Peroxisomes are subcellular organelles found in animals and plants. Peroxisomes contain enzymes for cholesterol and lipid metabolism and respiration.
A variety of chemical agents called peroxisome proliferators induce the proliferation of peroxisomes and increase the capacity of peroxisomes to metabolize fatty acids via increased expression of the enzymes required for the β-oxidation cycle. Peroxisome proliferators include unsaturated fatty acids, hypolipidemic drugs (Reddy, J. K., and Azarnoff, D. L., Nature 283:397–398, 1980), herbicides, leukotriene antagonists, and plasticizers (for a review, see Green, S., Biochem. Pharmacol. 43:393–400, 1992). Hypolipidemic drugs such as clofibrates have been found to lower triglycerides and cholesterol levels in plasma and to be beneficial in the prevention of ischemic heart disease in individuals with elevated levels of cholesterol (Havel, R. J. and Kane, J. P., Ann. Rev. Pharmac. 13:287–308, 1973). However, fibrate hypolipidemic drugs are also rodent hepatocarcinogens (Reddy, J. K., et al., Br. J. Cancer 40:476–482, 1979; Reddy, J. K., et al., Nature 283:397–398, 1980).
There are two hypotheses for peroxisome proliferation. The “lipid overload hypothesis” suggests that an increase in the intracellular concentration of fatty acids is the main stimulus for peroxisome proliferation (Nestel, P. J., Ann. Rev. Nutr. 10:149–167, 1990, and Phillipson, B. E., et al., N. Engld. J. Med. 312:1210–1216, 1985).
Another hypothesis postulates a receptor mediated mechanism. Peroxisome proliferator activated receptors (PPARs) have been isolated and cloned from various species (Isseman, I., and Green, S., Nature 347:645–650, 1990; Dreyer, C., et al., Cell 68:879–887, 1992; Gottlicher, M., et al., Proc. Natl. Acad. Sci. USA. 89:4653–4657, 1992; Sher, T., et al., Biochemistry 32:5598–5604, 1993; and Schmidt, A., et al., Mol. Endocrinol. 6:1634–16414–8, 1992). The ligand for PPARs is still unidentified.
PPARs belong to the nuclear hormone receptor superfamily. Some members of the superfamily are receptors for the classical steroid hormones; and others bind thyroid hormones, vitamin D3 and retinoic acid. However, the putative ligands for many remain to be identified, and such receptors have been termed orphan receptors. Nuclear hormone receptors are intracellular proteins that are ligand-dependent transcription modulators. They usually have a ligand binding domain that binds the cognate ligand with high affinity and specificity. They usually also have a DNA binding domain that recognizes short DNA motifs generally termed Hormone Response Elements (HREs).
Issemann and Green, Nature 347:645–650, 1990, cloned a mouse peroxisome proliferator activated receptor (mPPARα) gene from a mouse liver complementary DNA (cDNA) library. Chimeric receptors constructed using the DNA binding domain of either the estrogen or glucocorticoid receptor and the putative ligand binding domain of mPPAR are able to activate, respectively, an estrogen- or glucocorticoid-responsive gene in the presence of peroxisome proliferators. mPPARα protein binds to a specific peroxisome proliferator response element (PPRE) located 570 bp upstream of the rat acyl-CoA oxidase gene, which is a key marker of peroxisome proliferator action (Tugwood, J. D., et al. EMBO J. 11:433–439, 1992).
Göttlicher et al., Proc. Nat. Acad. Sci. USA 89:4653–4657, 1992, cloned a rat peroxisome proliferator activated receptor (rPPAR) gene from a rat liver cDNA library. PPARs from mouse and rat share 97% homology in amino acid sequence and a particularly well-conserved putative ligand-binding domain. Three members of the Xenopus nuclear hormone receptor superfamily (i.e., XPPARα, XPPARβ and XPPARγ) have also been found to be structurally and functionally related to the mPPARα (Dreyer et al., Cell 68:879–887, 1992). Schmidt et al., Molecular Endocrinology 6:1634–1641, 1992, cloned a steroid hormone receptor gene, hNUC1 (also known as hPPARβ), from a human osteosarcoma cell cDNA library. The homology between amino acid sequence of hNUC1 and that of mPPARα is 62%.
Chen et al., Biochem. Biophy. Res. Com. 196:671–677, 1993, cloned two mouse PPAR genes, mNUC1 and mPPARγ, from a neonatal mouse brain cDNA library and an adult mouse heart cDNA library, respectively.
Zhu et al., J. Biological Chemistry 268:26817–26820, 1993, cloned a mPPARγ gene from mouse liver cDNA library encoding a 475-amino acid protein with 75% amino acid similarity to XPPARγ, and 55% identity with mPPARα.
Tontonoz et al., Genes &Development 8:1224–34, 1994, not admitted to be prior art, cloned a mPPARγ2 gene from a mouse adipocyte cDNA library. mPPARγ2 protein is an adipocyte-specific transcription factor. It is highly expressed in white adipose tissue and is dramatically induced during differentiation of preadipocytes into adipocytes.
Kliewer et al., Proc. Natl. Acad. Sci. USA 91:7355–59, 1994, not admitted to be prior art, cloned mPPARγ and mPPARδ genes from a mouse liver cDNA library.
Two subtypes of PPAR receptors, α and β, have been characterized from humans (see, Sher et al., Biochemistry 32:5598–5604, 1993, and Schmidt et al., Molecular Endocrinology 6:1634–1641, 1992).