EBPa expression. The forced expression of
C/EBP a, P and Ô or PPARy can trigger the conversion of uncommitted fibroblasts to
adipocytes (Freytag eta/., 1994; Tontonoz et a/., 1994a; Yeh eta/., 1995).
The discovery that PPARy is an important adipogénie factor arose fi'om the
finding that PPAR activators, e.g., Wy-14643 and ETYA were able to convert
preadipocytes into mature adipocytes (Chawla et ai, 1994). The forced expression of
PPARy in NIH-3T3 cells resulted in the accumulation of lipids in these cells and their
conversion into adipocytes (Lemberger et a/., 1996). Several fibroblast cell lines, e.g.,
NIH-3T3, Swiss-3 T3, Balb/c-3T3, show the capability of undergoing adipogénie
conversion. Preadipocytes express only trace amounts of PPARy. On initiation of
29 differentiation, PPARy is expressed, followed by aP2, PEPCK and C/EBPa (Tontonoz et ai, 1994, 1994a, 1995a). Markers that are expressed in adipose tissue include aP2, adipsin and PPARy (Lemberger et ai, 1996).
The target adipose genes of PPARy are lipoprotein lipase (LPL, Schoojans et ai,
1996), fatty acid transporter protein (FATP), adipocyte fatty acid-binding protein (aP2), acyl-CoA synthetase (ACS), phophoenolpyruvate carboxy kinase (PEPCK) and malic enzyme (ME) (Catelein e/a/., 1994; Tontonoz e/a/., 1994, 1995; Schoojans er a/.,
1995).Thus, PPARy plays an important role in the fatty acid uptake (LPL, FATP, aP2),
NADPH synthesis for lipogenesis (ME), gluconeogenesis (PEPCK) and fatty acid estérification (ACS).
PPARy is shown to bind to a prostaglandin metabolite, 15-deoxy-A'^"- prostaglandin J; (Forman et ai, 1995; Kiiewer et ai, 1995) and a group of antidiabetic agents, the thiazolidinediones (Lehmann et ai, Berger et ai, 1996) Figure 1.7 shows chemical structures of a few drugs belonging to each class. Of the latter class of drugs,
BRL 49653 (Kj = 6 8 nM) and AD-5075 (Kj = 22 nM) are very potent ligands for PPARy;
their abilities to displace radiolabeled AD 5075 (Kp = 6 nM) Grom hamster PPARy i expressed in COS-1 cells correlates with their ability to lower plasma glucose and triglyceride concentrations (Berger et ai, 1996). Thus, thiazolidinediones may exert their antidiabetic effect through the PPARy. Upon binding PPARy, thiazolidinediones produce a conformational change in the receptor (Berger et al, 1996). These workers have shown that insulin and PPARy ligands can activate the receptor synergistically (Zhang et ai,
1996). Further, PPARy is phosphorylated in vivo by insulin stimulation of mitogen-
30 activated protein (MAP) kinase (Zhang e /a/., 1996; Hu e/a/., 1996). Some workers have
demonstrated that phosphorylation at Ser“^ inactivates the receptor (Hu et ai, 1996),
while others believe that phosphorylation is required for ligand-stimulated activation of the
receptor (Zhang et ai, 1996).
1.7 Differential activation of PPARs
As mentioned above, the ligand binding domains o f PPARs are poorly conserved
and therefore, it is expected that these receptors can be pharmacologically distinguished
from each other. The systems used to test compounds for their capabilities to activate
PPAR subtypes are usually transactivation systems, where the cDNA for the receptor under study is cotransfected into the cell with a second plasmid containing a PPRE upstream of a reporter gene. Wy-I4,64 is a more specific activator for PPARa (Kiiewer et ai, 1994; Lehmann e/a/., 1995; Yu e/a/., 1995); although at higher concentrations it can activate other subtypes. Clofibrate and LY 171883 also activated PPARa prefrentially, thus implicating the a isoform as the mediator of the peroxisome prolferative process (Kiiewer et ai, 1994; Yu et ai, 1995). This was further confirmed when Wy-
14,643 and clofibrate failed to cause peroxisome proliferation in mutant mice lacking the
PPARa gene (Lee e /ût/., 1995).
The thiazolidinediones are very specific PPARy activators (Lehmann et ai, 1995).
The EC5 0 for activation of PPARy lies in the nanomolar range. At 100-fold higher concentrations, these compounds can activate PPARô (Ibrahimi et ai, 1994; Forman et ai, 1995).
31 Fatty acids have been shown to activate PPARs; however, few studies have
demonstrated any isoform-selective activation by fatty acids. PPARa is activated by a
large variety of unsaturated fatty acids (C 1 0 -C2 2 ). Non-metabolizable fatty acids such as
the 3-thia fatty acids and 2-bromopalmitic acid are more potent than the parent fatty acids
(Gottlicher a/., 1992, 1993; Keller et a/., 1993). Linoleic acid and docosahexaneoic
acid (DHA) have been found to be good activators of PPARa, while only DHA activates
PPARy (Yu et ai, 1995). Recently, Kliewer et al (1997) have shown that oleic,
petroselenic, linolenic, linoleic and arachidonic acids can bind xPPARa with similar
affinities. The same study demonstrated that petroselenic, linoleic and arachidonic acid
can bind xPPARy equally well as they bind xPPARa, while linolenic and oleic acids
demonstrate lesser affinity for the y isoform. These workers found that 8 (S)-
hydroxyeicosatetraenoic acid to be a selective ligand for PPARa . Interestingly, in an
independent set of studies, Forman et ai (1997) have recently identified 8 (S)-
hydroxyeicosatetraenoic acid to be very potent at promoting the PPAR to bind to PPRE
(Forman et ai, 1997). Leukotriene 8 4 has been recently shown to bind PPARa
(Devchand et al, 1996); it was proposed that the PPARa was responsible for the uptake
and subsequent metabolism of this substance during the inflammation process. The ability
of this compound to interact with PPARy is unknown.
Prostaglandins of the PGJ; series, viz., A^^-PGJ; and 15-deoxy-A‘^‘‘‘-PGJ2 are
potent activators of PPARy, and bind to this receptor through a direct interaction
(Forman et ai, 1995; Kliewer et ai, 1995). Prostaglandins of the A, D and J series have been shown to activate all PPARs (Yu et ai, 1995). Therefore, it may be possible that
32 some prostaglandin metabolites may activate and bind a and Ô isoforms of PPAR.
Forman e/ ai (1997) demonstrated that although a number of fatty acids and prostaglandins could activate PPARô, only prostacyclin and iloprost can induce PPARÔ to bind the PPRE. Thus, ligands for PPARs are members of the prostaglandin and leukotriene family. These receptors are therefore also important mediators in the inflammatory pathway.
1,8 Statement of the problem
1.8.1 Objective and rationale
Clofibric acid and its congeners have been known to cause alterations in the levels of the hepatic peroxisomal and microsomal enzymes and changes in hepatic morphology.
The mechanism underlying this phenomenon involves the PPARa, a ligand-activated transcription factor. In mice, when the PPARa gene was subjected to targeted disruption, the hepatic effects of clofibric acid were abolished (Lee e/ ai, 1995).
There are no detailed reports of any extensive structure-activity relationships available for compounds that activate PPARa or for compounds that stimulate peroxisome proliferation. The most general characteristics possessed by this class of chemically diverse compounds is the presence of a lipophilic backbone and an acidic or a potential acidic function. Some investigators have performed computer-aided modelling studies to establish structure-activity parameters for peroxisome proliferators (Lake et ai, 1988).
They found that the structures of certain peroxisome proliferators, e.g., nafenopin,
33 clofibric acid and LY 171883, overlap sufiBciently, thus increasing the possibility that these
compounds bind to a common site. Eacho et al. (1996) have performed conformational
analyses on a series of leukotriene antagonists using the SYBYL software (Tripos
Associates, St. Louis, MO) to predict a hypothetical binding site for these compounds.
The nature of this binding site requires the compounds to assume a bent conformation,
thereby fitting into the binding pocket. They found that two other structurally diverse
peroxisome proliferators, Wy-14,643 and eicosatetraynoic acid could assume similar
conformations and could, theoretically, fit into this binding site.
The structure of clofibric acid was modified by the removal of the methyl groups at the 2-position to give 4-chlorophenoxyacetic acid, which is inactive in vivo in the mouse
(Lundgren et ai, 1987, 1987a) and active in in vitro hepatocyte cultures (Lake et al.,
1988). The reason for the inactivity of this compound in vivo is probably the rapid excretion. Similarly, substitution of the chlorine atom with a fluorine atom at the 4- position on the phenyl ring of clofibric acid results in a compound that is inactive in vivo due to rapid removal firom the body (AzamoflF et ai, 1976). Moving the chlorine atom on the phenyl ring from the 4-position to the 2- or 3-position resulted in inactive compounds, indicating that these compounds possess a complex structural requirement. There have
been few reports on what effect the substitution of various atoms on the 2 -position or on the phenyl ring of clofibric acid has on hepatic peroxisome proliferation. Our laboratory has previously examined structural analogs of clofibric acid containing different
substituents at the 2 -position in vivo and in vitro (Esbenshade et ai, 1990; Feller and
Intrasuksri, 1993). Harrison (1984) examined the activity of analogs of clofibrate where
34 the 2-position of the aromatic ring is joined to the acetic acid residue. This resulted in
inactive compounds resulting from the rigidity of the molecules. For the purpose of
structure-activity considerations, these compounds cannot be considered analogs of
clofibric acid due to their lack of flexibility (Bentley et al., 1993).
There are no existing studies on the effects on activity of substitution of the methyl group of clofibric acid with longer chain alkyl substituents or the effects of substitutents on the phenyl ring (Bentley et al, 1993). There have been surprisingly few reports of the effects of enantiomers on peroxisome proliferation, but most of the in vivo studies have reported a small effect or a lack of stereoselectivity on hepatic acyl-CoA oxidase (ACO), carnitine acetyl transferase and CYP4A1 activities (Ahmad and Caldwell, 1994; Chinje and
Gibson, 1991; Esbenshade et ai, 1990). In contrast, in vitro studies demonstrate existence of a high degree of stereoselectivity on peroxisomal ACO and microsomal
CYPIVAl activities (Esbenshade et al, 1990; Feller and Intrasuksri, 1993) and on activation of PPAR (Boie et al, 1993; Yu et ai, 1995). The lack of stereoselectivity in vivo is possibly due to pharmacokinetic effects; therefore, in vitro systems were used for the evaluation of the stereoselective effects of the CP AA analogs. In order to examine stereodependency of hepatic peroxisomal proliferative effects, we tested a series of chiral structural analogs of clofibric acid. Removal of one methyl group on the 2-position of clofibric acid creates a chiral center (Fig. 1.8). The resulting molecules used in these studies were asymmetric 2-substituted analogs of chlorophenoxyacetic acid (CPAA).
Substitution at the 2-position of CPAA included alkyl groups of varying chain lengths
(methyl, ethyl, «-propyl, isopropyl, w-hexyl) or a phenyl ring, to give a series of
35 stereoisomeric analogs. Another set of modifications involved substituting the chlorine
atom at the 4-position of the benzene ring with 4-chlorobenzyl- or a 4-(4-
chlorophenyl)benzyl- moiety. The 2-methyl substituted analog of CPAA has been previously studied in other systems; for the inhibition of platelet aggregation (Romstedt et ai, 1996) where the (+)-(R)-isomer was a potent antiaggregatory agent, and the inhibition of chloride conductance, where the (-)-(S)-isomer has greater inhibitory effects on chloride conductance in skeletal muscle (Heiny et ai, 1990). Examining the CPAA analogs for their effects on peroxisome proliferation will provide information as to the relationship between lipid-lowering, antiplatelet and myotonic effects of these compounds.
The CPAA analogs were tested for their effects on PPARa-mediated transactivation. The first set of studies was performed using a cotransfection assay, in which the cDNA for rat PPARa was transfected into CV-1 cells, along with a reporter plasmid containing a PPRE in its promoter region. Using this system, the effects of the
CPAA on the overexpressed receptor were evaluated. These studies were then performed using the endogenous PPAR in the H4IIEC3 cell line, which is known to be responsive to peroxisome proliferators (Osumi et ai, 1990). In this set of experiments, only the reporter plasmid containing the PPRE was introduced into the cells. The effects o f "these analogs on the levels of the enzyme peroxisomal acyl-CoA oxidase in H4IIEC3 cells were examined to determine if the transactivation of the receptor resulted in the subsequent induction of biochemical activity.
Even though it is known that the PPARa is essential to the peroxisome proliferative effects of clofibric acid and other fibric acid hypolipidemic agents, the binding
36 of these drugs to the PPARa has not been demonstrated. Certain investigators suggested
that the mode of peroxisome proliferation of fibric acids may involve binding to a
secondary protein such as the fatty acid binding protein (FABP)(Cannon and Eacho, 1991;
Issemann et ai, 1992). Therefore, in Chapter 3, the CPAA analogs have been evaluated
for binding to liver FABP (L-FABP). If the stereoselectivity observed for activation of
PPARa is retained for binding to FABP, this would imply that L-FABP is the target site
for binding for these CPAA analogs, and possibly other peroxisome proliferators. Chapter
3 also examines if potency of fatty acids like perfluorinated octanoic and butanoic acids to bind to L-FABP correlates to their ability to activate PPARa. This would give us an insight as to the mechanism by which these fatty acids cause peroxisome proliferation.
Devchand et al. (1996) using a bacterially expressed GST-xPPARa (ligand binding domain) fusion protein, demonstrated the binding of leukotriene B* (LTB^) to the ligand binding region of PPARa. This was the first report of binding studies performed using
PPARa. This system was utilized to evaluate the ability of the CPAA analogs to displace
LTB4 from PPARa. These studies would confirm if the mechanism of action of clofibric acid and its congeners involves direct interaction with the PPARa.
1.8.2 Significance
The demonstration of stereoselective activation of PPARa would indicate that there is a receptor-ligand interaction involved for the peroxisome proliferative effects of clofibric acid analogs. The elucidation of a structure-activity relationship would help in identifying the key structural features of the ligand molecule for activation of PPAR.
37 The PPARa has recently been found to be an important transcriptional factor in
lipid signaling. It is a key factor in establishing the link between lipid metabolism and
transcription of genes involved fatty acid metabolism. Such transcriptional regulation has
been observed in bacteria where the lac repressor binds to micromolar concentrations of allolactose to modify the levels of enzymes required for the catabolism of lactose. PPARa is thought to play an analogous role in higher organisms whereby the levels of the enzymes of fatty acid metabolism are regulated by the products of the metabolic pathways generated by these pathways (Forman et ai, 1997).
Devchand et al. (1996) have shown that PPARa plays an important role in the
metabolism of leukotriene B 4 , an important mediator of the inflammation process. Mice lacking the PPARa show a prolonged inflammed response to an inflammatory stimulus as compared to normal animals (Devchand et al., 1996). It was recently shown that the non steroidal anti-inflammatory agents such as ibuprofen can activate the PPAR (Lehmaim et ai, 1997). As discussed above, the fibric acid class of hypolipidémies exerts its hypolipidemic effects through the PPARa. This implies that identifying the nature of interaction between the receptor and this class of drugs would help in the design of more potent and selective hypolipidemic and antiinflammatory drugs.
38 Acetyl-CoA Acetyl-CoA i Cytosol Acetoacetyl-CoA Acetoacetyl-CoA ^Kcttjl-CaK ^ Aoetjrl-CoA HMG-CoA HMG-CoA ER * i p C Mevalonate — Mevalonate E i R Mevalonate-P 0 i X Mevalonate-PP 1 S Isopentenyl-PP o M i Dimethylallyl-PP E ipp j Geranyl-PP IPP I Famesyl-PP Famesyl-PP i Squ^ene
ER Lanosterol - Lanosterol i i Cholesterol Cholesterol
Figure 1.1. Pathway of peroxisomal cholesterol biosynthesis (Krisans, 1996)
39 Clofibrate Ciprofibrate
"Y" °
Nafenopin Wy-14,643
Figure 1.2. Chemical structures of fibric and non-fibric acid hypolipidemic agents.
40 Di(2-ethylhexyl)phthalate (DEHP)
Di(2-echylhexyl)adipate (DEHA)
a
Cl a
2,4-DichIorophenoxyacetic acid (2.4-D) 2,4,5-Trichlorophcnoxyacetic acid (2,4,5 T)
Figure 1.3. Chemical structures of plasticizers and phenoxy herbicides.
41 0 OH HO
L Y -171.883 Dehydrocpiandrosicrone
CO,H
Tetradecylthiopropionic acid or alkylthioacctic acid
CF3(CFj),C0jH CF,(CFj),CO,H
Perfluoro-n-butanoic acid (PFBA) Perfluoro-n-octanoic acid (PFOA)
:o,H CF,(CFj),C02H
Perfluoro-n-decanoic acid (PFDA)
MEDICA-16
Figure 1.4, Chemical structures of miscellaneous drugs and fatty acids that cause peroxisome proliferation.
42 DNA binding domain Ligand binding domain Transactivation domain
CYTOPLASM
NUCLEUS
PPAR
AGGTCA n AGGTCA TCCAGT n TCCAGT
Peroxisome Proliferator Response Element
Figure 1.5. Schematic representing the organization of the peroxisome proliferator- activated receptors (PPARs) into functional domains and the binding of the PPAR/retinoid X receptor (RXR) heterodimer to a peroxisome proliferator response element (PPRE).
43 OH
Leukotriene
OGH
GW2331
OH
:00H
8 (S)-Hydroxyeicosatetraenoic add (HETE)
Fig. 1.6. Structures of compounds that can bind PPARa.
44 OH
ÔH
Prostaglandin Jj
OH
ÔH
A'^-Prostaglandin
OH
lS-Deoxy-A‘^ "-Prostaglandin
Figure 1.7. Prostaglandin J;, metabolites and thiazolidinediones that are PPARy ligands
45 NH N
N
BRL 49653
HO
HN
AD-5075
Fig. 1.7. (Continued)
46 C O O H X
X R Analog
Cl—^ 2 ^ CH3 I
Cl—^2 ^ CH2CH3 II
Cl— CH2CH2CH3 III
Cl— CH(CH3)2 IV
Cl— (CH2) s C H 3 V
o
C l — V-CH2 CH 3 V II
C l — C % ^ ^ \ _ c h 2 C H 3
Figure 1.8. Structures of 2-(4-chlorophenoxy)acetic acid (CPAA) analogs.
47 Chapter 2
STEREOSELECTIVE EFFECTS OF CHIRAL CLOFIBRIC ACID ANALOGS ON RAT PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR a (rPPARa) ACTIVATION AND PEROXISOMAL FATTY ACID p-OXIDATION
2.1 Introduction
A large variety of chemicals, including the fibrate hypolipidemic drugs and industrial plasticizers (Reddy and Lalwani, 1983) are known to cause peroxisome proliferation. The putative mechanism for the occurrence of this phenomenon involves the activation of the peroxisome proliferator-activated receptor (PPAR). This protein, which is an orphan member of the nuclear receptor superfamily, mediates a number of pleiotropic effects by peroxisome proliferators (Issemaim and Green, 1990; Lee et ai, 1995). In this regard, a number of genes involved in lipid metabolism contain enhancers (peroxisome proliferator response elements, PPREs) which bind activated PPARs, thereby rendering the levels of the corresponding gene products sensitive to peroxisome proliferators
(reviewed in Wahli et ai, 1995; Schoojans et ai, 1996; Lemberger et ai, 1996). Further, one to three subtypes of PPAR (a, B and y) exist in species such as Xenopus laevis, mouse, rat and human (Issemann and Green, 1990; Dreyer et ai, 1993; Gôttlicher et ai,
1992; Kliewer et ai, 1994; Sher et ai, 1993). The expression of PPAR isoforms is tissue
48 specific, e.g., PPARy is primarily expressed in adipose tissue (Tontonoz et ai, 1994),
whereas PPARa is predominant in liver and kidney (Issemann and Green, 1990). The
discovery of PPREs on genes involved in lipid metabolism and adipogenesis, as well as the tissue specific expression of the various PPAR isoforms, suggests that the pharmacological manipulation of PPAR could be important in the treatment of conditions such as hypertriglyceridemia, hypercholesterolemia, obesity and diabetes (Forman et ai,
1995).
Although a diverse group of chemicals share the ability to activate PPAR, the most common known structural feature of peroxisome proliferators is the presence of an acidic function (or a potential acidic function). Although several reports have examined the structure-activity relationships for the different classes of peroxisome proliferators (Eacho et ai, 1989; Harrison, 1984; Lake et ai, 1987; Lundgren et ai, 1987), the exact chemical features that enable a drug molecule to activate PPAR have not been defined. Our work attempts to elucidate more precisely the stereochemical requirements of the fibric acid class of peroxisome proliferators to activate the rPPARa and, consequently, to stimulate fatty acyl Co A oxidase activity. It has been reported that antidiabetic drugs of the thiazolidinedione class as well as prostaglandins bind to PPARy with high affinity (Forman et ai, 1995; Lehmann e /a/., 1995). This implies that such high affinity binding sites for ligands exist on the other PPAR isoforms as well. Recently, two independent groups have
utilized radioligand binding assays to demonstrate the binding of leukotriene B 4 , certain unsaturated fatty acids and a novel fibrate hypolipidemic drug to the ligand binding domain of PPARa (Devchand e/a/., 1996; Kliewer e/a/. 1997). The binding of the
49 traditional fibric acid derivatives such as clofibrate and ciprofibrate to PPARa is yet to be
proven. While our experiments were in progress, Forman et al. (1997), using an indirect
method, showed that clofibric acid and ciprofibrate induce PPARa binding to a PPRE in
vitro.
We and others have previously studied the effects of enantiomeric ligands in vivo
on hepatic peroxisomal acyl CoA-oxidase, carnitine acetyl transferase (CAT) and
microsomal CÎPIVA1 activities (Ahmad and Caldwell, 1994; Chinje and Gibson, 1991;
Esbenshade et ai, 1990). Most in vivo studies report either a low or an absence of ligand
stereoselectivity. In contrast, in vitro studies demonstrate that steric effects have a major
influence on the stimulation of peroxisomal ACO and microsomal CYPIVAl activities
(Esbenshade et ai, 1990; Feller and Intrsuksri, 1993) and on the activation of PPAR (Boie
et ai, 1993). The lack of stereoselectivity in in vivo studies could be due to
pharmacokinetic effects, rather than the absence of a receptor-mediated event. It is also
possible that a steric conversion of the distomer to the eutomer could have occurred in vivo or that the chiral center on the ligand molecule was not involved in the interaction with the critical site on the receptor (Ariens, 1983).
In this study, we demonstrate the stereoselective activation of rPPARa by substituted analogs of 2-(4-chlorophenoxy)-acetic acid [CPAA] (Figure 1.8). Two transactivation assay systems were used; in the first system, CV-1 cells were co transfected with the expression plasmid for PPARa, along with the plasmid for the luciferase reporter downstream of a PPRE (peroxisome proliferator response element).
The other system utilized the endogenous PPAR present in H4IIEC3 cells, a rat hepatoma
50 cell line which has previously been shown to be responsive to peroxisome proliferators
(Osumi et ai, 1990). The PPRE-containing luciferase (LUC) reporter gene was
transfected into these cells to assay the activities of the CPAA analogs on the endogenous
PPAR. The abilities of these analogs to stimulate peroxisomal acyl-Co A oxidase activity
in H4IIEC3 cells was measured to determine the effects on the biochemical parameters of
peroxisome proliferation. Several sets of isomeric CPAA analogs were studied in these
systems. A few of these analogs were found to be highly stereoselective, almost
stereospecific in their actions, consistently in all systems in which they were tested. It has
often been found that the inactive isomer of a set of biologically stereoselective
compounds may bind to the receptor, but may not cause the subsequent necessary
transduction to elicit the final effect, i.e., the inactive isomer may act as an antagonist.
Therefore, we tested the inactive isomers of two analogs against known activators of
PPARa, ciprofibrate and 2-bromopalmitic acid. These compounds were chosen because they are known to activate PPARa potently and efficaciously, which would help in detecting small changes produced by the addition of a second drug, and, they represent two types of PPAR activators, viz., fibrate hypolipidemic xenobiotics and fatty acids.
Minor modifications in the chemical structure of CPAA analogs produce significant effects on the potency and stereochemistry of activation of this receptor and the subsequent peroxisomal enzyme induction, implying the potential existence of more rigid structural requirements for PPARa ligands . These results could be utilized to define the stereostructure-activity requisites for PPARa activation and may be useful to map the putative ligand binding site of this chemical class of peroxisome proliferators to PPARa.
51 2.2 Methods
2.2.1 Materials
The chemical structures of the isomeric CFA-related analogs used in this study are
given in Fig. 7 (Chapter I). All isomeric analogs were provided by Drs. Vincenzo
Tortorella, Antonio Longo and Fulvio Loiodice (University of Bari, Bari, Italy) and the
methods of preparation and determination of purity have been reported in a recent paper
(Rangwala et ai, 1997). Analog I is 2-(4-chlorophenoxy)-propanoic acid; analogs II-VI
have various alkyl or a phenyl substituent replacing the methyl group at the 2 carbon atom
of analog I; II is 2-(4-chlorophenoxy)-butanoic acid, HI is 2-(4-chlorophenoxy)-pentanoic acid, IV is 2-(4-chlorophenoxy)-3-methyl-butanoic acid, V is 2-(4-chlorophenoxy)- octanoic acid, VI is 2-(4-chlorophenoxy)-2-phenylacetic acid. Analogs VII and VIII contain modifications of the 4-chlorophenoxy group. VII is 2-(4-chlorobenzyloxy)- propanoic acid and Vm is 2-[4-(4'-chlorophenyl)-benzyloxy]-propanoic acid. The sodium salt of clofibric acid [2-(4-chlorophenoxy)-2-methyl-propanoic acid, CFA] was obtained fi'om Ayerst Laboratories (New York, NY). Ciprofibrate was obtained form Sterling
Winthrop Research Institute (Rensselaer, NY). All biochemicals and cell culture reagents were obtained fi'om Sigma Chemical Co. (St. Louis, MO) unless otherwise stated. Other reagents and chemicals used were of the highest purity available. All the plasmids used in the transfection assays were gifts of Dr. Daniel Noonan (University of Kentucky,
Lexington, KY).
52 2.2.2 Cell culture
H4UEC3 cells were obtained from American Type Culture Collection (Rockville,
MD). CV- 1 cells were a gift from Dr. Noonan’s laboratory (University of Kentucky,
Lexington, KY). All cell lines were cultured in the Dulbecco's Modified Eagle's Medium
(DMEM) (BioWhittaker, Walkersville, MD) supplemented with fetal bovine serum (10
%), L-glutamine (4 mM) and gentamicin (Sigma Chemical Company, St. Louis. MO) (50
pg/ml). The cells were grown at 37°C in a humidified atmosphere containing 95 % air: 5
% CO; in a water jacketed incubator. For routine maintenance, the cells were rinsed with
phosphate buffered saline (pH 7.4) and subsequently with trypsin-EDTA solution, and
then incubated at 37°C for 5-10 min. The detached cells were then resuspended in fresh
medium, and an aliquot of the cell suspension was used to innoculate the next passage.
During routine procedures, precautions were taken so as to prevent cross-contamination between various cell lines. For the acyl-CoA oxidase assays, cells were grown in a different culture medium which has been outlined in detail in section 2.2.5.
2.2.3 Co-transfection assay for CV-1 cells using calcium phosphate
coprecipitation
The rat PPARa receptor expression plasmid and the P-galactosidase normalization plasmid used were constructed by directionally cloning the cDNA structure for these genes downstream from the constitutive RS V-LTR promoter in the pRSV eukaryotic expression plasmid (Giguere et ai, 1986). CV-1 cells, grown as a monolayer in
Dulbecco's modified Eagle's medium (BioWhittaker, Inc., Walkersville, MD) containing
53 10% v/v fetal calf serum (Gibco BRL, Gaithersburg, MD), were plated 24 hr prior to
transfection at 70% confluency. The recombinant DNA constructs were transiently
transfected into CV-1 cells by calcium phosphate co-precipitation. Medium was removed
from the cells after 6 hr, cells were washed twice with phosphate-buffered saline (PBS)
and analogs were tested in triplicate over a suitable concentration range. Following a 38
hr incubation period, the cells were washed with PBS and lysed with 50 pi lysis buffer [25
mM Tris-phosphate, pH 7.8, 15% glycerol, 2% 3-[(3-cholamido-propyl)- dimethylammonio]-1 -propane-sulfbnate (CHAPS), 1% lecithin, 1% bovine serum albumin,
4 mM EGTA, 8 mM MgCl^, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride
(PMSF)]. Aliquots of cell extract (20 pi) were assayed for luciferase activity in 100 pi of assay buffer (10 mM MgCU, 1.66 mM ATP, 0.45 mM sodium luciferin, 90 mM potassium phosphate, pH 7.8) using a Dynatech luminometer (Model ML2250, Chantilly, VA). To determine the efficiency of transfection and to standardize the expression of activity, a pRSV-B-galactosidase expression plasmid was included in all co-transfections and analyzed as previously described (O’Brien et ai, 1996). Aliquots of each cell extract (30
pi) were mixed with 2 0 0 pi of a G-galactosidase substrate solution (100 mM NaH^PO^,
pH 7.8, 6.64 mM o-nitrophenyl 6 -D-galactopyranoside) and analyzed for G-galactosidase activity at 415 nm on an ELISA plate reader (Bio-Rad, Model 3550, Hercules, CA). Each transfection was run in triplicate, and the average luciferase response for the three independent transfections was normalized to the average G-galactosidase rate [average luciferase response/(average G-galactosidase response/minute)]. The data in each experiment were normalized relative to 1 mM clofibric acid. Data presented are the mean
± S.E.M. of a minimum of three independent experiments.
54 2.2.4 Transfection of H4IIEC3 cells
H4IEEC3 cells were grown in DMEM containing 10% v/v fetal calf serum (PCS)
and were plated 24 hour prior to transfection at a 20% confluency so as to obtain cells at a
50% confluency at the time of transfection. For electroporation, the cells were trypsinized, washed and resuspended in antibiotic-free medium at a density of 15 x 10^ cells/500 pi and electroporated using a BTX square electroporater (Model T 820,
Genetronics, San Diego, CA) at 190 V for 70 mseconds. Preliminary experiments established that these conditions, i.e., the voltage and cell density chosen, gave optimum transfection with the least possible cell mortality. Table 2.1 shows the results of one such preliminary experiment, where different voltages were utilized to transfect a fixed number of cells, in a constant volume, containing fixed amounts of plasmid DNA. Transfection efficiency increases with voltage up to 190 V, beyond which the cell mortality is a competing factor. The cells were plated in 48 cell well plates at a density of 5 x lO* cells
/500 pi of DMEM per well. After 24 hr, media was changed to DMEM containing charcoal-adsorbed PCS (HyClone Laboratories, Logan, UT, 10%v/v) and drugs were added onto these cells. At the end of another 24 hours, the media was aspirated, the cells were lysed using Packard lysis buffer (150 pi) and the luciferase activity of the cells was assayed by adding 35 pi of cell lysate to an identical quantity of reconstituted Luc-Lite® buffer (Luc-Lite® kit, Packard Instrument Co., Meriden, CT). Light emitted was measured using a Top-Count in the luminometer mode (Packard Instrument Company,
Meriden, CT). Data are expressed as average luciferase response ± S.E.M. (n = 4-5 experiments) normalized to the response to clofibric acid (CPA) within the same experiment (100 pM = 100%).
55 2.2.5 Cell treatment and assay for peroxisomal Q-oxidation
H4IIEC3 (rat hepatoma) cells were grown as described in section 2 above, except
that the media used for these cells was Swim's S-77 medium containing 5% fetal bovine
serum (Gibco BRL, Gaithersburg, MD) and 20% horse serum (Gibco BRL, Gaithersburg,
MD). For the peroxisomal fatty acyl-CoA assay the cells were grown in 24-welI cell
culture plates. The cells were incubated with drugs for 72 hr; media and drugs were
replaced at 36 hr. At 72 hr, cells were washed twice with 0.154 M KCl/50 mM TRIS-
HCl, pH 7.4 buffer and harvested by scraping into 500 pi of the same. The cells were then
sonicated, and aliquots of cell sonicate were used to assay protein and peroxisomal acyl
CoA-oxidase (ACO) activity. Protein content was determined by the method of Lowry et al. (1951), using bovine serum albumin as the standard. ACO activity was assessed by the fluorometric measurement of H^O; production as described (Walusimbi-Kisitu and
Harrison, 1983). The incubation mixture contained: 15 pg of enzyme protein, 60 pM palmitoyl CoA, 50 pM FAD, 1 pM scopoletin, 3 units peroxidase, 0.6 mg BSA, 60 mM
TRIS HCl, pH 8.3, in a total volume of 1 ml. The reaction was terminated with 4 ml of
0.1 M sodium borate buffer. Fluorescence was read at an excitation wavelength of 395 nm and an emission wavelength o f470 nm on a fluorescence spectrophotometer (Model
LS50B, Perkin Elmer, Buckinghamshire, U.K.). Enzyme activity was expressed as nmoleH^O/min/mg protein. Data within each experiment were normalized relative to 1 mM clofibric acid. Data presented are the mean ± S.E.M. of a minimum of three independent experiments (3-4 dishes of cells/experiment).
56 2.2.6 Data analysis
In all experiments, the data have been expressed as a percent of the maximal
response to clofibric acid within the same experiment, which was arbitrarily designated as
100 %. The ECjo values were defined as the concentrations of the drug required to produce a response equivalent to 50% of the maximal response by clofibric acid (CFA) (1 mM for activation in CV-1 cells and peroxisomal acyl-CoA oxidase activity in H4IIEC3 cells; 100 pM for PPAR activation studies in H4IIEC3 cells). In experiments where the
effects of compounds on ciprofibrate- and 2 -bromopalmitic acid-induced activation of
PPAR were examined, the data were normalized relative to the maximal response of ciprofibrate (30 pM) or 2-bromopalmitic acid (10 pM). In each experiment, the control and treatment groups were each tested in 3-4 individual dishes or wells of cells.
Comparisons among the means of enantiomers of each analog were performed using the
Student’s paired t test(Daniel, 1991) at the 5% level of significance. In case of comparisons between the means of different treatment groups (e.g., no treatment, one drug treatment, treatment with two drugs), one way analysis of variance followed by a
Student-Newman-Keul’s test was performed using SigmaStat software (Jandel
Corporation, San Rafael, CA, 1992).
2.3 Results
2.3.1 Effect of 2-substituted analogs of 2-(4-chlorophenoxy)acetic acid
(CPAA) on rPFARa activation
57 Enantiomers of various alkyl and phenyl substituted analogs of CPAA (structures
of analogs I-VI are given in Fig. 1.8) were evaluated for their ability to activate rPPARa
in CV- 1 cells (Figs. 2.1-2.4). Clofibric acid (CFA) was the standard against which all the
compounds were compared. The methyl and ethyl congeners of CPAA exhibited a limited degree of stereoselectivity for PPARa activation. For the methyl analog, the (+)-(R)-
enantiomer [EC% of 5.2 x 1 0 "* M] was more active than the (-)-(S)-enantiomer [EC% =
9.5 X 10"* M] (Fig. 2.1, top panel), whereas the (-)-(S)-enantiomer was more active for the ethyl analog [ECjoS of (+)-(R)- and (-)-(S)-enantiomers are 5.1 x 10 * M and 1.8 x 10"* M respectively] (Fig. 2.1, bottom panel). Notably, the isomeric //-propyl derivatives of
CPAA were highly stereoselective activators of rPPAR [(-)-(S) » (+)-(R), (-)-(S)-isomer,
ECso = 1 . 1 X 10"* M, (+)-(R)-isomer produced only 17% of the maximal response to CFA at the highest concentration tested (10*^ M)] (figure 2.2, top panel), whereas the corresponding enantiomers of the isopropyl derivative of CPAA were equally active
[ECjoS for the isomers are (+)-(R)-enantiomer = 1.4 x 10"* M, (-)-(S)-enantiomer = 1.7 x
10"* M] (Fig. 2.2, bottom panel). Of the series of 2-alkyl substituted compounds, the isomers of //-hexyl CPAA were the most potent activators of rPPARa, but they were only marginally stereodependent (Fig. 2.3, top panel) [(-)-(S)-isomer > (+)-(R)-isomer, EC%s are; (+)-(R)-isomer = 5.2 x 10'^ M, (-)-(S)-isomer = 2.3 x 10'* M]. Substituting a phenyl ring for the alkyl group at the 2 position of CPAA, diminished the activity of the compound; however, this phenyl analog of CPAA was highly stereoselective [(+)-(S)- isomer » (-)-(R)-isomer, ECjqS (-)-(R)-isomer produced only a 3% response at the highest concentration tested, (+)-(S)-isomer = 1.8 x 10"* M] (Fig. 2.3, bottom panel).
58 Thus, the stereoisomers of «-propyl and phenyl CPAA analogs exhibited the highest
stereoselectivity for the activation of rPPARa; (S)-isomer » (R)-isomer for both analogs.
2.3.2 Effect of the chemical modification of the 4-chlorophenoxy group of the
CPPA analogs on activation of rPPARa
Elongation of the 4-chlorophenoxy group of CPPA (EC;q > 5.3 x IO~‘M) to a 4-
chlorobenzyloxy moiety (analog VTI) resulted in a marked increase in potency (Fig. 2.4,
top panel, compared to Fig. 2.1, top). The (+)-(R)-isomer of this analog has an ECjo
value of 1.5 X 10'* M, while the EC^ of the (-)-(S)-isomer of this analog is 3.6 x 10’’ M.
Enlargement of the 4-chlorobenzyloxy group to a 4-(4'-chlorophenyl)-benzyloxy moiety
resulted in a decrease in activity; however, the enantioselectivity of this compound
improved [(+)-(R)-isomer > (-)-(S)-isomer; ECjoS are (+)-(R)-isomer = 5.4 x 10'* M, (-)-
(S)-isomer = 4.2 x ID'* M] (Fig. 2.4, bottom panel). Both 2-methyl substituted congeners
(analogs VII and VET), like 2-methyl CPAA, showed a reversal in stereoselectivity [(+)-
(R) > (-)-(S)] as compared to the other 2-alkyl or 2-phenyl substituted analogs.
2.3.3 Characterization of the PPAR in H4IIEC3 cells
The activation of PPARa observed in CV-1 cells by CPAA analogs is not host cell-specific. Nearly identical results were obtained when the endogenous receptor in
H4HEC3 cells was used. The PPAR isoform in H4IIEC3 cells was pharmacologically characterized using CFA, ciprofibrate and BRL 49653, a PPARy ligand (Fig. 2.5).
Compared to CV-1 cells, H4IIEC3 cells appear to be more sensitive to drug treatment for
59 PPAR activation; the highest concentration that could be tested in these ceils for clofibric
acid was 1 x 10"* M, in contrast with CV-1 cells, where the highest concentration of
clofibric acid tested was 1x10'^ M. Similarly, for all compounds tested in H4IIEC3 cells,
the highest concentration that could be utilized was about 1 0 -fold less than the highest
concentration tested in CV-1 cells. The ECjqS of clofibric acid and ciprofibrate were 2.1 x
10'* M and 1.8 x 10"^ M respectively. There exists a 10-fold difference in potency between
clofibric acid and ciprofibrate which is in agreement with prior results (Feller et ai, 1987;
Kocarek and Feller, 1989; Intrasuksri, 1991) demonstrating the greater potency of
ciprofibrate as compared to clofibric acid for stimulating peroxisomal and microsomal
enzymes in rat hepatocytes. The EC 5 0 for BRL 49653 is 2.7 x 10'* M, which is much
greater than its ECjo for PPARy, which is 60 nM (Willson et ai, 1997). This implies that
the endogenous PPAR in H4IIEC3 cells is predominantly the a isoform. As a control, the
PPRE-less plasmid (pBL-tk-luc) was also transfected into these cells; on treatment with
increasing concentrations of ciprofibrate, these cells did not show any concentration-
dependent increases in luciferase activity (Table 2.2). This indicates that the increase in luciferase activity seen in H4IIEC3 cells are mediated through the PPRE.
2.3.4 Activation of PPARa in H4IIEC3 ceils by 2-substituted analogs of
CPAA
A selected set of CPAA analogs was tested for its ability to increase transcriptional activation via the PPARa in H4IIEC3 cells. The results are expressed as a percent of the maximum CFA response at 1.0 x 10"* M. The isomers of the methyl analog (analog I)
60 could not produce more than a II % of a maximal CFA response at the highest
concentration tested (Fig. 2.6, top panel). This is in agreement with the results observed
in CV-1 cells, where these analogs exhibited activity at concentrations > 1 x 10“* M. The
//-propyl derivatives of CPAA (analog HI) (Fig. 2.6, bottom panel) were highly
stereoselective activators of the PPAR in H4IIEC3 cells; the (-)-(S)-isomer active (EC 5 0 ~
1.4 X IC’ M), whereas the (+)-(R)-isomer could only produce 9 % of the maximal
response to CFA at the highest concentration tested. The isopropyl derivatives (analog
IV) (Fig. 2.7, top panel) did not show a high degree of steroselectivity the EC 5 0 S for the
(+)-(R)- and (-)-(S)-isomers are 3.0 x 10*’ M and 5 . 1 x 10'* M respectively. The 2-phenyl substituted analogs of CP AA (analog VI) (Fig. 2.7, bottom panel) are highly
stereoselective, the (+)-(S)-enantiomer » (-)-(R)-enantiomer. The EC 5 0 of the active isomer is 1.6 x 10'* M, while the inactive isomer could not elicit more than a 5 % response.
2.3.5 Effects of modification of 4-chlorophenoxy group on activation of
PPARa in H4IIEC3 cells
Analogs VII and VUI were tested for activity in H4IIEC3 cells (Fig. 2.8). Analog
Vn with the 4-chlorobenzyloxy substituent in place of the 4-chlorophenoxy group, was
highly active in this system (Fig. 2.8, top panel). The (+)-(R)-isomer has an EC 5 0 of 4.1 x
10"® M, while the (-)-(S)-isomer has an EC 5 0 of 2.1 x lO"® M. Thus, the activity of this analog is 10-fold greater in H4IIEC3 cells than in CV-1 cells. When the 4- chlorobenzyloxy group was substituted with the 4-(4'-chlorophenyl)-benzyloxy moiety, the
61 activity decreased, but the stereoselectivity increased (Fig. 2.8, bottom panel). The (+)-
(R)-isomer exhibited an EC 5 0 of 1.7 x 10'* M, while the (-)-(S)-isomer could only produce
20 % of the maximal response to CFA. The stereoselectivity of these isomers is much more pronounced in H4HEC3 cells than in CV-1 cells.
2.3.6 Effects of isomeric CPAA analogs on peroxisomal ACO activity in
H4IIEC3 ceUs
Several isomeric CPAA analogs were evaluated for their capacity to increase peroxisomal ACO activity (Figs. 2.9 - 2.11). Both enantiomers of the methyl derivative were equally active [(-)-(S) - (+)-(R)] (Fig. 2.9, top panel), and were comparable to clofibric acid (CFA) in potency [ECjoS are: (-)-(S)-isomer 5.4 x 10“* M, the (+)-(R)-isomer produced only a 47 % response of that produced by 1 mM CFA at the highest concentration tested (10'^ M)]. The stereoisomers of the «-propyl analog exhibited a high degree of stereoselectivity [(-)-(S) » (+)-(R), EC^ for the (-)-(S)-isomer is 3.7 x 10'* M, the (+)-(R)-isomer could produce only a 24% response at the highest concentration tested
(10“* M)] (Fig. 2.9, bottom panel) as compared to those of the isopropyl analog [(-)-(S) >
(+)-(R), EC5 0 S are: (+)-(R)-isomer = 6 . 8 x 10"* M, (-)-(S)-isomer = 9.9 x 10'* M] (Fig.
2.10, top panel). The phenyl derivative of CPAA increased ACO activity in a stereodependent manner [(+)-(S) » (-)-(R), EC% for the (+)-(S)-isomer = 1.4 x 10"^ M, the (-)-(R)-isomer produced only a 21% response at the highest concentration tested]
(Fig. 2.10, bottom panel). The corresponding 4-chlorobenzyloxy- and the 4-(4'- chlorophenyl)-benzyloxy- substituted derivatives of 2-(4-chlorophenoxy)-propanoic acid
62 (CPPA) (analogs VII and VIII respectively) stimulated ACO activity in a stereodependent
manner (Fig. 2. II). The order of stereoselectivity is (-)-(S) > (+)-(R) [ECjoS are: (+)-(R)-
isomer = 1.7 x 10~* M, (-)-(S)-isomer = 5.5 x I0‘® M] for analog VII (Fig. 2.11, top panel).
For analog Vin, (+)-(R) » (-)-(S) [ECjo for the (+)-(R)-isomer is 7.1 x 10'’ M, the (-)-
(S)-isomer does not produce more than a 1 0 % response at the highest non-toxic
concentration tested, viz., 3 x 10"* M] (Fig 2.11, bottom panel).
2.3.7 Studies of the effects of inactive isomers of analogs III and VI on PPAR
activation in H4IIEC3 ceils
The effects of the inactive (+)-(R)-isomer of analog HI were examined on the
ciprofibrate- and 2-bromopalmitic acid-induced activation of rPPARa in H4IIEC3 cells
(Tables 2.3 - 2.5). Instead of blocking the activation of PPARa by ciprofibrate as
expected, (+)-(R)-III caused an dose-related increase in sensitivity of the response to
ciprofibrate (Fig. 2.12). As a result, the concentration-response curve of ciprofibrate
shifted slightly to the left, instead of the right, in the presence of increasing concentrations
of (+)-(R)-ni (Table 2.3). This shift to the left was also seen to a certain extent when the
inactive isomer of the phenyl-substituted analog of CPAA, (-)-(R)-VT, was tested for its
effects on ciprofibrate-induced PPARa activation, although the enhancement of the
response was not as apparent as it was for the «-propyl substituted CPAA analog, (+)-(R)-
in (Table 2.4). Further, the concentration-response curve of 2-bromopalmitic acid in
H4IIEC3 cells was not significantly affected by the presence of increasing concentrations of (+)-(R)-ra (Table 2.5).
63 2.4 Discussion
A series of chiral 2-(4-chlorophenoxy)-acetic acid (CPAA) analogs were
synthesized and used as probes to examine the structure-activity relationships for PPAR
activation and the consequential increase in peroxisomal ACO activity. Activation of
rPPARa was tested in two systems, in CV-1 cells where the receptor plasmid was
transfected into the cells, and in H4IIEC3 cells, which contain endogenous PPARa. This
is the first report of a study where the endogenous PPAR in a cell line has been utilized to
study the activation of the receptor. We have pharmacologically characterized the
receptor in H4IIEC3 cells as the a isoform. All the compounds tested for activation of
PPARa, in general, were more potent in the H4IIEC3 ceils than in CV-1 cells. This could be explained by the possibility that since H4DEC3 cells are hepatocytes and they contain endogenous PPARa, they may contain all the cellular machinery necessary for generating a response to these compounds. CV-1 cells, which are monkey kidney cells, contain very little endogenous PPARa, and therefore may lack the accessory transcription factors required to generate a response equal to that of H4IIEC3 cells at the various ligand concentrations.
Besides rPPARa activation, the efifects of these compounds on peroxisomal B- oxidation in H4IIEC3 cells were also determined. This was undertaken to ensure that the responses observed at the receptor level were consistent with the efifects at the biochemical level. For the six sets of stereoisomers evaluated, although a few dififerences were observed, the correlation between the activation of rPPARa and the increase in peroxisomal ACO activity was high ( / = 0.76). The maximal response produced by the
64 (-)-(S)-isomer of the //-propyl was much greater at ACO stimulation than in receptor
activation. Our studies demonstrate that even slight modifications of the 2-alkyl
substituent result in an alteration of the potency and/or stereoselectivity of the parent
compound. Removal of a single methyl group fi'om clofibric acid, as in 2-(4-
chlorophenoxy)propionic acid (CPPA), reduces the potency of the compound. As the
chain length of the 2 -substituent increases fi’om methyl to ethyl to propyl, so does the
ability of the compound to stimulate rPPARa and activate peroxisomal ACO activity. The methyl and ethyl isomers of CPAA show a limited degree of stereoselectivity. In all the systems studied, the enantiomers of the //-propyl analog were highly stereoselective in their response. In contrast, the enantiomers of the isopropyl analog were equally active or showed a small degree of stereoselectivity. This implies that the conformation of the
substituent at the 2 - position of clofibric acid analogs is critical for the peroxisome proliferative activities of these compounds. The increased stereoselectivity observed in the hepatocyte cell line for the isopropyl analogs may be explained due to differential binding or metabolism of these compounds in these cells as compared to the CV-1 cells.
The //-hexyl analog is also as potent as clofibric acid in activating rPPARa, but it is not stereoselective in its action. This is in contrast to results in primary rat hepatocyte cultures, where the (-)-(S)-enantiomer of this compound was more potent as a peroxisome proliferator than the (+)-(R)-enantiomer (Feller and Intrasuksri, 1993). Replacing the n~ hexyl group with a phenyl ring resulted in a reduced potency and stereospecific activation
[(+)-(S)-isomer » (-)-(R)-isomer] of the receptor in CV-1 cells.
65 The inactive isomers of the 2-«-propyI- and 2-phenyl-substituted analogs of CPAA
did not cause a block in the activation of PPARa activation due to ciprofibrate. In
contrast, especially at lower concentrations, these compounds caused an enhancement in
PPARa activation by ciprofibrate. This can be explained by the possibility that at these
concentrations the inactive compounds may bind to auxiliary proteins in the cells, e.g., liver fatty acid binding proteins, which may be putative binding/storage sites for ciprofibrate. As a result, ciprofibrate is displaced fi'om these sites, and its net effective concentration within the cell is increased, resulting in a enhancement of PPARa activation.
An agonist such as 2-bromopalmitic acid may bind such proteins with a greater affinity, and may not be displaced as easily as ciprofibrate firom these binding sites. As a result, the
PPARa activation due to 2-bromopalmitic acid is not enhanced by the presence of (+)-
(R)-ni, the inactive 2-w-propyl-substituted CPAA analog. The role of the liver fatty acid binding protein (L-FABP) in peroxisome proliferation caused by CPAA analogs is discussed in the next chapter.
The second set of structural modifications involved replacement of 4- chlorophenoxy group of CPPA with a 4-chlorobenzyloxy or a 4-(4'-chlorophenyl)- benzyloxy group (analogs VII and VUI respectively). These compounds are more potent activators of rPPARa than clofibric acid. The increased potency of analog VII as compared to analog VUI suggests that there may be an optimum size requirement for the chlorophenoxy group for activation of rPPARa. Furthermore, the enantiomers of analog
VUI stereoselectively activate the receptor in CV-1 cells, whereas in the H4IIEC3 cells analog VUI is stereospecific. This could be explained by pharmacokinetic differences in
66 the two cell lines. These CPPA analogs show a reversal in stereoselectivity with reference
to the other 2 -substituted analogs, indicating that the presence of a methyl group at the 2 - carbon position somehow alters the steric orientation of the ligand in the receptor binding pocket. Chinje and Gibson (1991) have shown a limited degree of stereoselectivity and relatively low potency for induction of liver CYP4A1 by the enantiomers of 2-[4-(4'- chlorophenyl)-benzyloxy]-2-phenylacetic acid in rats; in their studies (-)-R-isomer was more active than the (+)-(S)-isomer.
Our data are in agreement with our earlier results where we demonstrated that the loss of a methyl group from the a carbon atom of 2-substituted CPAA results in a decrease in the ability to stimulate peroxisomal palmitoyl CoA B-oxidase and microsomal laurate hydroxylase activities in vitro (Esbenshade et ai, 1990). Further, Kozuka et ai
(1991) have similarly reported that the removal of the methyl group from the a carbon atom of clofibrate results in a decreased capacity to increase peroxisomal and mitochondrial enzymes. These workers suggested that the presence of two methyl groups on the a carbon atom results in greater lipophilicity, and subsequently, greater activity.
This would explain the increase in activity seen with increasing chain length of the 2-alkyl side-chain of the CPAA analogs. In this regard, Lundgren et al. (1987) demonstrated that the presence of an ethyl group improves the peroxisome proliferative activities of a series o f 2-substituted ethyl hexanoic acid analogs. For the hexyl phthalate group of peroxisome proliferators. Lake et a/. (1987) have demonstrated that 2- or 3-ethyl substituents on the aliphatic chain result in the increased potency of these compounds to induce the cyanide- insensitive palmitoyl-CoA oxidation.
67 Stereoselectivity in response to a set of enantiomers of a drug may be due to the
differential binding of the molecules to their receptors. However, factors such as
pharmacokinetic properties, viz., differential uptake, transport, metabolism or protein
binding may be the cause of the stereoselectivity observed for a set of isomers (Ariens,
1983). The use of in vitro systems eliminates considerations such as tissue uptake and
distribution. It is possible that the stereoselective effects observed in response to our
CPPA isomers could be due to pharmacokinetic events other than the interaction of the
ligand molecule with the receptor. However, since these compounds exhibited similar
stereoselectivities in three different systems utilizing two different cell lines, viz., H4HEC3
(rat hepatoma), CV-1 (monkey kidney) cells, it is unlikely that the diverse pharmacokinetic environments in these cell lines would be responsible for the effects observed. We are not aware of any data concerning the stereoselective transport of these drugs into the cell. While it is known that the parent compound, clofibrate, is mainly excreted in the urine as the fi-ee acid or the glucuronide in man (Thorp and Waring, 1962) and rat (Baldwin et al., 1980), there are reports in the literature of chiral inversion of the aryl propionic acids (Caldwell et al, 1988) which are structurally similar to our compounds. The mechanism of the stereoselective conversion of these compounds involves the removal of the a-methine proton fi’om the acyl CoA thioester of the R- enantiomer of the aryl propionic acid to form an unsaturated intermediate. Racemization takes place via the non-stereoselective addition of protons across the double bond of the intermediate fi’om the aqueous media. However, for the CPPA isomers, the a-methine proton would not be as susceptible to removal due to the presence of the oxygen atom
68 between the aryl group and the aliphatic chain. Therefore, it is unlikely that a mechanism
similar to the aryl propionic acids is involved in the racemization of the CPPA isomers.
Further, as the size of the 2-substituent increases, the probability of racemic inversion of
these compounds decreases.
In conclusion, we propose that the 2-carbon atom position of clofibric acid plays
an important role in the docking of this ligand to a receptor protein. This consequently
results in the increased PPAR-mediated trancriptional activation of the ACO gene via the
PPRE. Altering the orientation of groups around this center influences the potency by which these compounds can activate rPPARa and stimulate peroxisomal B-oxidation. By demonstrating stereoselectivity for the activation of rPPARa, we hypothesize the existence of a protein binding site through which the fibric acid class of peroxisome proliferators exerts its efifects. This binding site may be on the PPAR itselfi similar to that on PPARy (Forman et ai, 1995; Lehmann et ai, 1995). On the other hand, fibric acids may bind to a site on an auxiliary protein, e.g., fatty acid binding protein (FABP), and thereby cause the release of a fatty acid which acts like the endogenous ligand for PPARa.
The avidities with which peroxisome proliferators bind L-FABP is of the same rank order as their potency as peroxisome proliferators (Cannon and Eacho, 1991). Developing a highly specific and selective fibric acid may help us to elucidate the nature of the binding site through which these compounds act as PPAR activators.
69 Voltage (V) Luciferase activity
0 60
180 1208.6
190 3423.8
2 0 0 2382.9
2 1 0 1331.4
2 2 0 358.1
Table 2.1. Representative experiment showing the effects of varying voltages on the transfection of a reporter luciferase plasmid into H4IIEC3 cells. The number of cells, the volume of electroporation and the amount of plasmid DNA were constant in all the samples.
70 Ciprofibrate (M) Luciferase activity (Mean ± S.E.M.)
Control (0) 264.3 ± 29.0
3.0 X 10'^ 239.1 ±0
1 .0 X 1 0 ^ 180.5 ± 1.4
3.0 X 10^ 237.6 ±7.1
1.0 X 10-* 365.7 ±34.3
3.0 X 10-* 249.0 ± 24.3
Table 2.2. Representative experiment showing the efifects of ciprofibrate on luciferase activity when the PPRE-less reporter plasmid was transfected into H4IIEC3 cells. Data presented are mean ± S.E.M. of duplicate determinations.
71 Treatment (tiM)* Percent normalized luciferase response (Ciprofibrate 30 pM = 100%) Ciprofibrate (+)-(R)-III
0.3 fiM - 8 . 6 ± 2 . 6
1 pM - 14.6 ± 4.2
3 pM - 37.9 ± 10.3
10 pM - 88.5 ± 13.5
30 pM - 1 0 0 ± 0
0.3 pM I pM 12.9 ± 6 . 6 1 pM 1 pM 23.2 ±8.9 3 pM 1 pM 28.6 ±3.9 10 pM 1 pM 130.6 ±35.4 30 pM 1 pM 155.8 ±38.5
0.3 pM 10 pM 28.9 ± 10.0 1 pM 10 pM 24.8 ± 8.4 3 pM 10 pM 84.9 ± 19.4* 10 pM 10 pM 142.1 ±42.9 30 pM 10 pM 190.6 ± 49.9
0.3 pM 100 pM 43.1 ± 12.4* 1 pM 100 pM 57.9 ± 17.7* 3 pM 100 pM 83.6 ± 18.9 10 pM 100 pM 134.7 ±31.1
30 pM 100 pM 131.6 ± 6 6 . 6
- 1 pM 2.7 ±3.8
- 10 pM 0 . 2 ± 2 . 1 - 100 pM 7.3 ± 3.5 ♦Values are significantly different from the corresponding ciprofibrate only control {P < 0.05) 'Compounds were incubated with one or both compounds for a period of 24 hr.
Table 2.3. Effects of (+)-(R)-isomer of analog EH («-propyl derivative of CPAA) on ciprofibrate-induced PPARa activation in H4IIEC3 cells. Values shown are a mean ± S.E.M. of 4 experiments, each in duplicate.
72 Treatment (jiM)* Percent normalized luciferase response (Ciprofibrate 30 pM = 100%) Ciprofibrate (>)-(R)-VI
0.3 pM - 8.4 ±3.4 1 pM - 10.3 ±4.7 3 pM - 28.4 ±3.6
10 pM - 78.1 ±6.4
30 pM - 1 0 0 ± 0
0.3 pM 1 pM 10.7 ±3.7 1 pM 1 pM 19.9 ± 1.0* 3 pM 1 pM 30.8 ±3.7 10 pM 1 pM 90.9 ± 19.6 30 pM 1 pM 129.5 ± 26.2
0.3 pM 10 pM 8.1 ±3.6 1 pM 10 pM 19.1 ±4.5 3 pM 10 pM 46.4 ± 5.2* 10 pM 10 pM 67.2 ± 13.5 30 pM 10 pM 105.8 ±2.8*
0.3 pM 100 pM 7.4 ±2.6 1 pM 100 pM 21.3 ±2.2* 3 pM 100 pM 38.6 ±5.2 10 pM 100 pM 68.5 ±3.8 30 pM 100 pM 115.9 ±7.8
_ 1 pM 1.6 ±2.9 - 10 pM 6.3 ± 1.1 - 100 pM 3.8 ± 1.9 •Values are significantly different firom the corresponding ciprofibrate only control {P < 0.05) ^Compounds were incubated with one or both compounds for a period of 24 hr.
Table 2.4. Effects of (-)-(R)-isomer of analog VI (2-phenyl derivative of CPAA) on ciprofibrate-induced PPARa activation in H4IIEC3 cells. Values shown are a mean ± S.E.M. of 4 experiments, each in duplicate.
73 Treatment (pM)" Percent normalized luciferase response (2- Bromopalmitic acid 10 pM = 100%) 2-Bromopaimitic (+)-(R)-in acid
0.1 pM 14.2 ±8.1 0.3 pM - 20.3 ±1.4 1 pM - 58.6 ± 10.0 3 pM - 81.0 ±8.9
10 pM - 1 0 0 ± 0
0.1 pM 1 pM 16.4 ±7.2 0.3 pM 1 pM 32.4 ±17.3 1 pM 1 pM 62.0 ±23.0 3 pM 1 pM 75.5 ±2.0 10 pM 1 pM 120.7 ±23.5
0.1 pM 10 pM 18.6 ±9.1 0.3 pM 10 pM 33.0 ±20.7 1 pM 10 pM 65.2 ±20.5 3 pM 10 pM 73.7 ± 15.6 10 pM 10 pM 90.1 ±3.5*
0.1 pM 100 pM 30.5 ±21.1 0.3 pM 100 pM 23.0 ±6.2 1 pM 100 pM 56.2 ± 20.6 3 pM 100 pM 90.2 ±51.4
10 pM 100 pM 1 0 2 . 1 ± 2 0 . 1
. 1 pM 2.0 ±4.4 - 10 pM 5.4±3.1
- 100 pM 2 1 . 8 ± 6 . 0 *Value is significantly different from the 2-bromopalmitic acid only control {P < 0.05) "Compounds were incubated with one or both compounds for a period of 24 hr.
Table 2.5. Effects of (+)-(R)-isomer of analog HI (n-propyl derivative of CPAA) on 2- bromopalmitic acid-induced PPARa activation in H4IIEC3 cells. Values shown are mean ± S.E.M. of 2-4 experiments, each in duplicate.
74 Fig. 2.1. Concentration-response curves for the activation of rPPARa in CV-1 cells by 2- substituted analogs of 2-(4-chIorophenoxy)acetic acid [CPAA] and clofibric acid (CFA). Inset text describes the substituent at the 2- position. Results are reported as luciferase activity normalized by the corresponding 6 -galactosidase rate and are expressed as a percentage of the maximum response produced by 1 mM clofibric acid (CFA). Each data point is a mean ± S.E.M. of 3-4 experiments, each in triplicate. Asterisks indicate statistical significance at P < 0.05.
75 i N» Percent normalized Percent normalized luciferase response luciferase response
L/1
S' S' GTQ CTQ O rs o o Ul p p n n n fp Q. P p
o O p P 2 2 Fig. 2.2. Concentration-response curves for the activation of rPPARa in CV-1 cells by 2- substituted analogs of 2-(4-chlorophenoxy)acetic acid [CPAA]. Inset text describes the substituent at the 2- position. Results are reported as luciferase activity normalized by the corresponding B-galactosidase rate and are expressed as a percentage of the maximum response produced by 1 mM clofibric acid (CFA). Each data point is a mean ± S.E.M. of 3-4 experiments, each in triplicate. Asterisks indicate statistical significance at P < 0.05.
77 I h*K> Percent normalized Percent normalized luciferase response luciferase response o LA oo O 8 to
0\ fO S' (M (W n n o o p p n n 00 « P «t-P p o p
w Fig. 2.3. Concentration-response curves for the activation of rPPARa in CV-1 ceils by 2- substituted analogs of 2-(4-chlorophenoxy)acetic acid [CPAA]. Inset text describes the substituent at the 2- position. Results are reported as luciferase activity normalized by the corresponding B-galactosidase rate and are expressed as a percentage of the maximum response produced by 1 mM clofibric acid (CFA). Each data point is a mean ± S.E.M. of 3-4 experiments, each in triplicate. Asterisks indicate statistical significance at P < 0.05.
79 s K> W Percent normalized Percent normalized luciferase response luciferase response
w LA O K) LA o LA O o LA O o
ç o %;
S" S' (TO VQ rs rs O O p P rs rs o00 rt rt P P
o o P P Fig. 2.4. Concentration-response curves for the activation of rPPARa in CV-1 cells by isomers of 2-(4-chlorobenzyloxy)-propanoic acid (top panel) and 2-[4-(4'-chlorophenyl)- benzyloxyj-propanoic acid (bottom panel). Results are reported as luciferase activity normalized to the P-galactosidase rate and are expressed as a percentage of the maximum response produced by I mM clofibric acid (CFA). Each data point is mean ± S.E.M. of 3 times, each in triplicate. Asterisks indicate statistical significance atP< 0.05.
81 I Percent normalized Percent normalized luciferase response luciferase response
Ü1 o cn o cn cn o o o o
CO 73 CD -
r . S' TO TO A A O 0 P 00 o N> A 1 “ D p
O o D p
c*> 300 « CFA s CIPRO .*= *3 3 BRL 49653 t3 4) 200 - tfi S S "a È uS o u s 100- e « L.u eu
•7 -6 5 -4 Log concentration (M)
Fig. 2.5. Concentration-response curves for the activation of rPPARa activity in H4HEC3 cells by clofibric acid (CFA), ciprofibrate (CIPRO) and BRL 49653. Results are expressed as a percentage of the maximum response produced by 1 mM clofibric acid. Each point on the CFA curve is a mean ± S.E.M. of 5-16 experiments, on the CIPRO curve is a mean ± S.E.M. of 4-6 experiments and on the BRL 49653 curve is a mean ± S.E.M. of 3 experiments, each in duplicate.
83 Fig. 2.6. Concentration-response curves for the activation of rPPARa in H4HEC3 cells by 2-substituted analogs of 2-(4-chlorophenoxy)acetic acid [CPAA] and clofibric acid (CFA). Inset text describes the substituent at the 2- position. Results are reported as luciferase activity and are expressed as a percentage of the maximum response produced by I mM clofibric acid (CFA). Each data point is mean ± S.E.M. of 4 experiments, each in duplicate. Asterisks indicate statistical significance at P < 0.05.
84 w b\ Percent normalized Percent normalized luciferase response luciferase response
N ) CJl "n J o hJ Ü1 O Ol o c n K)
S' era (M n n o o g g Ln00 « s m-p "1 P oI. O p P Fig. 2.7. Concentration-response curves for the activation of rPPARa in H4IIEC3 ceils by 2-substituted analogs of 2-(4-chlorophenoxy)acetic acid [CPAA]. Inset text describes the substituent at the 2- position. Results are reported as luciferase activity and are expressed as a percentage of the maximum response produced by 1 mM clofibric acid (CFA). Each data point is mean ± S.E.M. of 4 experiments, each in duplicate. Asterisks indicate statistical significance at P < 0.05.
86 Æ- w Percent normalized Percent normalized luciferase response luciferase response
K) cn o N3 en - v | o en o cn o en o e n o
K)
S' S' w OQ n O 0 O
0 0 R R n n a en a en 3 1 m- o o B B
- - Fig. 2.8. Concentration-response curves for the activation of rPPARa in H41IEC3 cells by isomers of 2-(4-chIorobenzyloxy)-propanoic acid (top panel) and 2-[4-(4'- chlorophenyl)-benzyloxy]-propanoic acid (bottom panel). Results are reported as luciferase activity and are expressed as a percentage of the maximum response produced by 1 mM clofibric acid (CFA). Each experiment was repeated 3-4 times, and each data point was performed in triplicate per experiment. Asterisks indicate statistical significance at P < 0.05.
88 w M Percent normalized Percent normalized luciferase response luciferase response ro CO Ol o cn o o o o o o o o o o 8 o
o> o> r S' (Ft) (Ft) n r> o O a D 00 n n vO n n P • P I % cn fh cn n p S'. o o p D Fig. 2.9. Concentration response curves for the stimulation of acyl CoA-oxidase activity in H4IIEC3 cells by 2-substituted analogs of 2-(4-chlorophenoxy)-acetic acid [CPAA]. Each data point is mean ± S.E.M. of 3 experiments, each in triplicate. Within each experiment, the results were normalized to the response to 1 mM clofibric acid (CFA). Inset text describes the nature of the substituent at the 2- position. Asterisks indicate statistical significance at P < 0.05.
90 2-METHYL > 100- -w R-(+) 08 75- S-(-) V M 08 Clofibric acid •o 50- o < 0 25- U 1
0-
T“ -5 -3 Log concentration (M)
150-
125-
100-
75- u 50-
25-
5 34 Log concentration (M)
Fig. 2.9.
91 Fig. 2.10. Concentration response curves for the stimulation of acyl CoA-oxidase activity in H4IIEC3 cells by 2-substituted analogs of 2-(4-chlorophenoxy)-acetic acid [CPAA]. Each data point is mean ± S.E.M. of 3 experiments, each in triplicate. Within each experiment, the results were normalized to the response to 1 mM clofibric acid (CFA). Inset text describes the nature of the substituent at the 2- position. Asterisks indicate statistical significance at P < 0.05.
92 100 2-ISOPROPYL - o - R-(+)
cn —-S-(-)
o
5 -4 -3 Log concentration (M)
100 2-PHENYL 75- - ^ S - ( + ) - o - R -(-) p 50- e 25-
-25-
5 4 3 Log concentration (M)
Fig. 2.10.
93 Fig. 2.11. Concentration response curves for the stimulation of acyl CoA-oxidase activity in H4HEC3 cells by isomers of 2-(4-chlorobenzyloxy)-propanoic acid (top panel) and 2- [4-(4'-chloropheny!)-benzyloxy]-propanoic acid (bottom panel). Each data point is mean ± S.E.M. of 3 experiments, each in triplicate. Within each experiment, the results were normalized to the response to I mM clofibric acid (CFA). Inset text describes the nature of the substituent at the 2- position. Asterisks indicate statistical significance at P < 0.05.
94 > 4-CHLOROBENZYLOXY u es ej %
o ^ < 0 V 1
<
Log concentration (M)
(4'-CHL0R0PHENYL)BENZYL0XY
Log concentration (M)
Fig. 2.11.
95 250-
200 -
150-
100-
50-
7 -6 5 -4 Log concentration (M)
(—Ciprofibrate -a—plus (+)-(R)-III 1 uM
plus (+)-(R)-m 10 uM - A - plus (+)-(R )-in 100 uM
o (+)-(R)-in alone
Fig. 2.12. Effects of increasing concentrations of (+)-(R)-III, the inactive isomer of the 2- substituted w-propyl analog of CPAA on the ciprofibrate-induced rPPARa activation. The solid squares represent the ciprofibrate concentration-response curve in the absence of any other drug. Each point is mean ± S.E.M. of 4 experiments, each in duplicate. Within each experiment, the data were normalized to the maximum réponse, that of ciprofibrate at 30 pM = 100%.
96 CHAPTERS
ROLE OF LIVER FATTY ACID BINDING PROTEIN (L-FABP) IN PEROXISOME PROLIFERATION
3.1 Introduction
Peroxisome proliferators are compounds that cause an increase in the size and
number of peroxisomes in rodent liver (Reddy and Lalwani, 1983). These efifeas are
accompanied by hepatomegaly and an increase in the levels of certain peroxisomal and
microsomal enzymes. It has been proposed that these effects may arise due to the
perturbation of lipid metabolism by these agents (Lock et ai, 1989).
A nuclear receptor was first cloned in 1990 (Issemann and Green, 1990) that was
shown to activate transcription in response to activation by peroxisome proliferators. The
potencies with which compounds activated this receptor, called the peroxisome
proliferator-activated receptor (PFAR), correlated with their abilities to induce peroxisomal enzymes. Since then, a variety of isoforms of this receptor have been cloned fi’om different species. The various isoforms show tissue-specific expression; the a isoform is predominantly expressed in the liver, the y isoform is adipose-specific and the ô isoform is ubiquitously expressed throughout development. In addition, response elements for these receptor have been found in a variety of genes involved in lipid
97 metabolism, including genes of the enzymes of the peroxisomal fatty acid P-oxidation
pathway as well as microsomal CKP4AI, mitochondrial HMG Co A synthase, malic
enzyme and phosphoenol pyruvate carboxykinase enzymes. Another gene shown to
contain a peroxisome proliferator response element is the liver fatty acid binding protein
(L-FABP). L-FABP is a 14 kDa protein that constitutes about 2-6% of the cytosolic
protein in hepatocytes (Bass, 1988). FABPs are present in various tissues other than the
liver, including heart, intestine and adipose tissue and are thought to play an important role
in the uptake and transport of fatty acids in the cell and regulating the free concentrations
of fatty acids in the cell, thereby protecting the cell from the deleterious effects of free
fatty acids (Bass, 1985). FABP is involved in the intracellular transport of fatty acids
between the various metabolic compartments (Peeters et ai, 1989; Appelkvist and
Dallner, 1980; Zanetti and Catala, 1990).
L-FABP is involved in the modulation of cell proliferation in hepatocytes (Sorof,
1994). Long-chain fatty acids, such as linoleic acid, arachidonic acid and other saturated and unsaturated fatty acids as well as prostaglandins PGEi and PGA,, which have growth modulatory effects, bind to L-FABP in vitro (Sorof, 1994 and references therein; Dutta-
Roy et ai, 1987; Khan and Sorof, 1990). Besides these agents, the hydroxy and hydroperoxy derivatives of arachidonic acid, the HETEs and HPETEs bind to L-FABP avidly, specifically, saturably, reversibly and rapidly (Sorof, 1994).
Bezafibrate was the first peroxisome proliferator shown to bind L-FABP (Brandes et al, 1990). Eacho and coworkers have shown that the binding avidities of several peroxisome proliferators for L-FABP in vitro correlate with their abilities to stimulate
98 peroxisomal enzymes in cultures hepatocytes (Cannon and Eacho, 1991; Eacho et ai,
1993). Compounds such as Wy-14,643 clofibric acid and nafenopin, as well as the
tetrazole-substituted acetophenones require the presence of L-FABP to exert their
mitogenic effects (Khan and Sorof, 1994).
Although PPARa has been implicated in mediating the effects of peroxisome
proliferators, the binding of most peroxisome proliferators to this nuclear receptor has not
been demonstrated. Mice lacking the PPARa gene have been shown to be refi’actory to
peroxisome proliferators (Lee et ai, 1995). Further, compounds that activate PPARs
vary widely in chemical structure. Recently, prostaglandin J; and its metabolites and
thiazolidinedione antidiabetic agents have been shown to bind the PPARy, while PPARa
has been shown to bind leukotriene 8 4 , certain unsaturated fatty acids (oleic, linoleic, linolenic and arachidonic acids) and a novel fibrate hypolipidemic agent, GW2331. It has been hypothesized that certain agents that activate PPAR in vitro may actually bind to a secondary protein such L-FABP thereby displacing certain substances, such as prostaglandins or long-chain unsaturated fatty acids fi’om their binding sites on FABP
(Issemann et ai, 1992). These displaced substances could then bind to the PPAR or may be metabolized into PPAR ligands. Even agents that have been shown to bind PPAR using in vitro assays may exert some proportion of their effects through this pathway. The fact that the L-FABP and the adipocyte fatty acid binding protein genes are both transcriptionally regulated by PPARs implicates these proteins in PPAR signaling pathways (Kaikaus et ai, 1993; Issemann et ai, 1992; Tontonoz et ai, 1994b).
99 In this study, we have compared the afiBnity of 2 -bromopaImitic acid (BPA), 12-
hydroxydodecanoic acid (12-OH) and dodecanoic acid (DA) to bind L-FABP. BPA has
been previously shown to inhibit the mobility of other fatty acids by binding to L-FABP
(Luxon, 1996). 1 2 -OH is formed due to laurate hydroxylase activity of CFP4AI, a
cytochrome P450 enzyme which is induced by peroxisome proliferators (Gibson et al.,
1982; Gibson, 1996). This hydroxylated fatty acid has been previously shown to activate
PPARa potently (Feller, unpublished results, 1994). BPA and 12-OH have been tested
along with a native long-chain fatty acid, DA. For comparison, we examined CFA, a
prototypical xenobiotic PPAR activator, for its afiBnity for L-FABP. This would help us
determine the potential significance of L-FABP in the eflfects manifested by two different
classes of peroxisome proliferators. The structures of BPA 1 2 -OH and DA are given in
Table 3.1.
A set of chiral 4-chlorophenoxyacetic analogs (CPAA) have been utilized to
examine the role of L-FABP in the phenomenon of peroxisome proliferation. These
compounds are known to stereoselectively activate PPARa and stimulate peroxisomal
fatty acyl-CoA oxidase activity (Feller et ai, 1996; Rangwala et al, 1997, Chapter 2).
The abilities of these compounds to displace tritiated oleic acid from L-FABP has been
evaluated. If the stereoselectivity and relative potency by which these compounds activate the PPARa is maintained for binding to L-FABP, then the binding to L-FABP is predictive of the peroxisome proliferative effects of these compounds.
We have examined the effects of nonmetabolizable perfluorinated fatty acids, e g, perfluorooctanoic acid and perfluorobutanoic acid to activate PPARa, and compared it
100 with their ability to bind L-FABP (For structures, see Table 3.1). Perfluorinated fatty
acids are potent peroxisome proliferators in vivo (Dceda et ai, 1985) as well as in vitro
(Intrasuksri and Feller, 1991). The aflBnities of these fatty acids for L- FABP as compared
to their potency to activate PPARa will provide an insight into the mechanism by which
fatty acids cause peroxisome proliferation, and the role of L-FABP in this phenomenon.
3.2 Methods
3.2.1 Materials
L-FABP was kindly provided to us by Dr. Jeflfrey Lawrence o f Eli Lilly and
Company (Indianapolis, IN) (See section 3.2.2.). [9,10-^H]oieic acid was purchased from
Sigma Radiochemicals (St. Louis, MO). Lipidex-1000, dodecanoic acid, 12- hydroxydodecanoic acid and oleic acid were obtained from Sigma Chemical Company (St.
Louis, MO). 2 -Bromopalmitic acid (BPA), perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA) and perfluorodecanoic acid (PFDA) were purchased from
Aldrich Chemical Company (Milwaukee, WI) . The sodium salt of clofibric acid [2-(4- chlorophenoxy)-2-methyl-propanoic acid, CFA] was obtained from Ayerst Laboratories
(New York, NY). Ciprofibrate was a gift of Sterling Winthrop Research Institute
(Rensselaer, NY). All biochemicals and cell culture reagents were obtained from Sigma
Chemical Co. (St. Louis, MO). Other reagents and chemicals used were of the highest purity available. All the plasmids used in the transfection assays were gifts of Dr. Daniel
Noonan (University of Kentucky, Lexington, KY).
101 3.2.2 Isolation and purification of L-FABP
The protein was isolated from rat liver homogenate and purified by the procedure
described by Wilkinson and Wilton (1986) using ['^Cjoleic acid as a tracer. The isolation
procedure involved subsequent size-exclusion chromatography, dialysis, ultrafiltration and
anion-exchange chromatography. The purity of the isolated protein was verified to be
greater than 95 % by SDS-polyacrylamide-gel electrophoresis (Lawrence and Eacho,
1997).
3.2.3 L-FABP binding assay
The binding buffer consisted of potassium phosphate (dibasic, KH 2 PO4 , 10 mM) solution. The pH of this buffer was adjusted to 7.4 using a 10 mM potassium phosphate
(monobasic, K 2 HPO4 ) solution. The isotope was freshly diluted before each experiment such that the binding incubation mixture contains [9, lO-^H] oleic acid at a concentration of
1 pM and at a specific activity of 1000 pCi/pmole : 10 pi of [9,10-^H]oleic acid at 500 pM and 9.2 Ci/mmol and 90 pi of cold oleic acid at 500 pM were added to 400 pi ethanol (100%). The binding incubation mixture consists of [9,10-^H] oleic acid (1 pM, 5 pi), delipidated FABP (60 pg/ml stock solution, 100 pi) and 5 pi of competitor drug in ethanol. The final volume of the incubation mixture was made up to 500 pi with the phosphate binding buffer, pH 7.4. Non-specific binding was determined using a 100-fold excess of cold oleic acid. Total binding was determined in the absence of any competitor.
The volume of ethanol in every assay tube was identical.
102 After addition of all the reagents, the tubes were vortexed and incubated for 15
min at 37°C, and then cooled on ice for 15 min. Ice-cold Lipidex-1000 (prepared as a
slurry, 1:1 vol/vol in phosphate bufier, pH 7.4) in a volume of 200 pi was then added.
The tubes were then vortexed every 2 min, 4 times, and then pelleted in a Eppendorf
microcentrifiige (Model 5415 C, Madison, WI) at 4°C for 2 min at 10,000 X g. A 200 pi aliquot of the supernatant was added to 3 ml of scintillation fluid (Econosafe-1, Fisher
Scientific Co., Atlanta, GA) and counted on a LKB-Wallac liquid scintillation counter
(Model 1219 Rackbeta, Gaithersburg, MD).
Data are expressed as a percent of the total [^HJoleic acid binding to L-FABP in the absence of competitor. Each point represents the average ± S.E.M. of at least 4 experiments. The average total binding for all the experiments was 19332 ± 848, while the average non-specific binding, calculated in the presence of 100 pM unlabeled oleic acid, for all the experiments was 5194 ± 344 (n = 30 experiments).
3.2.4 Transfection assay
H41IEC3 cells were grown in DMEM containing 10% v/v fetal calf serum (PCS) and were plated 24 hour prior to transfection at a 20% confluency so as to obtain cells at a
50% confluency at the time of transfection. For electroporation, the cells were trypsinized, washed and resuspended in antibiotic-fi’ee medium at a density of 15 x 10^ cells/500 pi and electroporated using a BTX square electroporater (Model T 820,
Genetronics, San Diego, CA) at 190 V for 70 mseconds. The conditions of electroporation were optimized as shown in chapter 2. The cells were plated in 48 cell
103 well plates at a density of 5 x lo^ cells /500 pi of DMEM per well. After 24 hr, media
was changed to DMEM containing charcoal-adsorbed PCS (HyClone Laboratories,
Logan, UT, 10%v/v) and drugs were added onto these cells. Stock solutions for all drugs
were prepared in ethanol (95 %). Appropriate vehicle only controls were performed in
each experiment. At the end of another 24 hours, the media was aspirated, the cells were
lysed using Packard lysis buffer (100 pi) and the luciferase activity of the cells was assayed
by adding 35 pi of lysate to an identical amount of reconstituted Luc-Lite® reagent (Luc-
Lite® kit, Packard Instrument Co., Meriden, CT). Light emitted was measured using a
Top-Count (Packard Instrument Company, Meriden, CT) in the luminometer mode. Data
are expressed as average luciferase response ± S.E.M. (« = 3 experiments) normalized to
the response to the maximal clofibric acid (CPA) response within the same experiment
(100 pM = 100 %).
3.2.5 Data analysis
Por the L-PABP binding experiments, non-specific binding, which was calculated
as the amount of radioligand ([^H]-oleic acid) bound to purified L-PABP in the presence
of a 100-fold excess cold ligand, was subtracted fi'om the data. Results were expressed as
a percent of total [^H]-oleic acid binding. The ICjo values for the compounds were defined as the concentration of drug required to reduce the binding of oleic acid to 50 % of the total binding. The values were calculated using the Cheng-Prusoflf equation (1973),
104 fr_
' l Æ
The Kj for oleic acid for L-FABP is 9.6 ^iM (Cannon and Eacho, 1991) and the
concentration of the radioligand used was 1 |xM.
Statistical significance between the IC 5 0 values of the pairs of stereoisomers were
determined by Student’s t test (Daniel, 1991). Each experiment was repeated a minimum
of 3 times; every concentration was tested as duplicate determinations.
In the PPAR activation experiments, the data have been expressed as a percent of
the maximal response to clofibric acid within the same experiment, which has been
arbitrarily designated as 1 0 0 %. The ECjo values were defined as the concentrations of
the drug required to produce a response equivalent to 50% of the maximal response by
CFA (100 pM) for PPAR activation studies in H4IIEC3 cells). Each experiment was
repeated a minimum of 4 times. In each experiment, the control and treatment groups
were each tested in 3-4 individual dishes or wells of cells.
3.3 Results
3.3.1 Evaluation of the affinities of clofibric acid (CFA) and ciprofibrate
(CIPRO) for binding liver fatty acid binding protein (L-FABP)
The IC5 0 S of two known peroxisome proliferators were determined, and based on these values the IQs were calculated (Fig. 3.1, Table 3.2). In our experiments, CFA has an
105 IC% of 1.2 X 10-* M(Kj = 5.7 x 10'^ M), whUe the IC» of CIPRO is 1.2 x 10'* M (Kj = 5.8
X 1Q-* M). The difference in affinities of these two drugs for L-FABP binding is 9.8-foId,
which is in agreement with the difference observed in the abilities of these compounds to
activate PPARa and stimulate peroxisomal enzymes both in vitro and in vivo (Chapter 2;
Intrasuksri, 1991; Kocarek and Feller, 1989).
3.3.2 Evaluation of the affinities of 2-bromopaImitic acid (BPA), 12-
hydroxydodecanoic acid (12-OH) and dodecanoic acid (DA) for binding to L-
FABP
The ability of a set of fatty acids, viz., BPA, 12-OH and DA, to inhibit binding of
[^H]-oleic acid to L-FABP was determined (Fig. 3.2). The IC^s and the corresponding
KjS for BPA, 12-OH and DA are given in Table 3.2. BPA was found to bind this protein with the highest afiBnity of any compound we tested; Kj = 8.5 x 10'^ M. DA and 12-OH bound L-FABP with similar afiBnities; DA exhibited a = 1.6 x 10'* M, while the for
12-OH was 1.1 X 10'* M. All of the fatty acids tested were more potent at binding L-
FABP as compared to CFA, which in these experiments had a Kj = 8 . 8 x 10'* M.
3.3.3 Determination of the potencies of the stereo isomeric analogs of 2-(4-
chiorophenoxy)acetic acid (CPAA) for binding to the L-FABP
The IC5 0 S and the KjS for the CPAA analogs tested for binding to L-FABP are given in Table 3.3. The stereoisomers of the 2-methyl substituted analog of CPAA
(analog I) inhibited the binding of [*H]-oleic acid to the fatty acid binding protein with
106 equal potency. The (+)-(R)-isomer had a K; = 1.3 x 10"* M and the (-)-(S)-isomer had a Kj
= 3.2 X 10“* M). CFA was slightly more potent at displacing tritiated oleic acid from L-
FABP than these isomers, with a Kj = 5.7 x 10'* M (Fig. 3.3, top panel). The n-propyl-
substituted analogs of CPAA (analog HI) did not show any signrScant degree of
stereoselectivity for binding to L-FABP; the KjS of the isomers were 4.1 x 10'® M for the
(+)-(R)-isomer and 1.0 x lO"* M for the (+)-(S)-isomer (Fig. 3.3, bottom panel). While both stereoisomers of 2-phenyl-substituted CPAA (analog VI) possessed identical affinities for binding to L-FABP [(+)-(S)-isomer, Kj = 1.9 x 10"’ M and (-)-(R)-isomer Kj
= 1.3 X 10'* M ] (Fig. 3.4, bottom panel), the stereoisomers of the 2-isopropyl substituted analog (analog IV) showed differences in binding to L-FABP (Fig. 3.4, top panel). For analog IV, the (+)-(R)-isomer (Kj = 2.1 x 10'* M) demonstrated a greater affinity for binding L-FABP than the (-)-(S)-isomer (Kj = 7.4 x 10'* M). When the chlorophenoxy ring of 2-methyl substituted CPA was replaced with the chlorobenzyloxy group (analog v n ), the stereoisomers of the resulting compound bound L-FABP with differing affinities
(Kj for (+)-(R)-isomer 7.4 x 10'* M and (-)-(S)-isomer 2.8 x 10'* M) (Fig. 3.5, top panel).
The enantiomers of the analog with the (4'-chlorophenoxy)benzyloxy substituent in place of the chlorophenoxy group of CPAA (analog VIII) exhibited KjS of 4.4 x 10* M) [(+)-
(R)-isomer] and 3.8 x 10'* M [(-)-(S)-isomer] (fig. 3.5, bottom panel).
3.3.4 Evaluation of the affinities of perfluorinated fatty acids to bind to L-
FABP
107 Perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA) inhibited the
binding of -oleic acid to L-FABP with KjS of 1.3 x 10*® M and 1.8 x 10'® M
respectively (Fig. 3.6). Both of these compounds are more potent than CFA, which has Kj
= 3.5 X 10*® M in these experiments. Perfluorobutanoic acid (PFBA) exhibits less aflBnity
for L-FABP than PFOA and PFDA with a Kj = 8.4 x 10 ® M. Perfluorooctanol (PFOL) binds to L-FABP very weakly, and inhibited the binding of [®H]-oleic acid by only 20 % at the highest concentration tested (1 mM).
3.3.5 Activation of PFARa in H4IIEC3 cells by perfluorinated fatty acids
Perfluorinated octanoic acid (PFOA), perfluorobutanoic acid (PFBA) and perfluorooctanol (PFOL) were evaluated for in H4IIEC3 cells for their abilities to activate
PPARa (Fig. 3.7). CFA and CIPRO, two known activators of PPARa were tested along with these perfluorinated fatty acids. CFA exhibited an EC jq of 3.5 x 10*® M in this system, while the EC% for CIPRO was 3.1 x 10"^ M. The EC;„ for PFOA was 2.6 x 10 ®
M. However, the maximal response for PFOA in this system was 173 % compared to that of CFA (100 %) at the same concentration of 1 x 10~* M. The response was much greater
in magnitude than the maximal CIPRO response of 118 % at the concentration of 3 x 1 0 "*
M. PFBA did not produce a 50 % response even at the highest concentration tested (10*^
M). The rank order of potencies (ECjq, M) of these compounds were CIPRO (3.1x10*^
M) » PFOA (2.6 X 10 ® M) k CFA (3.5 x 10 ® M) » PFBA (> 10*^ M). PFOL did not exhibit any significant activity in this system.
108 3.4 Discussion
An increase in the levels of fatty acids has been known to cause peroxisome proliferation. Physiological conditions that result in an overload in fatty acids (e.g., high fat diet, diabetes, starvation) have been known to cause an increase in peroxisomal enzymes (Ishii et ai, 1980; Neat et ai, 1980; Nedergaard et ai, 1980). PPAR, which has been implicated as the primary mediator of peroxisome proliferation, has been shown to be activated by saturated, monounsaturated and polyunsaturated fatty acids (Gottlicher et ai,
1992; Issemanne/a/., 1993; Keller eta/., 1993; Dreyer e/a/., 1993). Recently Kliewer e/ ai (1997) have shown certain long-chain unsaturated fatty acids to bind PPAR. Response elements for the PPAR have been found in several genes regulating the metabolism of fatty acids including fatty acid synthesis (malic enzyme, fatty acyl CoA synthase) and fatty acid
P-oxidation (enzymes o f the peroxisomal P-oxidation pathway) (reviewed in Wahli et ai,
1995; Schoojans et ai, 1996; Lemberger et ai, 1996). Thus, PPAR is an important physiological regulator of fatty acid levels in the organism.
Lipid-binding proteins include serum albumin, a-fetoprotein (in fetus), extracellular lipocalins and the cytosolic heat-shock proteins (Veerkamp and Maatman, 1995; Witiak et ai, 1977). Fatty acid binding proteins (FABPs) belong to a subfamily of proteins within this general category, which includes the cellular retinol binding proteins (CRBPs) and the cellular retinoic acid binding proteins (CRABPs) (for review, see Veerkamp and Maatman,
1995). Seven types of FABPs have been classified on the basis of their cDNA sequence, which have been classified by their tissue localization; liver, heart, intestine, adipocyte, ileum, myelin and epidermis. In a particular tissue, FABP expression may be restricted to
109 a particular ceil type, e.g., liver FABP is restricted to hepatocytes (Bass et al., 1989;
Fahimi et ai, 1990). Interestingly, PPARs are present in all tissues in which FABPs are
present. The liver FABP (L-FABP) and the adipocyte FABP (aP2) levels are known to be
regulated by PPAR via PPREs present within their promoter regions (Issemarm et ai,
1992; Tontonoz et ai, 1994b). Thus, these evidence point to an intimate relationship between the signaling pathways of the PPAR and the L-FABP. Further, Lawrence et al.
(1997) have shown that the association of fatty acids with the nucleus is L-FABP- dependent, and have also demonstrated the direct interaction of the L-FABP with nucleus in the presence of peroxisome proliferators. Recent reports implicate the L-FABP in the uptake and the incorporation of fatty acids as esters into specific intracellular lipid pools
(Murphy e/a/., 1996; Prows eta/., 1996).
The stereoisomers of a selected set of 4-chlorophenoxyacetic acid (CPAA) analogs were evaluated for their affinities for binding to L-FABP (For structures, see Fig. 1.8).
These compounds have exhibited a high stereoselectivity for activation of PPARa in CV-1 cells and H4IIEC3 cells and stimulation of peroxisomal fatty acyl-CoA oxidase activity
(Feller e/a/., 1996; Rangwalae/a/., 1997). Retention of a similar pattern of stereodependency for binding to L-FABP would indicate that the L-FABP is the primary protein target of these CPAA analogs. Our results have shown that no stereoselectivity is observed for binding of these compounds to L-FABP. For the two sets of stereoisomers that display some stereoselectivity for binding to L-FABP [e.g., 2-isopropyl (analog IV) and (4'-chlorophenyl)benzyloxy (analog VIU)], it is in the reverse direction with respect to the stereoselectivity these isomers exhibit in the PPAR activation and enzyme induction
110 studies. For instance, analog Vm, where the 4-chlorophenoxy group of CFA has been
substituted with the (4'-chlorophenyl)benzyIoxy group, in PPAR activation systems, the
(+)-(R)-isomer » (-)-(S)-isomer, but for binding to L-FABP, the (-)-(S)-isomer > (+)-
(R)-isomer. Thus, binding to L-FABP is not predictive for the peroxisome proliferative
effects of these compounds. This is in contrast with previous reports that have been
published, showing the maintenance of rank order of potencies between peroxisome
proliferative effects of drugs and the ability to bind PPAR (Cannon and Eacho, 1991;
Eacho et ai., 1993) and the dependence of peroxisome proliferators on the presence of L-
FABP for their mitogenic effects (Khan and Sorof 1994). On the basis of our results presented here, we propose that CFA and its congeners interact with some protein, besides L-FABP, to exert their peroxisome proliferative effects. This protein may be
PPARa itself, similar to the novel hypolipidemic agent GW2331 (Kliewer et ai, 1997).
The binding of CFA analogs to L-FABP may be physiologically significant in that it may help in the intracellular trafScking of these compounds.
2-Bromopalmitic acid (BPA) has a very high affinity for L-FABP (K; = 5.9 x 10’’
M) as compared to CFA (Kj = 8 . 8 x 10'* M), DA (K^ = 1.6 x 10'^ M) and 12-OH (K^ = 1.1
X 10‘* M ). Thus, in the cell, BPA would preferentially bind to L-FABP by displacing endogenous fatty acids. This would help in the efficient uptake and transportation of BPA in the cell. This may be one factor that could explain the high potency with which BPA acts as a PPAR activator, i.e., due to the accessibility of this fatty acid to the receptor.
Further, in chapter 2, the effects of the inactive isomer of 2-n-propyl-substituted CPAA analog [(+)-(R)-isomer of analog IE] was tested on the concentration-response curve of
111 CIPRO and BPA. The concentration-response curve of CIPRO was enhanced by the
presence of the (+)-(R)-HI, but there was no effect on the dose-response curve of BPA.
This could be explained by the fact that the inactive isomer may not be able to displace
BPA from L-FABP, but could displace CIPRO from L-FABP. As a result, the effective
cellular concentration of CIPRO available to PPAR may be higher, resulting in an apparent
increase in response to CIPRO.
Perfluorinated fatty acids such as PFOA and PFDA have been shown to stimulate peroxisomal fatty acyl-CoA oxidase (ACO) activity and microsomal laurate hydroxylase activity in vivo and in vitro (Dceda et ai, 1985; Intrasuksri and Feller, 1991). PFOL has been shown to stimulate enzyme activity in vivo (Ikeda et ai, 1985), but is inactive in our transactivation assay and is unable to cause a significant displacement of [^H]-oleic acid from L-FABP. Intrasuksri and Feller (1991) have previously shown this compound to be inactive in primary cultured rat hepatocytes, indicating that PFOL may require in vivo metabolic activation to exert its peroxisome proliferative effects. Our laboratory previously demonstrated that PFOA is 10-fold more potent at increasing peroxisomal acyl-
CoA oxidase (ACO) and microsomal laurate hydroxylase (LH) activity in primary cultures of rat heptocytes as compared to CFA (Intrasuksri and Feller, 1991). However, the present study shows the PFOA and CFA were almost equally potent at activating PPAR in
H411EC3 cells. These differences may possibly be due to the differential afiBnities of these two drugs for L-FABP. PFOA binds L-FABP with a greater afiBnity (Ki = 1.3 x 10’’) than
CFA (Kj = 8 . 8 X 10"’ M). Therefore, this perfluorinated fatty acid may be transported into the cell more efiBciently as compared to CFA. Primary cultures of rat hepatocytes may
112 contain a higher level of L-FABP than H4IIEC3 cells, therefore this may result in a more
efficient transport of PFOA across the cytosol, into the nucleus, where the PPAR resides.
However, final evidence for this hypothesis would require quantitating the expression of
L-FABP in H4IIEC3 cells in comparison to primary rat hepatocytes, which has not yet
been performed.
In support of the data presented here, are results obtained by Gottlicher et al.
(1993). These investigators evaluated a series of sulfiir-substituted mono- and
dicarboxylic acids for their ability to increase peroxisomal P-oxidation in rat liver and
activate a glucocorticoid receptor (GR)-PPAR chimera in CHO cells. A non-P-oxidizable sulfur-substituted derivative of hexadecanedioic acid, 3, 14-dithiahexadecanedioic acid, was very potent at inducing peroxisomal P-oxidation activity in rat liver. The same compound was very weak when tested in a GR-PPAR transactivation assay in CHO
(Chinese hamster ovary) cells. However, this compound was a potent activator of PPAR when primary cultures of rat hepatocytes were used as host cells for GR-PPAR instead of
CHO cells. It is possible that this thia-substituted fatty acid, similar to PFOA, is dependent on L-FABP to exert its peroxisome proliferative effects. Primary rat hepatocytes may contain higher levels of this protein, resulting in a higher activity of the chimeric receptor in these cells.
L-FABP has been shown to bind prostaglandins (Dutta-Roy et ai, 1987; Khan and
Soro^ 1990) and long-chain fatty acids (Sweetser et ai, 1987; Bass, 1988; Bass, 1993;
Bass, 1985). We have shown that compared to CFA, non-metabolizable fatty acids such as BPA and PFOA have a greater affinity for L-FABP. Therefore, fatty acids such as
113 BPA and PFOA, due to their high aflBnity for L-FABP, may be transported more efiBciently through the cytosol than fibrates and other peroxisome proliferators.
In conclusion, the presented data support the hypothesis that the ability of the analogs of clofibric acid to bind to L-FABP is not necessarily an accurate estimate of the ability of these compounds to activate PPARa and stimulate ACO activity. However, L-
FABP may play a significant role in the signaling pathways of fatty acids via the PPAR.
We speculate that this role may be that of an intracellular transporter, whereby the fatty acid is transported through the cell into the nucleus by L-FABP, thus increasing the possibility of the deliverance of these substrates to the PPAR.
114 Structures Names (abbreviations)
CH3 (CH2 )ioCOOH Dodecanoic acid (DA)
HOCH2 (CH2 )ioCOOH 12-hydroxydodecanoic acid (12-OH)
CH3 (CH2 )i2 CH2 CH(Br)COOH 2-Bromopalmitic acid (BPA)
CFj(CF2 )2 COOH Perfluorobutanoic acid (PFBA)
CF3 (CF2 )gC 0 0 H Perfluorooctanoic acid (PFOA)
CFj(CF2)sC00H Perfluorodecanoic acid (PFDA)
CF,(CF,),CH,OH Perfluorooctanol (PFOL)
Table 3.1. Chemical structures of fatty acid analogs. The abbreviations used are indicated in parentheses along with the names.
115 Compound n IC5 0 ± S.E.M. (M) Kj ± S.E.M.(M)
CFA 2 0 1 .8 x 1 0 -^ ±1.9x10-* 5.7 X 10 * ± 9.2 X 1 0 -*
CIPRO 4 1 .8 x 1 0 -* ± 2 . 1 x 1 0 -* 5.8x 10-*± l.Ox 1 0 -*
BPA 3 1.7 X 10^ ±5.2 X 10-' 8.5 X 10"' ± 2.6 X 1 0 -'
12-OH 3 2.2x 10-*± 1.8 X 10-* 1 . 1 X I 0 *±8.9x 1 0 '
DA 3 3.2x10'* ±1.3x10* 1.6 X 10 * ± 6.3 X 1 0 -*
Table 3.2. IC5 0 and values for clofibric acid (CFA), ciprofibrate (CIPRO), 2- bromopaimitic acid (BPA), 12-hydroxydodecanoic acid (12-OH) and dodecanoic acid (DA) for the displacement of -oleic acid fi"om liver fatty acid binding protein (L- FABP). Each drug was tested in duplicate in an experiment.
116 Compound n IC5 0 ± S.E.M. (M) Kj ± S.E.M.(M)
(+)-(R)-I 4 2.6 X 10-^ ±5.7 X 10-* 1.3 X 10"* ±2.8 X 10 *
(-HS)-I 4 6.5 X 10-" ±1.3 X 10-^ 3.2 X 10-^ ±6.2 X 10 *
(+).(R).ni 4 8.3x10-* ± 1 . 1 x 1 0 -* 4.1 X 10-* ± 5.6 X 10-*
(-)-(S)-m 3 2.1 X 10-* ± 4.7 X 10* l.Ox 10-*±2.3 X 10*
(+)-(R)-IV 4 4.3 xlO *± 1.1x10 * 2.1 X 10 * ± 5.3 X 10-*
(-)-(S)-IV 4 1.5 X 10"* ±3.4 X 10 * 7.4 X 10-* ± 1.7 X 10-*
(+)-(S)-VI 3 4.0x 10-*± I.Ox 10 * 1.9 X 10'* ±5.1 X 10-*
(-HR)-VI 3 2.6 X 10-* ±5.9 X 10-^ 1.3 X 10 * ± 2.9 X 10-*
(+)-(R)-vn 4 1.5 X 10-* ±6.2 X 10"^ 7.4 X 10* ± 3.0 X 10-*
(-)-(S)-vn 4 5.7x10-* ±5.3x10-* 2.8 X 10 * ± 2.6 X 10-*
(+)-(R)-vm 4 9.0x 10*±4.1 X 10-* 4.4 X 10-* ± 2.0 X 10-*
(-)-(S)-vm 4 7.7 X 10* ±2.0 X 10-* 3.8 X 10 * ± 9.7 X 10"*
Table 3.3. IC5 0 and Kj values for 2-(4-chlorophenoxy)acetic acid (CPAA) analogs for the displacement of [^H]-oleic acid from liver fatty acid binding protein. For structures, see Fig. 1.8, Chapter 1). Each drug was tested in duplicate in an experiment.
117 100
09 W 75 - *3 "oI m B « 2 5 - u 9i A,
6 4 35 Log concentration (M)
Figure 3.1. Concentration-response curves of clofibric acid (CFA) and ciprofibrate (CIPRO) for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L- FABP). Key: CIPRO, O; CFA, ■. Results are expressed as the percent of pH]-oleic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. CIPRO and CFA was tested together a total of 4 times (in duplicate each time).
118 100-
25-
-6 ■5 -4 -3 Log concentration (M)
Figure 3.2. Concentration-response curves of 12-hydroxydodecanoic acid (12-OH), 2- bromopalmitic acid (BPA), dodecanoic acid (DA) and clofibric acid (CFA) for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP). Key: 12- OH, □; BPA, ▲; DA, 0; and CFA, ■. Results are expressed as the percent of [^H]-oleic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. Each drug was tested in 3 independent experiments (in duplicate).
119 Figure 3.3. Concentration-response curves of 2 -substituted 2-(4-chIorophenoxy)acetic acid (CPAA) analogs I and III for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP). For structures, see Fig. 1.8. See inserts in each panel for the symbols for each compound. Top panel shows results for CFA and the enantiomers of the 2-methyl substituted CPAA analog (analog I), while the lower panel shows the enantiomers of the 2-n-propyl-substituted analog (analog EQ). Results are expressed as the percent of [^H]-oIeic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. Drugs were tested in 3-4 experiments (in duplicate).
1 2 0 2-M ETHYL « 100- « 01 ■o 75- S
50- CFA 25- o (+)-(R)-ISOMER (-)-(S)-ISOMER 0 T 1------r 3 -5 -4 -3 Log concentration (M)
100- » ^ * u O) 01 f f i l
g 2-/1-PROPYL \ 25- O (+)-(R)-ISOMER • (-HS)-ISOMER
0 - I 1 1 - 6 - 5 - 4 -3 Log concentration (M)
Fig. 3.3.
121 Figure 3.4. Concentration-response curves of 2-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs IV and VI for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP). See inserts in figure for the symbols of each compound. Top panel shows results for the enantiomers of the 2-isopropyl substituted CPAA analog (analog IV), while the lower panel shows the enantiomers of the 2-phenyl-substituted analog (analog VI). Results are expressed as the percent of [^H]-oleic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. Asterisks represent statistically significant differences between the isomers at P < 0.05. Drugs were tested in 3-4 experiments (in duplicate).
1 2 2 2-ISOPROPYL • (-)-(S)-isomer O (+)-(R)-isomer
0 - —r- T -5 -4 -3 Log concentration
125 a w 100- ’S oc 01 s 75- B .B B 50- V W 2-PHENYL u V 25- o (+)-(S)-ISOMER (-)-(R)-ISOMER 0 "T 1 -7 - 6 Log concentration (M)
Fig. 3.4.
123 Figure 3.5. Concentration-response curves of 2-(4-chlorophenoxy)acetic acid (CPAA) analogs Vn and Vm for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP). See inserts in figures for symbols of each compound. Top panel shows results for the enantiomers of the benzyloxy-substituted analog (analog VII), while the lower panel displays results for the enantiomers of the (4'-chlorophenyl)benzyloxy- substituted analog (analog VUI). Results are expressed as the percent of [^H]-oleic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. Asterisks represent statistically significant difference between the isomers atP < 0.05. Each drug was tested in 4 experiments (in duplicate).
124 <1 w In Percent [^H|-oleic acid Percent [ H]-oleic acid binding binding
CJl O N3 NON) O O l O Ol O l O Ol J 1...... I j_____ I
QTQ n o N) g LA fp & 3 125 w es u 100- u 01
S
w 25 -
6 5 4 3 Log concentration (M)
Figure 3.6. Concentration-response curves of perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA), perfluorodecanoic acid (PFDA), perfluorooctanol (PFOL) and clofibric acid (CFA) for the displacement of [^H]-oleic acid from liver fatty acid binding protein (L-FABP). Key: CFA ■; PFOA A; PFDA O; PFOL, ▲; PFBA # . Results are expressed as the percent of [^H]-oleic acid bound (mean ± S.E.M.) in the presence of increasing concentrations of competing drug. Each experiment was repeated 4 times (in duplicate).
126 « 200 - i a *S 150- 3
N 100- i I o 50- Z
Log concentration (IVf)
Figure 3.7. Concentration-response curves for perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA), perfluorooctanol (PFOL), clofibric acid (CFA) and ciprofibrate (CIPRO) for the activation of PPARa in H4UEC3 cells. Key; CFA ■; CIPRO, O; PFOA A; PFOL, ▲ and PFBA • • Results have been expressed as the luciferase response (mean ± S.E.M.) normalized to the maximal response to CFA (10*^ M) In the same experiment. Each drug was tested in 4 experiments (in duplicate).
127 CHAPTER 4
EVALUATION OF THE ABILITY OF FIBRIC ACID ANALOGS TO BIND TO THE LIGAND BINDING DOMAIN OF xPPARa
4.1 Introduction
Peroxisome proliferation, defined as the increase in the number and volume density
of peroxisomes, was initially seen in response to the administration of clofibric acid to
rodents (Paget, 1963; Hess et ai, 1965). Since then, a structurally diverse variety of
chemicals has been found to be peroxisome proliferators, including other fibric acid
hypolipidémies, fatty acids, phenoxy herbicides and phthalate esters.
Peroxisome proliferation has consistently showed a tissue and cell specificity. This
led Lalwani and co-workers (1987) to report the detection and semi-purification of a
"peroxisome proliferator binding protein" (PPBP) fi’om rat liver. Nafenopin, ciprofibrate
and clofibric acid were each individually immobilized on Sepharose 4B columns and the
cytosol containing the PPBP was passed through the column. The column was washed to
remove non-specifically bound protein, and it was found that the bound protein could be
eluted by incubation with an excess of ligand for a one hour period. However, other
laboratories could not reproduce these results (Milton et ai, 1988), and it was concluded that the ligands were binding non-specifically to plasma proteins.
128 The peroxisome proliferator activated receptor ( PPAR) was cloned from a mouse
liver cDNA library in 1990 by Issemarm and Green (1990). Using a transactivation assay,
it was shown that this protein was activated by peroxisome proliferators in the same rank order of potency with which these chemicals caused peroxisome proliferation in rodents.
These workers tried to demonstrate binding of [^H]-nafenopin to a chimera of ER-PPAR, but were unsuccessful. They concluded that this was due to a low aflBnity of nafenopin for the receptor. When the PPARa gene was disrupted by homologous recombination, the response to peroxisome proliferators was abolished, thus providing definitive evidence that this receptor is the primary mediator of the effects of peroxisome proliferators (Lee et al., 1995). The absence of studies demonstrating the binding of any of the peroxisome proliferators to the PPAR is attributable to the lack of a specific, high aflBnity ligand for this receptor.
Lehmann et al. (1995) first reported the binding of a thiazolidinedione, BRL 49653 to the Y isoform of the PPAR (see Fig. 5 in Chapter 1). Shortly after, other ligands for the
Y isoform of the PPAR were reported by two groups (Kliewer et al., 1995; Forman et al.,
1995). In independent studies, these workers demonstrated that PPARy bound a metabolite ofPGJ^, 15-deoxy-A'^"-prostaglandin 3% (Fig. 5). This prostaglandin was proposed to be an endogenous ligand for this isoform of the receptor. All the drugs that were shown to bind PPARy also stimulated transcriptional activity of the receptor and increased the adipogénie conversion of fibroblasts. Thus, it became apparent from these studies that the PPARs, like other nuclear hormone receptors, were indeed ligand- activated transcription factors.
129 In 1996, Devchand et ai illustrated that leukotriene B 4 (LTB4 Fig. 6 in Chapter I)
was able to bind to a fusion protein consisting of the ligand binding domain of the
Xenopus PPARa and glutathione-S-transferase. The for LTB 4 was 90 nM. This was
the first report of a high aflBnity ligand for the PPARa. These workers showed that
PPARa was responsible for the increased metabolism of LTB4; in animals lacking the
protein, the inflammation response was prolonged due to a slower uptake and increased
persistence of the leukotriene. Using a similar binding assay, Kliewer et ai (1997)
reported that a novel fibrate, GW2331 (see Fig. 6 ), acts as a high aflBnity ligand for both
PPARs a and y. The K^ values for this drug for binding to a GST-xPPARa and GST- xPPARy fusion proteins are 140 nM and 300 nM respectively. Using competition binding assays, these workers showed that certain fatty acids, namely, oleic, petroselenic, linoleic, linolenic and arachidonic acids bind PPARa. However, none of the classical hypolipidemic peroxisome proliferators were tested by these authors in their binding assay.
Forman et al. (1997) utilized a electrophoretic mobility shift assay system to demonstrate the binding of hypolipidemic drugs, fatty acids and eicosanoids to the PPARa. In vitro- translated mouse PPARa and human RXRa were used in sub-microgram quantities. The
DNA used in this assay was a Klenow-labeled acyl-CoA oxidase PPRE. Since the proteins are present in very small amounts, the binding of the proteins to the DNA is contingent on the presence of the PPAR activator (Forman et ai, 1997). Clofibrate, ciprofibrate, Wy 14,643 (for structures, refer to Fig. 1 in Chapter 1) and several other compounds including fatty acids and eicosanoids were able to induce the binding of the receptors to the PPRE.
130 In this set o f experiments, the CPAA analogs (Fig. 1.8 in Chapter 1) were
examined for their ability to bind to the ligand binding domain of xPPARa. The phenyl
and the n-propyl CPAA analogs activate PPARa and stimulate ACO activity in a highly
stereoselective manner (Rangwala et ai, 1997; Feller et ai, 1996; Chapter 2). We have
showed that the binding to L-FABP is not predictive of the peroxisome proliferative
effects of these compounds, as suggested by some workers (Cannon and Eacho, 1991;
Issemann et ai, 1992). Therefore, our hypothesis was that these compounds would bind
to PPARa with a rank order of stereoselectivity that was similar to that observed in the transactivation and the ACO assay systems. Using the system established by Devchand et ai (1996), the GST-xPP ARaLBD fusion protein was isolated and purified from E. coli ceils and the ability o f clofibric acid, ciprofibrate and two sets of CPAA isomers to inhibit binding of [^H]-LTB.* to the protein was examined.
4.2 Methods
4.2.1 Materials
The biochemicals used and their sources used were the following: tryptone, yeast extract, glycine, bromphenol blue (Fisher Biotech, Fair Lawn, NJ), ampicillin, isopropyl-B-
D-thiogalactoside (IPTG), human y-globulin, glutathione (free acid), ammonium persulfate, N,N,N’,N'-tetramethylethylenediamine (TEMED), 30 % acrylamide-0.8 % bisacrylamide solution. Tris base, sodium dodecyl sulfate (SDS), Tris hydrochloride, glycerol, HEPES, sodium chloride, Triton-X 100 (Sigma Chemical Company, St. Louis,
131 MO). Clofibric acid and ciprofibrate were gifts obtained from Ayerst and Sterling
Winthrop Research Institute (Rensselaer, NY) respectively. Unlabeled LTB 4 was obtained
from Cayman Chemicals (Ann Arbor, MI). The CPAA analogs were synthesized by Dr.
Vincenzo Tortorella (University of Bari, Bari, Italy). Other chemicals used were of
reagent grade.
4.2.2 Expression of xPPARa(LBD>-GST fusion protein
The plasmid for the fusion protein pGEX-xPPARa(LBD) was provided by Dr.
Walter Wahli (University o f Lausanne, Lausanne, Switzerland). The fusion protein
contains amino acids 171-474 of the wild type xPPARa protein. The E. coli strain BL21
DE3 pLysS (Novagen, Inc., Madison, WI) was transformed with this plasmid. The
transformed bacteria were selected for ampicillin resistance. A single clone was used to
innoculate a starter culture which was grown overnight at 30°C in a shaking incubator.
The media used was LB containing ampicillin (50 pg/ml), and the volume of the starter
culture was 40 ml. The starter culture was then used to innoculate 300 ml cultures, which
were grown for 6 hr. At this point, the cultures were induced with 0.2 mM EPTG and
grown for another 6 hr. The cultures were then centrifuged at 2300 x g in an lEC CR-
6000 centrifuge (International Instrument Company, Needham Heights, MA) at 4°C for
35 min to sediment the cells. The supernatant media was discarded and the cells were frozen at -70 °C until further processing.
132 4.2.3 Purification of xPPARa(LBD)>GST fusion protein
The cells were thawed and resuspended in ice-cold PBS (pH 7.3) in a volume such
that 50 pi of PBS was added per ml of culture. The resuspended cells were placed in 10
ml aUquots in 15-ml capacity tubes and were subjected to four rapid freeze-thaw cycles by
placing the tubes in a dry ice-ethanol bath and 37°C water bath alternately. The lysis of
the cells was evidenced by a increase in viscosity of the suspension. The tubes were then
centrifuged at 31,000 x g in a Sorvall RC SC Plus centrifuge (DuPont, Wilmington, DE)
for 1 hr at 4° C . The supernatant, containing the fusion protein, was carefully decanted
and filtered through a 0.45 pm syringe filter (Acrodisc filters, Gelman Sciences, Ann
Arbor, MI). The filtrate was placed on ice.
The filtrate containing the fusion protein was then passed through a glutathione-
Sephadex® column (Pharmacia Biotech, Piscataway, NJ). The column was washed three
times with 20 ml aliquots of PBS. The fusion protein was eluted by the addition of 2 ml of
Tris-HCI (50 mM, pH 8 ) buffer containing reduced glutathione (10 mM). For efficient
elution to occur, the column was incubated for 1 0 min each time with the glutathione
containing buffer. The elution was repeated three times, to obtain a total volume of 6 ml of eluate containing the fusion protein. Generally, the latter two fi-actions contained greater amounts of the protein, and were subjected to the desalting process to remove the excess amount of glutathione which could interfere with the stability of the LTB^.
Desalting of the protein solution was done using size exclusion chromatography. The protein in the glutathione containing buffer was passed through a PD-10 Sephadex® (G-
25 grade) column (Pharmacia Biotech, Piscataway, NJ). The column was washed and
133 eluted with Tris-HCI bufifer (50 tnM, pH 8 ) as per the manufacturer’s instructions. Due to the larger size of the protein as compared to the glutathione, glutathione molecules were trapped in the pores of the Sephadex®, while the protein was collected in the eluate.
4.2.4 Identification of purified protein using SDS-PAGE
Protein content was analyzed using the Coomassie blue protein assay (Pierce
Laboratories, Rockford, IL), using human y-globulin as a standard. The protein was further analyzed and semi-quantitated using denaturing (SDS), discontinuous polyacrylamide gel electrophoresis (PAGE) based on the method of Laemmli (1970)
(Ausubel et ai, 1995). The sample was diluted with 6 X SDS sample buffer in a ratio of
5:1, vortexed gently, and heated at 100° C for 5 min. The composition of the sample bufifer is given in the Appendix. The samples, which were maintained at 60°C for ease of pipetting, were then loaded on to the wells of a vertical slab acrylamide gel placed in a gel bath (Model 200, Aquebogue Machine Shop, Aquebogue, NY). The gel consisted of a stacking gel (3.9 % acrylamide) above a separating gel (7.5 % acrylamide). The separating gel occupies about the bottom two-thirds of the gel volume. The composition of the gels and the electrophoresis bufifer used is given in the Appendix. The current source used was Biorad Model 1000/500 Power Supply (Biorad Laboratories, Hercules,
CA). The gel was run at a constant current of 20 mAmp through the stacking gel and a constant current of 30 mAmp through about two-thirds of the separating gel. The gel was carefully removed, fixed and stained using the Pharmacia PlusOne Silver staining kit
(Pharmacia Biotech, Piscataway, NJ). The protocol followed was that as provided by the
134 manufacturer. Using the estimates of protein concentrations obtained from the Coomassie
protein assay, increasing amounts of the protein were run on the gel along with the
corresponding identical concentrations of a standard albumin solution (Pierce
Laboratories, Rockford, IL). A standard protein marker (Rainbow® colored protein marker, 14,300 - 220,000 kDa, Amersham Corp., Arlington Heights IL) was also included on the same gel to determine the size of the isolated protein.
4.2.5 Ligand binding assay
The ligand binding assay involved the incubation of the [^HJ-LTB^ (50 nM)
(specific activity 202 Ci/mmol, Amersham Corporation, Arlington Heights, IL) and 25 pg of protein in the presence of different concentrations of competitors in a 50 pi reaction
containing 50 mM Tris, 1 0 mM MgCl; and 100 mM NaCl (pH 8.0) for 3.5 hr on ice, in the dark. At the end of the incubation period the bound ligand was separated from the unbound by passing the incubation mixture through a NAP-5 Sephadex® (G-25 grade) column (Pharmacia Biotech, Piscataway, NJ) which has a void volume of approximately
600 pi. The column was then washed two times with 300 pi aliquots of elution buffer containing 10 mM HEPES, 1 mM EDTA, 50 mM NaCl, 5 % glycerol and 0.05 % Triton-
X 100 (pH 7.4). The protein-bound radioligand was eluted in a third 300 pi wash fraction of the elution buffer. The bound radioligand was quantitated by liquid scintillation counting. The sample was added to 3.2 ml of Econosafe-1 (Fisher Scientific Company,
Atlanta, GA) and counted on a LKB-Wallac liquid scintillation counter (Model 1219
Rackbeta, Gaithersburg, MD).
135 4.2.6 Data analysis
For the ligand binding assay, results were determined as the counts per minute of
[^H]-leukotriene 8 4 bound to the eluted protein and are expressed as the percent of [^H]-
leukotriene 8 4 displaced in the presence of the competing drug. Non-specific binding was
determined in the presence of ciprofibrate (CIPRO) 10 mM; data were corrected for non
specific binding using this value. Data were statistically analyzed by a direct comparison
among the means using a Student's t-test (Daniel, 1991). The KjS for the competitors
were calculated using the Cheng and Prusofif equation (1973),
K.=- I . A
where IC5 0 is the concentration of the competitor required to reduce the binding of the radioligand by 50 %, [L] is the concentration of the radioligand used and for the radioligand is known from saturation binding experiments. In this case, [L] is 50 nM and
Kj is 90 nM for LTB4 .
4.3 Results
4.3.1 Isolation and purification of GST-xPPARa(LBD)
The GST-xPPARa(LBD) protein was isolated and purified using affinity and size exclusion chromatography. As per the results of the Coomassie protein assay using y- globulin as a standard, the typical yield of protein on completion of the isolation and
136 desalting procedure was 5 mg/ml. The protein was further analyzed by SDS-PAGE. A
silver stained sample gel picture is shown in Fig. 4.1. Lane I represents a sample from the
washes of the glutathione-Sephadex® column after loading the fusion protein-containing
extract. Lanes 2-5 contained decreasing amounts of bovine serum albumin (BSA), lane 6
contained the Rainbow® colored molecular weight marker (molecular weights of the
standards are shown on the right hand side of the gel), whereas lanes 7-10 contained
amounts of fusion protein correspondingly identical to the amounts of BSA present in
lanes 2-5. The band present in lane 1 confirmed that none of the fusion protein was lost while washing the glutathione-Sephadex® column. Lanes 2-4, containing 2700 ng, 900 ng and 300 ng of BSA respectively appear as distinct bands, while lane 5, which contains a
100 ng of BSA was not detectable using this staining procedure. Lanes 7-9 contain 2700 ng, 900 ng and 300 ng of the fusion protein respectively; the différences in the intensity of the bands representing identical amounts of albumin and the fusion protein may be due to a different sensitivity of the two proteins to silver staining (DeSilva et ai, 1995). The absence of any other bands besides the one at approximately 60 kDa indicated that this protein solution contained GST-xPPARa(LBD) and is free of any proteinaceous contaminants.
4.3.2 Ability of clofibric acid (CFA), ciprofibrate (CIPRO) and LTB 4 to
inhibit binding of to PFARa
Our initial experiments were undertaken to establish whether CFA, CIPRO and
LTB4 were able to competitively inhibit [^H]-LTB4 to this fusion protein. Fig. 4.2A shows
137 that CFA at a concentration of 10 mM did not inhibit LTB 4 binding to xPPARa(LBD).
However, CIPRO can reduce the total amount of bound to GST-
xPPARa(LBD) from an average of 3398 ± 70 CPM to an average of 754 ± 109 CPM (n =
10 experiments, mean ± S.E.M.). Unlabeled LTB 4 at the highest concentration tested, 50
pM, decreased the total counts from an average of 1507 ± 149 to an average of 830 ± 90
CPM (n = 3 experiments) (Fig. 4.2B). This represents a 45 % decrease, which is in
agreement with the findings of Devchand et a/. (1996). Fig 4.2C displays results o f a
representative experiment. The binding of labeled LTB 4 to GST-xPPARa(LBD)
decreases with an increase in the concentration of CIPRO, reaching a minimum of 22.2 %
in the presence of 1 0 mM CIPRO.
Devchand et al. (1996) attempted displacing the tritiated LTB4 with unlabeled
LTB4 . They found that the total displacement of the the radiolabeled compound took a
7000-fold excess of unlabeled LTB4 . The concentration required to displace 50 % of
[^H]-LTB4 was estimated at 50 pM. We used this information to calculate the for
CIPRO,
Using this equation, the relative of CIPRO was calculated (Table 4.1).
The Cheng and Prusoff equation (1973) was used to calculate the Kj for CIPRO, which is presented in Table 4.1.
138 4.3.3 Binding of n-propyi and phenyl 2-(chIorophenoxy)acetic acid (CPAA)
analogs to xPPARa (LBD)-GST
Fig. 4.3 shows the effects of the stereoisomers of the n-propyi CPAA isomers
(analog HI) on the binding of the LTB 4 to the fusion protein. Both enantiomers were
almost equally potent in competing with LTB^. At a concentration of 3 mM, both isomers
inhibited to 83 % of the total binding; at 10 mM, binding was reduced
to 30 % of the total binding.
The phenyl isomers (analog VI) showed some degree of stereoselectivity in
binding to xPPARa (LBD)-GST (Fig. 4.4). The (+)-(S) isomer was significantly less
potent than the (-)-(R) isomer at 3 mM {P < 0.05). However, at 10 mM, both isomers
equally abolished binding of the LTB 4 to the fusion protein.
The Kj values for these CPAA analogs, calculated using the Cheng and Prusoff equation (1973) (section 4.2.6.), are given in Table 4.1. The relative values for the analogs were calculated using the equation presented in section 4.2.2. and are presented in
Table 4.1.
4.4 Discussion
The observation that two sets of 4'-chlorophenoxyacetic acid analogs (CPAA) were highly stereoselective at activating PPARa and stimulating acyl CoA-oxidase activity led to the utilization of these compounds as probes for the elucidation of their mechanism of action. Previously, we have shown that liver fatty acid binding protein (L-FABP) is not
139 the protein binding site that can discriminate between the stereoisomers of these
compounds (Chapter 3). In this set of studies, these CPAA analogs, along with
ciprofibrate (CIPRO) and clofibric acid (CFA) have been evaluated for their ability to
inhibit leukotriene 8 4 (LTB4 ) binding to the ligand binding domain of xPPARa, expressed
as a fusion protein with glutathione-S-transferase (GST), using the ligand binding assay described by Devchand et al. (1996). The ligand binding assay utilizes the entire fusion protein, without cleavage of the GST portion. The non-specific binding observed in this assay is mainly due to the binding of the radioligand to GST (Devchand et ai, 1996).
Inhibition of [^H]-LTB 4 binding to fusion protein in the presence of unlabeled
ligand was attempted. As discussed in section 4.3.2., a concentration of 50 pM o f LTB4 decreased the binding of the radiolabel to the fusion protein by 45 %, which is in agreement with the results of Devchand etal. (1996). This concentration, 50 pM, is well above the value of the ligand. Our studies indicate that CIPRO binds to the ligand binding domain of xPPARa. The concentration of CIPRO required to produce a 50% decrease in ligand binding can be intrapolated to be approximately 5 mM. This is the first
report of binding between this drug and PPARa, however, the IC 5 0 of CIPRO for
inhibiting LTB 4 binding to xPPARa does not correspond to the ECjo of CIPRO for activating PPARa and stimulating ACO activity both in vivo and in vitro. Further, in these systems, CFA has been shown to be about 10-fold less potent than CIPRO (Feller et ah, 1987; Kocarek and Feller, 1989; Intrasuksri, 1991; Chapter 2). In our studies, we have not been able to demonstrate the binding of CFA with xPPARa at the highest concentration of CFA tested (10 mM). Further, Devchand et al. (1996) tested the
140 compound Wy-14,643 in this assay for its ability to bind to xPPARa. They found that 500
pM of Wy-14,643 was required to effect a 45 % displacement of LTB 4 from
xPPARa(LBD). Wy-14,643 is one of the most potent peroxisome proliferators and
selective PPARa activators known (Kliewer et ai, 1994); its EC% for activation of
rPPARa is 1.5 pM, while that for CFA when tested under the same conditions is 310 pM
(Issemann and Green, 1990). The high concentrations of competitors required to inhibit
binding of the radioligand could be attributed to the experimental conditions, that may allow for the dissociation of the unlabeled ligands, and hence, an underestimation of the Kj values. Therefore, relative Kj values were determined, which approximate the ECjoS of
CIPRO, CFA and the CPAA analogs in functional experiments much more closely than
the calculated values (Table 4. 1 ).
The «-propyl CPAA isomers tested bind to the protein without any stereoselectivity. This is in contrast with the findings in the PPARa activation and the
ACO induction studies, where the (-)-(S)-isomer of this compound is much more potent than the (+)-(R)-isomer. For the phenyl CPAA analogs, the (-)-(R)-isomer is significantly more potent than the (+)-(S) isomer at inhibiting binding of [^Hj-LTB^ to PPARa. These compounds showed reverse chiral selectivity in the PPARa activation and increase in
ACO activity, where (+)-(S)-isomer » (-)-(R)-isomer. Our results imply that the (R)- isomers of the CPAA analogs, which are inactive at activating PPARa and stimulating
ACO activity, can bind to the PPARa, but do not result in the conformational change required to produce an increase in transcriptional activity, and subsequently, stimulation of enzyme activity. Particularly, the (-)-(R)-isomer of the phenyl CPAA analog, which is
141 relatively more potent at displacing LTB^ from the fusion protein, may prove to be a very
eflBcient antagonist of the receptor. The inactive isomers of both of these analogs,
however, did not block the rPPARa activation mediated by CIPRO or 2-bromopalmitic
acid (BP A) (see Chapter 2). In contrast, the inactive isomer of the n-propyl analog
enhanced the maximal luciferase response of CIPRO in the transactivation assay. As
discussed in the preceeding chapters, this could be due to the displacement of CIPRO by
these inactive compounds from intracellular binding sites. Also, there may exist certain
feedback mechanisms in these cell systems that cause an increased expression of the
receptor when it is bound by an inactive compound. Thus, increased cellular PPARa
levels result in an enhancement of the response in the presence of the activating
compound. Hence, it appears that the regulation of PPAR activation in the cell may be complex, and the interpretation of in vitro binding data will require caution because of these considerations.
PPARa has been recently found to mediate the hypolipidemic effects of fibric acids by regulating the expression of the apolipoproteins A-I, A-II and C-m (for review, see
Schoojans et ai, 1996; Chapter I). Identifying novel, specific ligands for this receptor can lead to the discovery of effective antihyperlipidemic drugs. PPARa is also responsible for
the increased metabolism of LTB4 , leading to a decrease in the levels of this mediator during inflammation (Devchand e/nr/., 1996). Interestingly, the non-steroidal antiinflammatory agents such as indomethacin, flufenamic acid, fenoprofen and ibuprofen have been found to be activators of PPAR (Lehmann et ai, 1997). It is possible that these agents exert a certain proportion of their activity through the PPARa. Thus, PPARa
142 represents a possible target for the design of anti-inflammatory agents. The structural characteristics that result in better hypolipidemic activity than anti-inflammatory activity and vice-versa remain to be established. The compounds studied here may provide leads to studying the interactive amino acids in the ligand binding site of PPARa, and possibly help in the design of potent hypolipidemic or antiinflammatory agents.
143 Competitor n Kj (mM) Relative Kq (pM)
CIPRO 2 1.8 ±0.5 5.0 ± 1.3
(+)-(R)-in 2 4.7 ± 1.0 13.0 ±2.7
(-)-(S)-m 2 4.7 ± 1.6 13.0 ±4.4
(+)-(S)-VI 2 3.2 ±0.3 8.9 ±0.8
(-)-(R)-VI 2 1.9 ±0.3 5.3 ±0.8
Table 4.1. fQ values for ciprofibrate (CIPRO) and 2-«-propyl and 2-phenyl-substituted 2- (4-chIorophenoxy)acetic acid (CPAA) for displacing LTB^ fi’om the rf*PARa (LBD)-GST fiision protein. KjS were calculated using the Cheng-Prusoff equation (1973), and relative Kj values calculated using formula in section 4.3.2.
144 Molecular weight standards
BSA 66K
GST-xPPAR(LBD) 46K
123456789 10
Figure 4.1. SDS-Polyacrylamide gel electrophoretic (SDS-PAGE) analysis of the fusion protein, xPPARa(LBD)-GST. Key: lane 1, ^utathione-Sephadex® column wash; lane 2,
BSA 2700 ng; lane 3, BSA 900 ng; lane 4, BSA 300 ng; lane 5, 100 ng, lane 6 , Rainbow® protein molecular weight marker; lane 7, fusion protein, 2700 ng; lane 8 , fusion protein 900 ng; lane 9, fusion protein, 300 ng and lane 10, fusion protein, 100 ng. Molecular mass sizes of the standard markers are given on the right side of the gel. The size of the fusion protein is approximately 60 kDa.
145 Figure 4.2. A. Effect of clofibric acid (CFA, 10 mM, « = 2 experiments) and ciprofibrate (CIPRO, I mM n = l experiments; 10 mM, « = 10 experiments) on the binding of [^H]-
LTB4 to xPPARa(LBD)-GST. B. Effects of unlabeled LTB 4 (50 pM) on the binding of
[^H]-LTB4 to xPPARa(LBD)-GST. Data presented are mean ± S.E.M. of 3 experiments. The effects of CIPRO 10 mM in the these experiments (n = 2 experiments) is shown for comparison. C. Representative experiment demonstrating the concentration-dependent displacement of [^H]-LTB 4 by CIPRO. For reference, the displacement of the radiolabel by unlabeled LTB 4 (50 pM) in the same experiment has been included.
146 k> CPM CPM \j\ KJ\ § 8I I 1 __j____ No competitor n Total CFA 10 mM mmwâ CIPRO 1 mM CIPRO 1 mM i ; CIPRO 10 mM
4 k CIPRO 3 mM CPM
SSSSÎS CIPRO 7 mM
CIPRO lOmM No competitor
LTB4 50 uM LTB4 50 uM
lîT CIPRO lOmM o o o Suipuiq |B)Oi % 100 2-/1-PROPYL
75-
50-
25-
E E E E CO o CO o
Figure 4.3. Inhibition of [^HJ-LTB^ binding to PPARa by the stereoisomers of the 2-n- propyi-substituted 2-(4-chlorophenoxy)acetic acid (CPAA) analogs (analog III). Results are expressed as a percent of the total binding. All data were corrected for non-specific binding, calculated in the presence of CIPRO 10 mM. Data presented are mean ± S.E.M. of 5 experiments (10 mM concentration) or 2 experiments (3 mM concentration).
148 100 Ü 2-PHENYL [=□ (+)-(S)
75- * n T u 50- m m u w CL C/3 25-
0 - - n E E E E CO o CO o
Figure 4.4. Inhibition of [^HJ-LTB, binding to PPARa by the stereoisomers of the 2- phenyl-substituted 2-(4-chIorophenoxy)acetic acid (CPAA) analogs (analog VI). Results are expressed as a percent of the total binding. All data were corrected for non-specific binding, calculated in the presence of CIPRO 10 mM. Data presented are mean ± S.E.M. of 3 experiments. Asterisk indicates statistically significant difference between the stereoisomers at P < 0.05.
149 CHAPTERS
SUMMARY AND CONCLUSIONS
This work attempted to elucidate the mechanism of peroxisome proliferation. A set of chiral 4-chlorophenoxyacetic acid (CPAA) analogs were selected to determine the role of stereoselectivity in this phenomenon. The compounds chosen were structural analogs of clofibric acid (CFA); one of the methyl groups at the 2-position of CFA was removed to create a chiral center. Various substituents, such as methyl, ethyl, «-propyl, isopropyl, «-hexyl and phenyl groups were substituted at this position to obtain a series of structurally related compounds. Besides these structural variations, the 4-chlorophenoxy group of the 2-methyl substituted CPAA analog was substituted with a 4-chlorobenzyloxy group and a 4-(4'-chlorophenyl)benzyloxy group.
Two sets of these isomers, 2-(4-chlorophenoxy)-pentanoic acid and -octanoic acid,
i.e., the analogs possessing the «-propyl and the «-hexyl substituents at the 2 -position, were examined for their abilities to stimulate peroxisomal acyl-CoA oxidase (ACO) and microsomal laurate hydroxylase activities in primary rat hepatocytes (Feller and
Intrasuksri, 1991), and were found to be highly stereoselective. We wanted to study the effects of these compounds on activation of the a isoform of the peroxisome proliferator-
150 activated receptor (PPARa), as this receptor has been found to be the primary mediator of
the peroxisome proliferative responses to these compounds. A transactivation system,
where an expression plasmid for PPARa was co-transfected along with a luciferase
reporter gene under control of a peroxisome proiiferator response element (PPRE) into
CV-1 cells, was utilized to investigate the effects of the chiral CPAA analogs on
transcriptional activity of the receptor. Such a system would enable us to detect the direct
effects of these compounds on receptor activity, without interference from the endogenous
PPARs. In these experiments, we found that the all of the compounds tested resulted in
PPARa activation in a concentration-dependent manner. Removal of one methyl group
from the 2-position of clofibric acid resulted in a decrease in activity. Of the series of analogs, enantiomers of 2-«-propyl and 2-phenyl CPAA were highly stereoselective at activating the PPARa. The 2-isopropyl substituted-CPAA compounds did not demonstrate any stereoselectivity. Generally, the greater the length of the alkyl group, the greater was the potency of the CPAA analog at activating PPARa. The stereoisomers of the 2-n-hexyl CPAA analog, although not very stereoselective, were potent activators of
PPARa. Substitution of the «-hexyl group with a phenyl ring at the 2-position decreased the potency of the compound, but increased its stereoselectivity dramatically. The rank order of stereoselectivity observed for the CPAA analogs was (S) > (R), except for the 2- methyl substituted analogs, all of which exhibited a reversal in stereoselectivity, i.e., for these compounds, (R) isomer > (S) isomer. Increasing the length of the chlorophenoxy
151 group to a chlorobenzyloxy group increased the potency; further elongation to a 4-(4'-
chlorophenyI)benzyloxy group led to a decrease in the potency but an increase in the
stereoselectivity of the enantiomers.
The abilities of the CPAA analogs to transactivate the endogenous PPAR in
H4IIEC3 cells was studied. This was done by transfecting only the PPRE-containing
reporter gene into H4IIEC3 cells, a rat hepatoma cell line. This cell line has been
previously found to be responsive to peroxisome proliferators (Osumi et ai, 1990). The
endogenous receptor was pharmacologically characterized as PPARa. The rank orders of
potency and stereoselectivity observed for the CPAA analogs for activating PPARa in
CV-1 cells were mostly conserved for the activation of the PPAR in H4IIEC3 cells. A
few minor differences in the potencies of the compounds were observed; however, these
could be explained by pharmacokinetic differences in the two cell systems. Overall,
H4IIEC3 cells were more sensitive to the effects of peroxisome proliferators than CV - 1
cells.
Once the effects of the CPAA analogs at the molecular level of PPAR had been
characterized, we sought to examine the effects of these analogs on events downstream of
activation of the receptor, such as on acyl-CoA oxidase (ACO) activity. ACO has been
used as an enzyme marker for peroxisome proliferation. H4IIEC3 cells were used as the biological model as the activation of the receptor in these cells had already been
characterized. Using a sensitive fluorometric assay, the stimulation of ACO by the CPAA analogs was examined. The correlation between the ability of these compounds to activate PPARa and stimulate ACO was very high; the order of potency and
152 stereoselectivity mirrored those seen in the activation studies. Hence, it was concluded
from these studies that the activation of PPARa is related to the increase in peroxisomal
fatty acid P-oxidation activity.
The liver fatty acid binding protein (L-FABP) represents a potential binding site
for peroxisome proliferators, as the rank order of potencies of peroxisome proliferators
and their binding to L-FABP are very similar. Using our stereoselective analogs as
probes, we wanted to investigate if binding to L-FABP is predictive of the peroxisomal
proliferative effects of the CPAA analogs. However, we observed that these compounds
do not enantioselectively bind to L-FABP, and, therefore, L-FABP is not the
discriminatory cellular binding site for these compounds. All of these compounds can
displace [^H]-oleic acid from the L-FABP, and may possibly interact with this protein in
vivo. Interestingly, non-metabolizable fatty acids such as 2-bromopalmitic acid and
perfluorinated fatty acids bind L-FABP with a higher affinity than the CFA and its
congeners. Since L-FABP has been hypothesized to be a fatty acid transporter within the
cell, this protein may be involved in the intracellular trafficking of peroxisome
proliferators. As fatty acids bind L-FABP more efficaciously, they may be transported to the nucleus, and to the PPAR, more rapidly and efficiently as compared to xenobiotics.
This explains the differences seen in the ability of perfluoroctanoic acid (PFOA) to
stimulate ACO activity in primary hepatocyte cultures and its ability to activate PPARa in
CV- 1 cells or in H4UEC3 cells. Thus, L-FABP may play an important role in the signaling pathway of PPAR as a transporter protein.
153 The discovery of LTB 4 and the GW2331 as ligands ioxXenoptis PPARa led us to
test our CPAA analogs for binding to PPARa. The ligand binding domain of xPPARa,
bacterially expressed as a fusion protein, was isolated, purified and utilized in this binding
assay. [^HJ-LTB^ was used as the radioligand. Ciprofibrate was found to inhibit the
binding of radiolabel to PPAR with a Kj of 3.52 mM, but CFA did not inhibit binding of
radiolabel, even at the highest concentration tested (10 mM). The 2-«-propyl- and the 2-
phenyl-substituted CPAA analogs, the two sets of stereoisomers that were very
stereoselective in the PPARa activation and the ACO enzyme assays, were tested for their potential to inhibit binding of radiolabeled LTB4 to the fusion protein. Surprisingly, both stereoisomers of the «-propyl compound were equally active at binding xPPARa(LBD).
However, for the phenyl-substituted analogs, although some degree of stereoselectivity was observed between the enantiomers for binding xPPARa, it was in the reverse direction to that observed as a PPARa activator. In activation and ACO stimulation studies, the phenyl substituted analogs were less potent activators of the receptor than the
«-propyl CPAA isomers, but in the binding studies, the phenyl isomers seem to bind the receptor more potently. The results of the binding studies imply that the inactive isomers of these analogs may bind to the PPAR, but may not cause a conformational change of the receptor so as to enable transcriptional regulation to occur.
A summary of the major conclusions of the dissertation research is presented below:
1. The peroxisome proliferation caused by clofibric acid and its congeners is mediated via a direct interaction with PPARa. These compounds could be used to develop probes in order to study the ligand-interactive sites of PPARa.
154 2. The 2-position of clofibric acid is important for interaction with PPARa as
modifications in structure or introduction of a chiral center at this position dramatically
aflfect the potency of the molecule as a PPAR activator. In general, the longer chain
length of alkyl substituent at the 2-position is correlated to a greater potency for PPARa
activation. Changing the M-propyl group to an isopropyl moiety results in a loss in a
dramatic loss in stereoselectivity, whereas replacement of a «-hexyl group at this position
with a phenyl group reduces the potency, but increases stereoselectivity.
3. The chlorophenoxy group of clofibric acid is also involved in the interaction with
the binding site. Increasing the size of this group to chlorobenzyloxy increases the
potency, but increasing the size further to (4'-chlorophenyl)benzyloxy does not result in a
further increase in potency. Thus, only a substituent of a certain maximal size can be
accommodated in the binding pocket of the receptor.
4. The inactive isomers of the 2-phenyl and the 2-«-propyl substituted analogs, which
bind to the receptor but do not cause transactivation, can be used for the design of PPARa
antagonists.
5. The role of L-FABP as a transporter for peroxisome proliferators can be further investigated, by studying the activation of the PPAR by fatty acids and xenobiotic peroxisome proliferators in the presence or absence of L-FABP in a variety of cell systems.
6 . The effects of the CPAA analogs on the activation of other isoforms of PPAR would give us an idea of the nature of the binding site of these isoforms, and their similarities to PPARa.
155 Thus, in conclusion, the information provided here could be utilized for the
elucidation of structure-activity relationships for activators of PPARa, and this would help
in the design of novel, potent hypolipidemic and antiinflammatory agents. As research uncovers new roles for the PPAR in cellular physiology, the potential of this receptor as a target for pharmacological manipulation will increase.
156 APPENDIX
Method of preparation is stated if there are special considerations.
4X Tris chloride/SDS, pH 8.8
Dissolve 1.5 M Tris base (91 g) in 300 ml water. Adjust pH to 8 . 8 with 1 N HCl. Add water to a total volume of 500 ml. Filter solution through a 0.45 pm filter, add 0.4 % w/v
( 2 g) SDS, and store at 4°C.
4X Tris chloride/SDS, pH 6.8
Dissolve 0.5 M Tris base (6.05 g) in 40 ml water. Adjust pH to 6 . 8 with 1 N HCl. Add water to a total volume of 100 ml. Filter solution through 0.45 pm filter, add 0.4 % w/v
(0.4 g) SDS, and store at 4°C.
SDS sample buffer, 6X
0.28 M 4X Tris chloride/SDS, pH 6 . 8
30 % v/v glycerol
1 % w/v SDS
0.5MDTT
157 0 . 0 0 1 2 % w/v bromphenol blue
Prepared in distilled, deionized water.
The buffer was stored in 0.5 ml aliquots at -70“C.
Stacking gel
3.9 % acrylamide (1.3 ml of a stock of 30 % acrylamide/0.8 % bisacrylamide)
2.5 ml 4X Tris chloride/SDS, pH 6 . 8
6 . 1 ml distilled, deionized water added to make up a total volume of 1 0 ml.
Add 50 pi of 10 % ammonium persulfate and 10 pi of N,N,N',N'-
tetramethylethylenediamine (TEMED). Swirl to mix and use immediatly.
Separating gel
7.5 % acrylamide (7.5 ml of a stock of 30 % acrylamide/0.8 % bisacrylamide solution)
7.5 ml of 4X Tris chloride/SDS, pH 8 . 8
Add 100 pi of 10% w/v ammonium persulfate and 20 pi of TEMED. Swirl to mix and pour immediatly.
Electrophoresis buffer (5X)
0.125 M Tris base
0.96 M glycine
0.5 % w/v SDS
Prepared in distilled, deionized water.
This buffer was diluted to IX before use. Store at 4“ till use.
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