Insulin Receptor and Glucose Transporters in the Mammalian Cochlea.

Insulin receptors are expressed on nerve cells in the mammalian brain, but little is known about insulin signaling and the expression of the insulin receptor (IR) and glucose transporters in the cochlea. We performed immunohistochemistry and gene/protein expression analysis to characterize the expression pattern of the IR and glucose transporters in the mouse organ of Corti (OC). We also performed glucose uptake assays to explore the action of insulin and the effects of pioglitazone, an insulin sensitizer, on glucose transport in the OC. Western blots of protein extracts from OCs showed high expression of IR and glucose transporter 3 (GLUT3). Immunohistochemistry demonstrated that the IR is specifically expressed in the supporting cells of the OC. GLUT3 was found in outer and inner hair cells, in the basilar membrane (BM), the stria vascularis (SV), Reissner's membrane and spiral ganglion neurons (SGN). Glucose transporter 1 (GLUT1) was detected at low levels in the BM, SV and Reissner's membrane, and showed high expression in the SGN. Fluorescence glucose uptake assays revealed that hair cells take up glucose and that addition of insulin (10 nM or 1 µM) approximately doubled the rate of uptake. Pioglitazone conferred a small but nonsignificant potentiation of glucose uptake at the highest concentration of insulin. Gene expression analysis confirmed expression of IR, GLUT1 and GLUT3 mRNA in the OC. Pioglitazone significantly upregulated IR and GLUT1 mRNA expression, which was further increased by insulin. Together, these data show that insulin-stimulated glucose uptake occurs in the OC and may be associated with upregulation of both the IR and GLUT1.

Pasireotide prevents nuclear factor of activated T cells nuclear translocation and acts as a protective agent in aminoglycoside-induced auditory hair cell loss.

Hearing impairment is a global health problem with a high socioeconomic impact. Damage to auditory hair cells (HCs) in the inner ear as a result of aging, disease, trauma, or toxicity, underlies the majority of cases of sensorineural hearing loss. Previously we demonstrated that the Ca2+ -sensitive neuropeptide, somatostatin (SST), and an analog, octreotide, protect HCs from gentamicin-induced cell death in vitro. Aminoglycosides such as gentamicin trigger a calcium ion influx (Ca2+ ) that activates pro-apoptotic signaling cascades in HCs. SST binding to the G-protein-coupled receptors (SSTR1-SSTR5) that are directly linked to voltage-dependent Ca2+ channels inhibits Ca2+ channel activity and associated downstream events. Here, we report that the SST analog pasireotide, a high affinity ligand to SSTRs 1-3, and 5, with a longer half-life than octreotide, prevents gentamicin-induced HC death in the mouse organ of Corti (OC). Explant experiments using OCs derived from SSTR1 and SSTR1and 2 knockout mice, revealed that SSTR2 mediates pasireotide's anti-apoptotic effects. Mechanistically, pasireotide prevented a nuclear translocation of the Ca2+ -sensitive transcription factor, nuclear factor of activated T cells (NFAT), which is ordinarily provoked by gentamicin in OC explants. Direct inhibition of NFAT with 11R-VIVIT also prevented the gentamicin-dependent nuclear translocation of NFAT and apoptosis. Both pasireotide and 11R-VIVIT partially reversed the effects of gentamicin on the expression of downstream survival targets (NMDA receptor and the regulatory subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase, PI3K). These data suggest that SST analogs antagonize aminoglycoside-induced cell death in an NFAT-dependent fashion. SST analogs and NFAT inhibitors may therefore offer new therapeutic possibilities for the treatment of hearing loss.

Effects of pioglitazone and fenofibrate co-administration on bone biomechanics and histomorphometry in ovariectomized rats.

Pioglitazone, the peroxisome proliferator-activated receptor-gamma (PPAR-γ) agonist is an effective therapy for type 2 diabetes, but has been associated with increased risk for bone fracture. Preclinical studies suggest that PPAR-α agonists (e.g., fenofibrate) increase bone mineral density/content, although clinical data on bone effects of fibrates are lacking. We investigated the effects of pioglitazone (10 mg/kg/day) and fenofibrate (25 mg/kg/day) on bone strength and bone histomorphometric parameters in osteopenic ovariectomized (OVX) rats. An additional group of rats received a combination of pioglitazone + fenofibrate to mimic the effects of a dual PPAR-α/γ agonist. The study consisted of a 13-week treatment phase followed by a 6-week treatment-free recovery period. Pioglitazone significantly reduced biomechanical strength at the lumbar spine and femoral neck compared with rats administered fenofibrate. Co-treatment with pioglitazone + fenofibrate had no significant effect on bone strength in comparison with OVX vehicle controls. Histomorphometric analysis of the proximal tibia revealed that pioglitazone suppressed bone formation and increased bone resorption at both cancellous and cortical bone sites relative to OVX vehicle controls. In contrast, fenofibrate did not affect bone resorption and only slightly suppressed bone formation. Discontinuation of pioglitazone treatment, both in the monotherapy and in the combination therapy arms, resulted in restoration of bone formation and resorption rates, demonstrating reversibility of effects. The above data support the concept that dual activation of PPAR-γ and PPAR-α attenuates the negative effects of PPAR-γ agonism on bone strength.

Farnesoid X receptor protects human and murine gastric epithelial cells against inflammation-induced damage.

Bile acids from duodenogastric reflux promote inflammation and increase the risk for gastro-oesophageal cancers. FXR (farnesoid X receptor/NR1H4) is a transcription factor regulated by bile acids such as CDCA (chenodeoxycholic acid). FXR protects the liver and the intestinal tract against bile acid overload; however, a functional role for FXR in the stomach has not been described. We detected FXR expression in the normal human stomach and in GC (gastric cancer). FXR mRNA and protein were also present in the human GC cell lines MKN45 and SNU5, but not in the AGS cell line. Transfection of FXR into AGS cells protected against TNFα (tumour necrosis factor α)-induced cell damage. We identified K13 (keratin 13), an anti-apoptotic protein of desmosomes, as a novel CDCA-regulated FXR-target gene. FXR bound to a conserved regulatory element in the proximal human K13 promoter. Gastric expression of K13 mRNA was increased in an FXR-dependent manner by a chow diet enriched with 1% (w/w) CDCA and by indomethacin (35 mg/kg of body weight intraperitoneal) in C57BL/6 mice. FXR-deficient mice were more susceptible to indomethacin-induced gastric ulceration than their WT (wild-type) littermates. These results suggest that FXR increases the resistance of human and murine gastric epithelial cells to inflammation-mediated damage and may thus participate in the development of GC.

Compounds targeting OSBPL7 increase ABCA1-dependent cholesterol efflux preserving kidney function in two models of kidney disease.

Impaired cellular cholesterol efflux is a key factor in the progression of renal, cardiovascular, and autoimmune diseases. Here we describe a class of 5-arylnicotinamide compounds, identified through phenotypic drug discovery, that upregulate ABCA1-dependent cholesterol efflux by targeting Oxysterol Binding Protein Like 7 (OSBPL7). OSBPL7 was identified as the molecular target of these compounds through a chemical biology approach, employing a photoactivatable 5-arylnicotinamide derivative in a cellular cross-linking/immunoprecipitation assay. Further evaluation of two compounds (Cpd A and Cpd G) showed that they induced ABCA1 and cholesterol efflux from podocytes in vitro and normalized proteinuria and prevented renal function decline in mouse models of proteinuric kidney disease: Adriamycin-induced nephropathy and Alport Syndrome. In conclusion, we show that small molecule drugs targeting OSBPL7 reveal an alternative mechanism to upregulate ABCA1, and may represent a promising new therapeutic strategy for the treatment of renal diseases and other disorders of cellular cholesterol homeostasis.

Hematopoietically expressed homeobox is a target gene of farnesoid X receptor in chenodeoxycholic acid-induced liver hypertrophy.

UNLABELLED: Farnesoid X receptor (FXR/Fxr) is a bile acid-regulated nuclear receptor that promotes hepatic bile acid metabolism, detoxification, and liver regeneration. However, the adaptive pathways under conditions of bile acid stress are not fully elucidated. We found that wild-type but not Fxr knockout mice on diets enriched with chenodeoxycholic acid (CDCA) increase their liver/body weight ratios by 50% due to hepatocellular hypertrophy. Microarray analysis identified Hex (Hematopoietically expressed homeobox), a central transcription factor in vertebrate embryogenesis and liver development, as a novel CDCA- and Fxr-regulated gene. HEX/Hex was also regulated by FXR/Fxr and CDCA in primary mouse hepatocytes and human HepG2 cells. Comparative genomic analysis identified a conserved inverted repeat-1-like DNA sequence within a 300 base pair enhancer element of intron-1 in the human and mouse HEX/Hex gene. A combination of chromatin immunoprecipitation, electromobility shift assay, and transcriptional reporter assays demonstrated that FXR/Fxr binds to this element and mediates HEX/Hex transcriptional activation. CONCLUSION: HEX/Hex is a novel bile acid-induced FXR/Fxr target gene during adaptation of hepatocytes to chronic bile acid exposure.

Effects of LTB4 receptor antagonism on pulmonary inflammation in rodents and non-human primates.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Pioglitazone Ameliorates Gentamicin Ototoxicity by Affecting the TLR and STAT Pathways in the Early Postnatal Organ of Corti.

Noise trauma, infection, and ototoxic drugs are frequent external causes of hearing loss. With no pharmacological treatments currently available, understanding the mechanisms and pathways leading to auditory hair cell (HC) damage and repair is crucial for identifying potential pharmacological targets. Prior research has implicated increased reactive oxygen species (ROS) and inflammation as general mechanisms of hearing loss common to diverse causes. Novel targets of these two key mechanisms of auditory damage may provide new paths toward the prevention and treatment of hearing loss. Pioglitazone, an oral antidiabetic drug from the class of thiazolidinediones, acts as an agonist of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) and is involved in the regulation of lipid and glucose metabolism. PPAR-γ is an important player in repressing the expression of inflammatory cytokines and signaling molecules. We evaluated the effects of pioglitazone in the mouse Organ of Corti (OC) explants to characterize its influence on signaling pathways involved in auditory HC damage. The OC explants was cultured with pioglitazone, gentamicin, or a combination of both agents. Pioglitazone treatment resulted in significant repression of interferon (IFN)-α and -gamma pathways and downstream cytokines, as assessed by RNA sequencing and quantitative PCR gene expression assays. More detailed investigation at the single gene and protein level showed that pioglitazone mediated its anti-inflammatory effects through alterations of the Toll-like receptor (TLR) and STAT pathways. Together, these results indicate that pioglitazone significantly represses IFN and TLR in the cochlea, dampening the activity of gentamicin-induced pathways. These data support our previous results demonstrating significant protection of auditory HCs in the OC explants exposed to pioglitazone and other PPAR-targeted agents.

Dual PPARα/γ agonist aleglitazar confers stroke protection in a model of mild focal brain ischemia in mice.

Peroxisome proliferator-activated receptors (PPARs) control the expression of genes involved in glucose homeostasis, lipid metabolism, inflammation, and cell differentiation. Here, we analyzed the effects of aleglitazar, a dual PPARα and PPARγ agonist with balanced affinity for either subtype, on subacute stroke outcome. Healthy young adult mice were subjected to transient 30 min middle cerebral artery occlusion (MCAo)/reperfusion. Daily treatment with aleglitazar was begun on the day of MCAo and continued until sacrifice. Blood glucose measurements and lipid profile did not differ between mice receiving aleglitazar and mice receiving vehicle after MCAo. Aleglitazar reduced the size of the ischemic lesion as assessed using NeuN immunohistochemistry on day 7. Sensorimotor performance on the rotarod was impaired during the first week after MCAo, an effect that was significantly attenuated by treatment with aleglitazar. Smaller lesion volume in mice treated with aleglitazar was accompanied by a decrease in mRNA transcription of IL-1ß, Vcam-1, and Icam-1, suggesting that reduced proinflammatory signaling and reduced vascular inflammation in the ischemic hemisphere contribute to the beneficial effects of aleglitazar during the first week after stroke. Further experiments in primary murine microglia confirmed that aleglitazar reduces key aspects of microglia activation including NO production, release of proinflammatory cytokines, migration, and phagocytosis. In aggregate, a brief course of PPARα/γ agonist aleglitazar initiated post-event affords stroke protection and functional recovery in a model of mild brain ischemia. Our data underscores the theme of delayed injury processes such as neuroinflammation as promising therapeutic targets in stroke. KEY MESSAGES: PPARα/γ agonist aleglitazar improves stroke outcome after transient brain ischemia. Aleglitazar attenuates inflammatory responses in post-ischemic brain. Aleglitazar reduces microglia migration, phagocytosis, and release of cytokines. Beneficial effects of aleglitazar independent of glucose regulation. Aleglitazar provides extended window of opportunity for stroke treatment.

Pioglitazone Represents an Effective Therapeutic Target in Preventing Oxidative/Inflammatory Cochlear Damage Induced by Noise Exposure.

Recent progress in hearing loss research has provided strong evidence for the imbalance of cellular redox status and inflammation as common predominant mechanisms of damage affecting the organ of Corti including noise induced hearing loss. The discovery of a protective molecule acting on both mechanisms is challenging. The thiazolidinediones, a class of antidiabetic drugs including pioglitazone and rosiglitazone, have demonstrated diverse pleiotrophic effects in many tissues where they exhibit anti-inflammatory, anti-proliferative, tissue protective effects and regulators of redox balance acting as agonist of peroxisome proliferator-activated receptors (PPARs). They are members of the family of ligand regulated nuclear hormone receptors that are also expressed in several cochlear cell types, including the outer hair cells. In this study, we investigated the protective capacity of pioglitazone in a model of noise-induced hearing loss in Wistar rats and the molecular mechanisms underlying this protective effects. Specifically, we employed a formulation of pioglitazone in a biocompatible thermogel providing rapid, uniform and sustained inner ear drug delivery via transtympanic injection. Following noise exposure (120 dB, 10 kHz, 1 h), different time schedules of treatment were employed: we explored the efficacy of pioglitazone given immediately (1 h) or at delayed time points (24 and 48 h) after noise exposure and the time course and extent of hearing recovery were assessed. We found that pioglitazone was able to protect auditory function at the mid-high frequencies and to limit cell death in the cochlear basal/middle turn, damaged by noise exposure. Immunofluorescence and western blot analysis provided evidence that pioglitazone mediates both anti-inflammatory and anti-oxidant effects by decreasing NF-κB and IL-1ß expression in the cochlea and opposing the oxidative damage induced by noise insult. These results suggest that intratympanic pioglitazone can be considered a valid therapeutic strategy for attenuating noise-induced hearing loss and cochlear damage, reducing inflammatory signaling and restoring cochlear redox balance.

Effects of peroxisome proliferator activated receptors (PPAR)-γ and -α agonists on cochlear protection from oxidative stress.

Various insults cause ototoxicity in mammals by increasing oxidative stress leading to apoptosis of auditory hair cells (HCs). The thiazolidinediones (TZDs; e.g., pioglitazone) and fibrate (e.g., fenofibrate) drugs are used for the treatment of diabetes and dyslipidemia. These agents target the peroxisome proliferator-activated receptors, PPARγ and PPARα, which are transcription factors that influence glucose and lipid metabolism, inflammation, and organ protection. In this study, we explored the effects of pioglitazone and other PPAR agonists to prevent gentamicin-induced oxidative stress and apoptosis in mouse organ of Corti (OC) explants. Western blots showed high levels of PPARγ and PPARα proteins in mouse OC lysates. Immunofluorescence assays indicated that PPARγ and PPARα proteins are present in auditory HCs and other cell types in the mouse cochlea. Gentamicin treatment induced production of reactive oxygen species (ROS), lipid peroxidation, caspase activation, PARP-1 cleavage, and HC apoptosis in cultured OCs. Pioglitazone mediated its anti-apoptotic effects by opposing the increase in ROS induced by gentamicin, which inhibited the subsequent formation of 4-hydroxy-2-nonenal (4-HNE) and activation of pro-apoptotic mediators. Pioglitazone mediated its effects by upregulating genes that control ROS production and detoxification pathways leading to restoration of the reduced:oxidized glutathione ratio. Structurally diverse PPAR agonists were protective of HCs. Pioglitazone (PPARγ-specific), tesaglitazar (PPARγ/α-specific), and fenofibric acid (PPARα-specific) all provided >90% protection from gentamicin toxicity by regulation of overlapping subsets of genes controlling ROS detoxification. This study revealed that PPARs play important roles in the cochlea, and that PPAR-targeting drugs possess therapeutic potential as treatment for hearing loss.

Induction of cytochrome P450 3A4 and P-glycoprotein by the isoxazolyl-penicillin antibiotic flucloxacillin.

Clinical findings indicate that co-administration of the isoxazolyl-penicillin flucloxacillin with cyclosporine may reduce the plasma concentrations of cyclosporine. We have explored in the present study if induction of cytochrome P450 3A4 or P-glycoprotein may offer a mechanistic explanation of the observed effects. Flucloxacillin is neither an inhibitor nor a substrate of drug metabolizing cytochrome P450 isoenzymes (CYP3A4, 1A2, 2C9, 2C19 and 2D6) or P-glycoprotein as shown by an in vitro assay for CYP inhibition, a fluorescent indicator assay for P-glycoprotein inhibition and a functional P-glycoprotein ATPase assay. However, incubation of human LS 180 colorectal adenocarcinoma cells with flucloxacillin led to a dose-dependent induction of MDR1 as well as of CYP3A4 mRNA, which was also confirmed in primary human hepatocytes. At high concentrations, flucloxacillin activated the human Pregnane-X-Receptor, PXR, a ligand-dependent transcription factor that is the target of many drugs that induce CYP3A4, with consequences for the metabolism of other drugs. Liver microsomes from control rats or rats, which received for 3 consecutive days 100 mg/kg of oral flucloxacillin, were used to study the metabolism and metabolite pattern of midazolam, a model substrate of CYP 3A4. There was a trend towards a higher intrinsic microsomal clearance of midazolam using microsomes from flucloxacillin treated rats. In addition, there was a significant increase in the formation of the principal midazolam metabolites 1-hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy midazolam as compared to controls. These findings indicate that flucloxacillin has the potential to induce expression of both CYP3A4 as well as P-glycoprotein, most likely through activation of the nuclear hormone receptor PXR. This would offer an explanation for the observed clinical drug-drug interactions between the antibiotic and cyclosporine.

Efficacy of SSH PCR in isolating differentially expressed genes.

BACKGROUND: Suppression Subtractive Hybridization PCR (SSH PCR) is a sophisticated cDNA subtraction method to enrich and isolate differentially expressed genes. Despite its popularity, the method has not been thoroughly studied for its practical efficacy and potential limitations. RESULTS: To determine the factors that influence the efficacy of SSH PCR, a theoretical model, under the assumption that cDNA hybridization follows the ideal second kinetic order, is proposed. The theoretical model suggests that the critical factor influencing the efficacy of SSH PCR is the concentration ratio (R) of a target gene between two cDNA preparations. It preferentially enriches "all or nothing" differentially expressed genes, of which R is infinite, and strongly favors the genes with large R. The theoretical predictions were validated by our experiments. In addition, the experiments revealed some practical limitations that are not obvious from the theoretical model. For effective enrichment of differentially expressed genes, it requires fractional concentration of a target gene to be more than 0.01% and concentration ratio to be more than 5 folds between two cDNA preparations. CONCLUSION: Our research demonstrated theoretical and practical limitations of SSH PCR, which could be useful for its experimental design and interpretation.

Minireview: Challenges and opportunities in development of PPAR agonists.

The clinical impact of the fibrate and thiazolidinedione drugs on dyslipidemia and diabetes is driven mainly through activation of two transcription factors, peroxisome proliferator-activated receptors (PPAR)-α and PPAR-γ. However, substantial differences exist in the therapeutic and side-effect profiles of specific drugs. This has been attributed primarily to the complexity of drug-target complexes that involve many coregulatory proteins in the context of specific target gene promoters. Recent data have revealed that some PPAR ligands interact with other non-PPAR targets. Here we review concepts used to develop new agents that preferentially modulate transcriptional complex assembly, target more than one PPAR receptor simultaneously, or act as partial agonists. We highlight newly described on-target mechanisms of PPAR regulation including phosphorylation and nongenomic regulation. We briefly describe the recently discovered non-PPAR protein targets of thiazolidinediones, mitoNEET, and mTOT. Finally, we summarize the contributions of on- and off-target actions to select therapeutic and side effects of PPAR ligands including insulin sensitivity, cardiovascular actions, inflammation, and carcinogenicity.

Examining the safety of PPAR agonists - current trends and future prospects.

INTRODUCTION: The peroxisome proliferator-activated receptor (PPAR)-α and -γ agonists, fibrates and glitazones, are effective treatments for dyslipidemia and type 2 diabetes mellitus, respectively, but exhibit class-related, as well as compound-specific safety characteristics. AREAS COVERED: This article reviews the profiles of PPAR-α, PPAR-γ, and dual PPAR-α/γ agonists with regard to class-related and compound-specific efficacy and adverse effects. We explore how learnings from first-generation drugs are being applied to develop safer PPAR-targeted therapies. EXPERT OPINION: The finding that rosiglitazone may increase risk for cardiovascular events has led to regulatory guidelines requiring demonstration of cardiovascular safety in appropriate outcome trials for new type 2 diabetes mellitus drugs. The emerging data on the possibly increased risk of bladder cancer with pioglitazone may prompt the need for post-approval safety studies for new drugs. Since PPAR-α and -γ affect key cardiometabolic risk factors (diabetic dyslipidemia, insulin resistance, hyperglycemia, and inflammation) in a complementary fashion, combining their benefits has emerged as a particularly attractive option. New PPAR-targeted therapies that balance the relative potency and/or activity toward PPAR-α and -γ have shown promise in retaining efficacy while reducing potential side effects.

6-Alkoxy-5-aryl-3-pyridinecarboxamides, a new series of bioavailable cannabinoid receptor type 1 (CB1) antagonists including peripherally selective compounds.

We identified 6-alkoxy-5-aryl-3-pyridinecarboxamides as potent CB1 receptor antagonists with high selectivity over CB2 receptors. The series was optimized to reduce lipophilicity compared to rimonabant to achieve peripherally active molecules with minimal central effects. Several compounds that showed high plasma exposures in rats were evaluated in vivo to probe the contribution of central vs peripheral CB1 agonism to metabolic improvement. Both rimonabant and 14g, a potent brain penetrant CB1 receptor antagonist, significantly reduced the rate of body weight gain. However, 14h, a molecule with markedly reduced brain exposure, had no significant effect on body weight. PK studies confirmed similarly high exposure of both 14h and 14g in the periphery but 10-fold lower exposure in the brain for 14h. On the basis of these data, which are consistent with reported effects in tissue-specific CB1 receptor KO mice, we conclude that the metabolic benefits of CB1 receptor antagonists are primarily centrally mediated as originally believed.

Comparative molecular profiling of the PPARα/γ activator aleglitazar: PPAR selectivity, activity and interaction with cofactors.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors that control the expression of genes involved in a variety of physiologic processes, through heterodimerization with retinoid X receptor and complex formation with various cofactors. Drugs or treatment regimens that combine the beneficial effects of PPARα and γ agonism present an attractive therapeutic strategy to reduce cardiovascular risk factors. Aleglitazar is a dual PPARα/γ agonist currently in phase III clinical development for the treatment of patients with type 2 diabetes mellitus who recently experienced an acute coronary event. The potency and efficacy of aleglitazar was evaluated in a head-to-head comparison with other PPARα, γ and δ ligands. A comprehensive, 12-concentration dose-response analysis using a cell-based assay showed aleglitazar to be highly potent, with EC(50) values of 5 nM and 9 nM for PPARα and PPARγ, respectively. Cofactor recruitment profiles confirmed that aleglitazar is a potent and balanced activator of PPARα and γ. The efficacy and potency of aleglitazar are discussed in relation to other dual PPARα/γ agonists, in context with the published X-ray crystal structures of both PPARα and γ.

Comparative transcriptional network modeling of three PPAR-α/γ co-agonists reveals distinct metabolic gene signatures in primary human hepatocytes.

AIMS: To compare the molecular and biologic signatures of a balanced dual peroxisome proliferator-activated receptor (PPAR)-α/γ agonist, aleglitazar, with tesaglitazar (a dual PPAR-α/γ agonist) or a combination of pioglitazone (Pio; PPAR-γ agonist) and fenofibrate (Feno; PPAR-α agonist) in human hepatocytes. METHODS AND RESULTS: Gene expression microarray profiles were obtained from primary human hepatocytes treated with EC(50)-aligned low, medium and high concentrations of the three treatments. A systems biology approach, Causal Network Modeling, was used to model the data to infer upstream molecular mechanisms that may explain the observed changes in gene expression. Aleglitazar, tesaglitazar and Pio/Feno each induced unique transcriptional signatures, despite comparable core PPAR signaling. Although all treatments inferred qualitatively similar PPAR-α signaling, aleglitazar was inferred to have greater effects on high- and low-density lipoprotein cholesterol levels than tesaglitazar and Pio/Feno, due to a greater number of gene expression changes in pathways related to high-density and low-density lipoprotein metabolism. Distinct transcriptional and biologic signatures were also inferred for stress responses, which appeared to be less affected by aleglitazar than the comparators. In particular, Pio/Feno was inferred to increase NFE2L2 activity, a key component of the stress response pathway, while aleglitazar had no significant effect. All treatments were inferred to decrease proliferative signaling. CONCLUSIONS: Aleglitazar induces transcriptional signatures related to lipid parameters and stress responses that are unique from other dual PPAR-α/γ treatments. This may underlie observed favorable changes in lipid profiles in animal and clinical studies with aleglitazar and suggests a differentiated gene profile compared with other dual PPAR-α/γ agonist treatments.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor γ through spatial relocalization at helix 7 of its ligand-binding domain.

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that promotes differentiation and cell survival in the stomach. PPARγ upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPARγ is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPARγ signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPARγ and enhanced nuclear translocation and ligand-independent transcription of PPARγ target genes. In contrast, Cav1 overexpression sequestered PPARγ in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPARγ's ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPARγ and to inhibit cell proliferation. Ligand-activated PPARγ also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPARγ regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPARγ to ligands, limiting proliferation of gastric epithelial cells.

Recent progress in liver X receptor-selective modulators.

The liver X receptors LXRalpha and LXRbeta regulate the expression of genes promoting cellular cholesterol efflux and the formation of HDL particles, and are atheroprotective. However, LXRalpha and LXRbeta also regulate the expression of genes involved in lipogenesis and hypertriglyceridemia. The identification of efficacious LXR modulators that are devoid of undesirable side effects remains a significant challenge for drug development. The X-ray structures of many LXR protein/small-molecule complexes have revealed that the ligand-binding pockets of LXRalpha and LXRbeta, despite being highly conserved, are large and flexible; these properties have allowed the design of a wide range of ligands with varied selectivity profiles. This review discusses the latest medicinal chemistry strategies used to derive novel LXR modulators with the potential for enhanced therapeutic utility and safety, and summarizes the current status of compounds that have progressed into clinical development.