Differential expression of peroxisome proliferator-activated receptor-alpha, -beta, and -gamma during rat embryonic development.

The expression patterns of the three different peroxisome proliferator-activated receptor (PPAR) isotypes have been determined during rat embryonic development by in situ hybridization. The expression of PPARalpha starts late in development, with increasing levels in organs such as liver, kidney, intestine, and pancreas, in which it will also be present later in adulthood to regulate its specific target genes. PPARalpha is also transiently expressed in the embryonic epidermis and central nervous system. PPARgamma presents a very restricted pattern of expression, being strongly expressed in brown adipose tissue, in which differentiation it has been shown to participate. Like PPARalpha, it is also expressed transiently in the central nervous system. Interestingly, PPARalpha, -beta and -gamma are coexpressed at high levels in brown adipose tissue. Finally, the high and ubiquitous expression of PPARbeta suggests some fundamental role(s) that this receptor might play throughout development.

Pancreatic islet adaptation to fasting is dependent on peroxisome proliferator-activated receptor alpha transcriptional up-regulation of fatty acid oxidation.

The cellular response to fasting and starvation in tissues such as heart, skeletal muscle, and liver requires peroxisome proliferator-activated receptor-alpha (PPARalpha)-dependent up-regulation of energy metabolism toward fatty acid oxidation (FAO). PPARalpha null (PPARalphaKO) mice develop hyperinsulinemic hypoglycemia in the fasting state, and we previously showed that PPARalpha expression is increased in islets at low glucose. On this basis, we hypothesized that enhanced PPARalpha expression and FAO, via depletion of lipid-signaling molecule(s) for insulin exocytosis, are also involved in the normal adaptive response of the islet to fasting. Fasted PPARalphaKO mice compared with wild-type mice had supranormal ip glucose tolerance due to increased plasma insulin levels. Isolated islets from the PPARalpha null mice had a 44% reduction in FAO, normal glucose use and oxidation, and enhanced glucose-induced insulin secretion. In normal rats, fasting for 24 h increased islet PPARalpha, carnitine palmitoyltransferase 1, and uncoupling protein-2 mRNA expression by 60%, 62%, and 82%, respectively. The data are consistent with the view that PPARalpha, via transcriptionally up-regulating islet FAO, can reduce insulin secretion, and that this mechanism is involved in the normal physiological response of the pancreatic islet to fasting such that hypoglycemia is avoided.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.

Chemical probes that differentially modulate peroxisome proliferator-activated receptor alpha and BLTR, nuclear and cell surface receptors for leukotriene B(4).

Peroxisome proliferator-activated receptor alpha (PPARalpha)is a nuclear receptor for various fatty acids, eicosanoids, and hypolipidemic drugs. In the presence of ligand, this transcription factor increases expression of target genes that are primarily associated with lipid homeostasis. We have previously reported PPARalpha as a nuclear receptor of the inflammatory mediator leukotriene B(4) (LTB(4)) and demonstrated an anti-inflammatory function for PPARalpha in vivo (Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996) Nature 384, 39-43). LTB(4) also has a cell surface receptor (BLTR) that mediates proinflammatory events, such as chemotaxis and chemokinesis (Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624). In this study, we report on chemical probes that differentially modulate activity of these two LTB(4) receptors. The compounds selected were originally characterized as synthetic BLTR effectors, both agonists and antagonists. Here, we evaluate the compounds as effectors of the three PPAR isotypes (alpha, beta, and gamma) by transient transfection assays and also determine whether the compounds are ligands for these nuclear receptors by coactivator-dependent receptor ligand interaction assay, a semifunctional in vitro assay. Because the compounds are PPARalpha selective, we further analyze their potency in a biological assay for the PPARalpha-mediated activity of lipid accumulation. These chemical probes will prove invaluable in dissecting processes that involve nuclear and cell surface LTB(4) receptors and also aid in drug discovery programs.

Sex difference in hepatic peroxisome proliferator-activated receptor alpha expression: influence of pituitary and gonadal hormones.

Peroxisome proliferator-activated receptor (PPAR) alpha is a nuclear receptor that is mainly expressed in tissues with a high degree of fatty acid oxidation such as liver, heart, and skeletal muscle. Unsaturated fatty acids, their derivatives, and fibrates activate PPARalpha. Male rats are more responsive to fibrates than female rats. We therefore wanted to investigate if there is a sex difference in PPARalpha expression. Male rats had higher levels of hepatic PPARalpha mRNA and protein than female rats. Fasting increased hepatic PPARalpha mRNA levels to a similar degree in both sexes. Gonadectomy of male rats decreased PPARalpha mRNA expression to similar levels as in intact and gonadectomized female rats. Hypophysectomy increased hepatic PPARalpha mRNA and protein levels. The increase in PPARalpha mRNA after hypophysectomy was more pronounced in females than in males. GH treatment decreased PPARalpha mRNA and protein levels, but the sex-differentiated secretory pattern of GH does not determine the sex-differentiated expression of PPARalpha. The expression of PPARalpha mRNA in heart or soleus muscle was not influenced by gender, gonadectomy, hypophysectomy, or GH treatment. In summary, pituitary-dependent hormones specifically regulate hepatic PPARalpha expression. Sex hormones regulate the sex difference in hepatic PPARalpha levels, but not via the sexually dimorphic GH secretory pattern.

Developmental and tissue-specific involvement of peroxisome proliferator-activated receptor-alpha in the control of mouse uncoupling protein-3 gene expression.

Uncoupling protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor-α (PPARα) on the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPARα-null mice both in basal conditions and under the stimulus of starvation. In contrast, UCP3 mRNA is down-regulated in adult heart both in fed and fasted PPARα-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARα-null mice. In neonates, PPARα-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. The murine UCP3 promoter is activated by fatty acids through either PPARα or PPARδ but not by PPARγ or retinoid X receptor alone. PPARδ-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPARα. However, among transcripts from other PPARα and PPARδ target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPARα-null neonates. Thus, PPARα-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart.

Peroxisome proliferator-activated receptor alpha expression is regulated by estrogen receptor alpha and modulates the response of MCF-7 cells to sodium butyrate

Peroxisome proliferator-activated receptors are ligand-activated transcription factors with a potential role in cancer. We investigated peroxisome proliferator-activated receptor alpha expression in breast cancer cell lines and showed a relationship between mean peroxisome proliferator-activated receptor alpha and estrogen receptor alpha mRNA levels in estrogen receptor alpha positive breast cancer cells. Transfection of estrogen receptor alpha into the estrogen receptor alpha negative cell line, MDA-MB-231 decreased peroxisome proliferator-activated receptor a mRNA and conversely inhibition of estrogen receptor alpha by ICI-182 780 in estrogen receptor a positive, MCF-7 cells increased peroxisome proliferator-activated receptor a mRNA levels. Estrogen receptor alpha levels can be modulated by histone deacetylase inhibitors and such agents are in clinical trials for cancer treatment. We found the histone deacetylase inhibitor, sodium butyrate, increased peroxisome proliferator-activated receptor alpha mRNA levels within 4 h of treatment. Peroxisome proliferator-activated receptor a modulation was independent of estrogen receptor alpha, as a similar increase was observed in the estrogen receptor a negative MDA-MB-231 cells. To further investigate the relationship between sodium butyrate and peroxisome proliferator-activated receptor alpha expression, we created an MCF-7 cell line that conditionally over-expresses human peroxisome proliferator-activated receptor alpha. Over-expression of the peroxisome proliferator-activated receptor protected MCF-7 cells from sodium butyrate-mediated inhibition of proliferation and attenuated sodium butyrate-mediated induction of histone deacetylase 3 mRNA, indicating that elevated levels of peroxisome proliferator-activated receptor alpha may reduce the sensitivity of cells to histone deacetylase inhibitors. The estrogen receptor alpha dependence of peroxisome proliferator-activated receptor alpha levels may be significant since estrogen receptor alpha negative breast cancer cells are associated with a more aggressive phenotype. Our studies also suggest that peroxisome proliferator-activated receptor alpha levels may be a marker of breast cancer cell sensitivity to histone deacetylase inhibitors. (c) 2004 Elsevier Ltd. All rights reserved.

Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

The direct peroxisome proliferator-activated receptor target fasting-induced adipose factor (FIAF/PGAR/ANGPTL4) is present in blood plasma as a truncated protein that is increased by fenofibrate treatment.

The fasting-induced adipose factor (FIAF, ANGPTL4, PGAR, HFARP) was previously identified as a novel adipocytokine that was up-regulated by fasting, by peroxisome proliferator-activated receptor agonists, and by hypoxia. To further characterize FIAF, we studied regulation of FIAF mRNA and protein in liver and adipose cell lines as well as in human and mouse plasma. Expression of FIAF mRNA was up-regulated by peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARbeta/delta agonists in rat and human hepatoma cell lines and by PPARgamma and PPARbeta/delta agonists in mouse and human adipocytes. Transactivation, chromatin immunoprecipitation, and gel shift experiments identified a functional PPAR response element within intron 3 of the FIAF gene. At the protein level, in human and mouse blood plasma, FIAF was found to be present both as the native protein and in a truncated form. Differentiation of mouse 3T3-L1 adipocytes was associated with the production of truncated FIAF, whereas in human white adipose tissue and SGBS adipocytes, only native FIAF could be detected. Interestingly, truncated FIAF was produced by human liver. Treatment with fenofibrate, a potent PPARalpha agonist, markedly increased plasma levels of truncated FIAF, but not native FIAF, in humans. Levels of both truncated and native FIAF showed marked interindividual variation but were not associated with body mass index and were not influenced by prolonged semistarvation. Together, these data suggest that FIAF, similar to other adipocytokines such as adiponectin, may partially exert its function via a truncated form.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Protease-activated receptor 2 and [alpha]-crystallin : interactions and functional implications

Magdeburg, Univ., Fak. für Naturwiss., Diss., 2010

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.