New molecular aspects of regulation of mitochondrial activity by fenofibrate and fasting

Fenofibrate and fasting are known to regulate several genes involved in lipid metabolism in a similar way. In this study measuring several mitochondrial enzyme activities, we demonstrate that, in contrast to citrate synthase and complex II, cytochrome c oxidase (COX) is a specific target of these two treatments. In mouse liver organelles, Western blot experiments indicated that mitochondrial levels of p43, a mitochondrial T3 receptor, and mitochondrial peroxisome proliferator activated receptor (mt-PPAR), previously described as a dimeric partner of p43 in the organelle, are increased by both fenofibrate and fasting. In addition, in PPAR alpha-deficient mice, this influence was abolished for mt-PPAR but not for p43, whereas the increase in COX activity was not altered. These data indicate that: (1) PPAR alpha is involved in specific regulation of mt-PPAR expression by both treatments; (2) fenofibrate and fasting regulate the mitochondrial levels of p43 and thus affect the efficiency of the direct T3 mitochondrial pathway.     

PPARalpha agonists stimulate progastrin production in human colorectal carcinoma cells

The three subtypes of peroxisome proliferator activated-receptors (PPARalpha, delta and gamma) control the storage and metabolism of fatty acids. Treatment of rats with the PPARalpha ligand ciprofibrate increases serum gastrin concentrations, and several lines of evidence suggest that non-amidated gastrins act as growth factors for the colonic mucosa. The aim of the present study was to investigate the expression of PPARs and the effect of PPAR ligands on gastrin production and cell proliferation in human colorectal carcinoma (CRC) cell lines. mRNAs for all three PPAR subtypes were detected by PCR in all CRC cell lines tested. The concentrations of progastrin, but not of glycine-extended or amidated gastrin, measured by radioimmunoassay in LIM 1899 conditioned media and cell extracts were significantly increased by treatment with the PPARalpha ligand clofibrate. Similar increases in progastrin were seen following treatment with the PPARalpha ligands ciprofibrate and fenofibrate, but not with bezafibrate, gemfibrozil or Wy 14643. The PPARgamma agonist rosiglitazone had no significant effect on progastrin production. The PPARalpha ligand clofibrate also stimulated proliferation of the LIM 1899 cell line. We conclude that some PPARalpha ligands increase progastrin production by the human CRC cell line LIM 1899, and that clofibrate increases proliferation of LIM 1899 cells. These studies have revealed a relationship between PPARs and gastrin, two regulatory molecules implicated in the pathogenesis of CRC.     

n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.     

Involvement of PPARα and PPARγ in apoptosis and proliferation of human hepatocarcinoma HepG2 cells

Peroxisome proliferator‐activated receptors (PPARs) mediate the effects of various ligands, known as peroxisome proliferators, a heterogeneous class of compounds including industrial chemicals, pharmaceuticals, and biomolecules such as fatty acids and eicosanoids. Among peroxisome proliferators, fibrate derivatives are considered specific ligands for PPARα, whereas eicosanoids, such as PGJ2, for PPARγ. The study aimed to clarify the relation between PPARs and apoptosis or proliferation on the same type of cells, using clofibrate as specific ligand of PPARα and PGJ2 as specific ligand of PPARγ. The cells used were human hepatocarcinoma HepG2 cells. The results showed that PPARα protein content increased in HepG2 cells treated with clofibrate, causing apoptosis in a time‐ and concentration‐dependent way, as evidenced by the citofluorimetric assay and determination of BAD, myc and protein phosphatase 2A protein content. It also emerged that PPARγ increased in the same cells when treated with a specific ligand of this PPAR; in this case the increase of PPARγ did not cause an increase of apoptosis, but a time‐ and concentration‐dependent inhibition of cell proliferation, evidenced by decreased cell numbers and increased number of cells in the G0/G1 phase of the cycle. It may be concluded that PPARα is chiefly related to apoptosis and PPARγ to cell proliferation. Copyright © 2010 John Wiley & Sons, Ltd.     

Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

BACKGROUND: Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. METHODS: Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP), 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls).The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. RESULTS: Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. CONCLUSION: The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.     

Role of PPARgamma, a nuclear hormone receptor in neuroprotection

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. PPAR-alpha is involved in wound healing, stimulation of lipid and folic acid catabolism, inflammation control, inhibition of ureagenesis and peroxisome proliferation. The PPARgamma/delta is involved wound healing, cell proliferation, embryo implantation, adipocyte differentiation, myelination alteration and apoptosis. The PPARgamma is involved in fat, lipid and calorie utilization, sugar control, inflammation control and macrophage (MQ) matutation. Homocysteine (Hcy) binds to nuclear peroxisome proliferator activated receptor. Increase in PPAR expression decreases the level of nitrotyrosine and increases endothelial nitric oxide concentration, decreases metalloproteinase activity and expression as well as elastinolysis and reverses Hcy-mediated vascular dysfunction. The PPARgamma initially recognized as a regulator of adipocyte development has become a potential therapeutic target for the treatment of diverse disorders. In addition, the activation of PPARgamma receptor ameliorates neurodegenerative disease. This review focuses on the recent knowledge of PPARgamma in neuroprotection and deals with the mechanism of neuroprotection of central nervous system disorder by PPARgamma.     

Detection of heat-stable delta 3,delta 2-enoyl-CoA isomerase in rat liver mitochondria and peroxisomes by immunochemical study using specific antibody

The subcellular distribution of delta 3,delta 2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the peroxisomal isomerase, revealing a single band with an apparent molecular weight of 30,000. However, the isomerase was induced by clofibrate administration mainly in the mitochondrial fraction. These results suggest that delta 3,delta 2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.     

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.     

Apoptosis induced by clofibrate in Yoshida AH-130 hepatoma cells: role of HMG-CoA reductase

Clofibrate is a hypolipidemic drug belonging to the peroxisome proliferator (PP) family. PPs are well-recognized hepatocarcinogens, though only for rodents and not for humans. Their oncogenicity is usually ascribed to mitogenic or antiapoptotic action. However, we have reported that clofibrate can trigger fast and extensive apoptosis in rodent and human tumor cell lines. The present study examines the possible mechanisms involved in clofibrate-induced apoptosis in AH-130 hepatoma cells. The results show that the apoptogenic effect of clofibrate does not depend on induction of peroxisome proliferator activated receptors (PPARs), but on interference with HMG-CoA reductase (HMGR), a key enzyme that regulates cholesterol biosynthesis and production of isoprenoid units for protein farnesylation. The level and activity of HMGR mRNA are reduced in clofibrate-treated AH-130 cells and apoptosis can be partially prevented by addition of mevalonate. Moreover, cholesterol and cholesterol ester content decreases early in mitochondria, and cytocrome c is released in the cytosol. On the contrary, perturbations at the level of protein farnesylation are not important in determining the fast apoptogenic effect, since treatment of AH-130 cells with an inhibitor of farnesyltransferase induces apoptosis only after 4 h. In conclusion, inhibition of HMGR and decreased cholesterol content are crucial events in clofibrate-induced apoptosis in AH-130 hepatoma cells.     

PPAR expression and function during vertebrate development

The peroxisome proliferator activated receptors (PPARs) are ligand activated receptors which belong to the nuclear hormone receptor family. As with other members of this superfamily, it is thought that the ability of PPAR to bind to a ligand was acquired during metazoan evolution. Three different PPAR isotypes (PPARalpha, PPARbeta, also called 6, and PPARgamma) have been identified in various species. Upon binding to an activator, these receptors stimulate the expression of target genes implicated in important metabolic pathways. The present article is a review of PPAR expression and involvement in some aspects of Xenopus laevis and rodent embryonic development. PPARalpha and beta are ubiquitously expressed in Xenopus early embryos but become more tissue restricted later in development. In rodents, PPARalpha, PPARbeta and PPARgamma show specific time- and tissue-dependent patterns of expression during fetal development and in the adult animals. PPARs are implicated in several aspects of tissue differentiation and rodent development, such as differentiation of the adipose tissue, brain, placenta and skin. Particular attention is given to studies undertaken by us and others on the implication of PPARalpha and beta in rodent epidermal differentiation.     

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.     

Natural ligands of PPARgamma: are prostaglandin J(2) derivatives really playing the part?

The peroxisome proliferator-activated receptor (PPAR) family was discovered from an orphan nuclear receptor approach, and thereafter, three subtypes were identified, namely PPARalpha, PPARbeta or PPARgamma and PPARgamma. The two former seem to regulate lipid homeostasis, whereas the latter is involved, among others, in glucose homeostasis and adipocyte differentiation. PPARs were pharmacologically characterised first using peroxisome proliferators such as clofibrates, which demonstrate moderate affinity (efficiency at micromolar concentrations) and low PPARalpha/delta versus PPARgamma specificity. Hence, several laboratories have started the search for potent and subtype-specific natural PPAR activators. In this respect, prostaglandin (PG)-related compounds were identified as good PPARgamma agonists with varying specificity, the most notable PPAR ligand being 15-deoxy-Delta12-14-PGJ2 (15d-PGJ2). Recently, an oxidized phosphatidylcholine was identified as a potent alternative (patho)physiological natural ligand of PPARgamma. In the present review, we discuss the different PPARgamma-dependent and -independent biological effects of the PG PPARgamma ligands and the concern about their low potency in molecular models as compared with thiazolidinediones (TZDs), a family of potent (nanomolar) synthetic PPARgamma ligands. Finally, the oxidized lipids are presented as a novel and interesting alternative for discovering potent PPARgamma activators in order to understand more in details the implications of PPARgamma in various pathophysiological conditions.