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Peroxisome proliferator-activated receptors (PPARs) act as metabolic sensors and central regulators of fat and glucose homeostasis (1). Furthermore, PPARγ has been implicated as major catabolic regulator of bone mass in [...]

Peroxisome proliferator-activated receptor (PPAR) [delta] is a member of the nuclear hormone receptor superfamily. PPAR[delta] may ameliorate metabolic diseases such as obesity and diabetes. However, PPAR[delta]'s role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPAR[delta] decreases intestinal adenoma growth in [Apc.sup.Min/+] mice and inhibits tumor-promoting effects of a PPAR[delta] agonist GW501516. More importantly, we found that activation of PPAR[delta] up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPAR[delta] agonists for treatment of metabolic disorders in patients who are at high risk for colorectal cancer, but also support the rationale for developing PPAR[delta] antagonists for prevention and/or treatment of cancer. apoptosis | colorectal cancer

The peroxisome proliferator-activated receptor [gamma] (PPAR[gamma]) mediates the activity of the insulin-sensitizing thiazolidinediones and plays an important role in adipocyte differentiation and fat accretion. The analysis of PPAR[gamma], functions in mature adipocytes is precluded by lethality of PPAR[[gamma].sup.-/-] fetuses and tetraploid-rescued pups. Therefore we have selectively ablated PPAR[gamma] in adipocytes of adult mice by using the tamoxifen-dependent Cre-E[R.sup.T2] recombination system. We show that mature PPAR[gamma]-null white and brown adipocytes die within a few days and are replaced by newly formed PPAR[gamma]-positive adipocytes, demonstrating that PPAR[gamma], is essential for the in vivo survival of mature adipocytes, in addition to its well established requirement for their differentiation. Our data suggest that potent PPAR[gamma] antagonists could be used to acutely reduce obesity. conditional somatic mutagenesis | tamoxifen-dependent | adipocyte maintenance | obesity | nuclear receptor

Roles of PPAR(beta)/(delta) in keratinocyte inflammation response are discussed. Immediately after skin injury comes release of inflammatory signals. Using cultures of primary keratinocytes from wild-type and PPAR(beta)/(delta)(super.-/-) mice has been helpful in finding that such signals induce keratinocyte differentiation through influencing TNF-alpha and IFN-gamma.

Author(s): Sander Kersten; Béatrice Desvergne; Walter Wahli (corresponding author) The PPARs were first cloned as the nuclear receptors that mediate the effects of synthetic compounds called peroxisome proliferators on gene [...]

Author(s): Beatrice Desvergne [1] The identification of nuclear receptors defined a new paradigm: small lipophilic hormones specifically bind their cognate receptor and the complex directly regulates the expression of multiple [...]

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EGVEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ+/- and PPAR mice. Major processes of placental development that are known to be controlled by PPAR, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ+/- mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion. [ABSTRACT FROM AUTHOR]

The article discusses a study which investigated the effect of dietary fatty acids on cardiac gene expression and examines the functional consequences. The researchers used GW501516 from Alexis, Wy14643 from Eagle Picher Technologies laboratories and Trilinolein from Larodan Free Chemicals. A single oral gavage of synthetic triglycerides (TGs) were given to SV129 mice. It was found that stimulation of cardiac Angt14 gene expression by dietary fatty acids protects the heart against lipid overload.

Peroxisome proliferator-activated receptor (PPAR) δ is a member of the nuclear hormone receptor superfamily. PPARδ may ameliorate metabolic diseases such as obesity and diabetes. However, PPARδ's role in colorectal carcinogenesis remains controversial. Here, we present genetic and pharmacologic evidence demonstrating that deletion of PPAR6 decreases intestinal adenoma growth in ApcMin/+ mice and inhibits tumor-promoting effects of a PPARδ agonist GW501516. More importantly, we found that activation of PPAR& up-regulated VEGF in colon carcinoma cells. VEGF directly promotes colon tumor epithelial cell survival through activation of PI3K-Akt signaling. These results not only highlight concerns about the use of PPAR6 agonists for treatment of metabolic disorders in patients who are at high risk for colorectat cancer, but also support the rationale for developing PPARδ antagonists for prevention and/or treatment of cancer. [ABSTRACT FROM AUTHOR]

Studies the peroxisome-proliferator-activated receptors (PPAR) and cancers. Functions of PPAR, nuclear hormone receptors that mediate the effects of fatty acid and their derivatives; Regulation of cell proliferation, differential and survival; Controlling carcinogenesis in various tissues; Links between each PPAR isotype and carcinogenesis.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors that belong to the steroid/thyroid/retinoic acid receptor superfamily. All their characterized target genes encode proteins that participate in lipid homeostasis. The recent finding that antidiabetic thiazolidinediones and adipogenic prostanoids are ligands of one of the PPARs reveals a novel signaling pathway that directly links these compounds to processes involved in glucose homeostasis and lipid metabolism including adipocyte differentiation. A detailed understanding of this pathway could designate PPARs as targets for the development of novel efficient treatments for several metabolic disorders. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor α(PPARα) 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 PPARα target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARα, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARβ/δ during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARα. Binding of PPARβ/δ to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARβ/δ 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 PPARβ/δ. In contrast, the data did not support activation of PPARα by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARβ/δ target genes and show that upregulation of gene expression by PPARβ/δ is sensitive to plasma FFA levels. In contrast, this is not the case for PPARα, revealing a novel mechanism for functional differentiation between PPARs. [ABSTRACT FROM AUTHOR]

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)β/δ occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARβ/δ expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-β1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARβ/δ accelerate wound closure. The results reveal a dual role of TGF-β1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARβ/δ expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-β1 at wound sites. [ABSTRACT FROM AUTHOR]

Abstract: The peroxisome proliferator-activated receptors (PPAR) α, β/δ and γ belong to the nuclear hormone receptor superfamily. As ligand-activated receptors, they form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs). PPARs are activated by fatty acids and their derivatives, whereas RXR is activated by 9-cis retinoic acid. This heterodimer binds to peroxisome proliferator response elements (PPRE) residing in target genes and stimulates their expression. Recent reports now indicate that PPARs and RXRs can function independently, in the absence of a hetero-partner, to modulate gene expression. Of importance, these non-canonical mechanisms underscore the impact of both cofactors and DNA on gene expression. Furthermore, these different mechanisms reveal the increasing repertoire of PPAR ‘target’ genes that now encompasses non-PPREs containing genes. It is also becoming apparent that understanding the regulation of PPAR expression and activity, can itself have a significant influence on how the expression of subgroups of target genes is studied and integrated in current knowledge. [Copyright &y& Elsevier]

Activation of the transcription factor PPARg by the n-3 fatty acid docosahexaenoic acid (DHA) is implicated in controlling proinflammatory cytokine secretion, but the intracellular signaling pathways engaged by PPARγ are incompletely characterized. Here, we identify the adapter-encoding gene SOCS3 as a critical transcriptional target of PPARγ. SOCS3 promoter binding and gene transactivation by PPARγ was associated with a repression in differentiation of proinflammatory T-helper (TH)17 cells. Accordingly, TH17 cells induced in vitro displayed increased SOCS3 expression and diminished capacity to produce interleukin (IL)-17 following activation of PPARγ by DHA. Furthermore, naïve CD4 T cells derived from mice fed a DHA-enriched diet displayed less capability to differentiate into TH17 cells. In two different mouse models of cancer, DHA prevented tumor outgrowth and angiogenesis in an IL-17-dependent manner. Altogether, our results uncover a novel molecular pathway by which PPARγ-induced SOCS3 expression prevents IL-17-mediated cancer growth. [ABSTRACT FROM AUTHOR]

Successful pregnancy depends on well coordinated developmental events involving both maternal and embryonic components. Although a host of signaling pathways participate in implantation, decidualization, and placentation, whether there is a common molecular link that coordinates these processes remains unknown. By exploiting genetic, molecular, pharmacological, and physiological approaches, we show here that the nuclear transcription factor peroxisome proliferator-activated receptor (PPAR) δ plays a central role at various stages of pregnancy, whereas maternal PPARδ is critical to implantation and decidualization, and embryonic PPARδ is vital for placentation. Using trophoblast stem cells, we further elucidate that a reciprocal relationship between PPARδ-AKT and leukemia inhibitory factor-STAT3 signaling pathways serves as a cell lineage sensor to direct trophoblast cell fates during placentation. This novel finding of stage-specific integration of maternal and embryonic PPARδ signaling provides evidence that PPARδ is a molecular link that coordinates implantation, decidualization, and placentation crucial to pregnancy success. This study is clinically relevant because deferral of on time implantation leads to spontaneous pregnancy loss, and defective trophoblast invasion is one cause of preeclampsia in humans. [ABSTRACT FROM AUTHOR]

The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARγ and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARγ target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARγ coregulators that includes Med1 and PGC-1α, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation. [ABSTRACT FROM AUTHOR]

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor α (PPARα) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARα helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARα helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARα transcription activation potential. Finally, we disclose important functions of residues in PPARα AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARα constitutive activity, without changing PPARα ligand-dependent activation potential. [ABSTRACT FROM AUTHOR]

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor α (PPARα) of the mouse GRHPR gene in liver. Mice fed with a PPARα ligand or in which PPARα activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARα present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARα ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARα-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARα regulation. Overall, these data indicate a species-specific regulation by PPARα of GRHPR, a key gene of the glyoxylate cycle. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-α or PPAR-δ activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-γ activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-γ activation stimulates keratinocyte differentiation. Additionally, PPAR-γ activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-γ activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-γ activators are mediated by PPAR-γ, we next examined animals deficient in PPAR-γ. Mice with a deficiency of PPAR-γ specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-γ-deficient epidermis. Although PPAR-γ activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-γ-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-γ activators is mediated by PPAR-γ. In contrast, PPAR-γ activators inhibited inflammation in both PPAR-γ-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-γ activators does not require PPAR-γ in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-γ activators maybe useful in the treatment of cutaneous disorders. [ABSTRACT FROM AUTHOR]

Rationale: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart., Objective: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences., Methods and Results: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding., Conclusions: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

Little is known about the role of the transcription factor peroxisome proliferator-activated receptor (PPAR) beta/delta in liver. Here we set out to better elucidate the function of PPARbeta/delta in liver by comparing the effect of PPARalpha and PPARbeta/delta deletion using whole genome transcriptional profiling and analysis of plasma and liver metabolites. In fed state, the number of genes altered by PPARalpha and PPARbeta/delta deletion was similar, whereas in fasted state the effect of PPARalpha deletion was much more pronounced, consistent with the pattern of gene expression of PPARalpha and PPARbeta/delta. Minor overlap was found between PPARalpha- and PPARbeta/delta-dependent gene regulation in liver. Pathways upregulated by PPARbeta/delta deletion were connected to innate immunity and inflammation. Pathways downregulated by PPARbeta/delta deletion included lipoprotein metabolism and various pathways related to glucose utilization, which correlated with elevated plasma glucose and triglycerides and reduced plasma cholesterol in PPARbeta/delta-/- mice. Downregulated genes that may underlie these metabolic alterations included Pklr, Fbp1, Apoa4, Vldlr, Lipg, and Pcsk9, which may represent novel PPARbeta/delta target genes. In contrast to PPARalpha-/- mice, no changes in plasma free fatty acid, plasma beta-hydroxybutyrate, liver triglycerides, and liver glycogen were observed in PPARbeta/delta-/- mice. Our data indicate that PPARbeta/delta governs glucose utilization and lipoprotein metabolism and has an important anti-inflammatory role in liver. Overall, our analysis reveals divergent roles of PPARalpha and PPARbeta/delta in regulation of gene expression in mouse 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 mode of action of nuclear receptors in living cells is an actively investigated field but much remains hypothetical due to the lack, until recently, of methods allowing the assessment of molecular mechanisms in vivo. However, these last years, the development of fluorescence microscopy methods has allowed initiating the dissection of the molecular mechanisms underlying gene regulation by nuclear receptors directly in living cells or organisms. Following our analyses on peroxisome proliferator activated receptors (PPARs) in living cells, we discuss here the different models arising from the use of these tools, that attempt to link mobility, DNA binding or chromatin interaction, and transcriptional activity.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.