Synthesis and in vitro cytotoxicity of benzoxazole-based PPARα/γ antagonists in colorectal cancer cell lines.

A series of benzoxazole-based amides and sulfonamides were synthesized and evaluated for their human peroxisome proliferator-activated receptor (PPAR)α and PPARγ activity. All tested compounds showed a dual antagonist profile on both PPAR subtypes; based on transactivation results, seven compounds were selected to test their in vitro antiproliferative activity in a panel of eight cancer cell lines with different expression rates of PPARα and PPARγ. 3f was identified as the most cytotoxic compound, with higher potency in the colorectal cancer cell lines HT-29 and HCT116. Compound 3f induced a concentration-dependent activation of caspases and cell-cycle arrest in both colorectal cancer models. Docking experiments were also performed to shed light on the putative binding mode of this novel class of dual PPARα/γ antagonists.

First-in-human Phase I Trial of TPST-1120, an Inhibitor of PPARα, as Monotherapy or in Combination with Nivolumab, in Patients with Advanced Solid Tumors.

PURPOSE: TPST-1120 is a first-in-class oral inhibitor of peroxisome proliferator-activated receptor α (PPARα), a fatty acid ligand-activated transcription factor that regulates genes involved in fatty acid oxidation, angiogenesis, and inflammation, and is a novel target for cancer therapy. TPST-1120 displayed antitumor activity in xenograft models and synergistic tumor reduction in syngeneic tumor models when combined with anti-PD-1 agents. EXPERIMENTAL DESIGN: This phase I, open-label, dose-escalation study (NCT03829436) evaluated TPST-1120 as monotherapy in patients with advanced solid tumors and in combination with nivolumab in patients with renal cell carcinoma (RCC), cholangiocarcinoma (CCA), or hepatocellular carcinoma. Objectives included evaluation of safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity (RECIST v1.1). RESULTS: A total of 39 patients enrolled with 38 treated (20 monotherapy, 18 combination; median 3 prior lines of therapy). The most common treatment-related adverse events (TRAE) were grade 1-2 nausea, fatigue, and diarrhea. No grade 4-5 TRAEs or dose-limiting toxicities were reported. In the monotherapy group, 53% (10/19) of evaluable patients had a best objective response of stable disease. In the combination group, 3 patients had partial responses, for an objective response rate of 20% (3/15) across all doses and 30% (3/10) at TPST-1120 ≥400 mg twice daily. Responses occurred in 2 patients with RCC, both of whom had previously progressed on anti-PD-1 therapy, and 1 patient with late-line CCA. CONCLUSIONS: TPST-1120 was well tolerated as monotherapy and in combination with nivolumab and the combination showed preliminary evidence of clinical activity in PD-1 inhibitor refractory and immune compromised cancers. SIGNIFICANCE: TPST-1120 is a first-in-class oral inhibitor of PPARα, whose roles in metabolic and immune regulation are implicated in tumor proliferation/survival and inhibition of anticancer immunity. This first-in-human study of TPST-1120 alone and in combination with nivolumab supports proof-of-concept of PPARα inhibition as a target of therapeutic intervention in solid tumors.

Selective PPARα Modulator Pemafibrate and Sodium-Glucose Cotransporter 2 Inhibitor Tofogliflozin Combination Treatment Improved Histopathology in Experimental Mice Model of Non-Alcoholic Steatohepatitis.

Ballooning degeneration of hepatocytes is a major distinguishing histological feature of non-alcoholic steatosis (NASH) progression that can lead to cirrhosis and hepatocellular carcinoma (HCC). In this study, we evaluated the effect of the selective PPARα modulator (SPPARMα) pemafibrate (Pema) and sodium-glucose cotransporter 2 (SGLT2) inhibitor tofogliflozin (Tofo) combination treatment on pathological progression in the liver of a mouse model of NASH (STAM) at two time points (onset of NASH progression and HCC survival). At both time points, the Pema and Tofo combination treatment significantly alleviated hyperglycemia and hypertriglyceridemia. The combination treatment significantly reduced ballooning degeneration of hepatocytes. RNA-seq analysis suggested that Pema and Tofo combination treatment resulted in an increase in glyceroneogenesis, triglyceride (TG) uptake, lipolysis and liberated fatty acids re-esterification into TG, lipid droplet (LD) formation, and Cidea/Cidec ratio along with an increased number and reduced size and area of LDs. In addition, combination treatment reduced expression levels of endoplasmic reticulum stress-related genes (Ire1a, Grp78, Xbp1, and Phlda3). Pema and Tofo treatment significantly improved survival rates and reduced the number of tumors in the liver compared to the NASH control group. These results suggest that SPPARMα and SGLT2 inhibitor combination therapy has therapeutic potential to prevent NASH-HCC progression.

Upregulation of peroxisome proliferator-activated receptor-α and the lipid metabolism pathway promotes carcinogenesis of ampullary cancer.

Ampullary cancer is a rare periampullary cancer currently with no targeted therapeutic agent. It is important to develop a deeper understanding of the carcinogenesis of ampullary cancer. We attempted to explore the characteristics of ampullary cancer in our dataset and a public database, followed by a search for potential drugs. We used a bioinformatics pipeline to analyze complementary (c)DNA microarray data of ampullary cancer and surrounding normal duodenal tissues from five patients. A public database from the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) was applied for external validation. Bioinformatics tools used included the Gene Set Enrichment Analysis (GSEA), Database for Annotation, Visualization and Integrated Discovery (DAVID), MetaCore, Kyoto Encyclopedia of Genes and Genomes (KEGG), Hallmark, BioCarta, Reactome, and Connectivity Map (CMap). In total, 9097 genes were upregulated in the five ampullary cancer samples compared to normal duodenal tissues. From the MetaCore analysis, genes of peroxisome proliferator-activated receptor alpha (PPARA) and retinoid X receptor (RXR)-regulated lipid metabolism were overexpressed in ampullary cancer tissues. Further a GSEA of the KEGG, Hallmark, Reactome, and Gene Ontology databases revealed that PPARA and lipid metabolism-related genes were enriched in our specimens of ampullary cancer and in the NCBI GSE39409 database. Expressions of PPARA messenger (m)RNA and the PPAR-α protein were higher in clinical samples and cell lines of ampullary cancer. US Food and Drug Administration (FDA)-approved drugs, including alvespimycin, trichostatin A (a histone deacetylase inhibitor), and cytochalasin B, may have novel therapeutic effects in ampullary cancer patients as predicted by the CMap analysis. Trichostatin A was the most potent agent for ampullary cancer with a half maximal inhibitory concentration of < 0.3 µM. According to our results, upregulation of PPARA and lipid metabolism-related genes are potential pathways in the carcinogenesis and development of ampullary cancer. Results from the CMap analysis suggested potential drugs for patients with ampullary cancer.

Discovery and Structure-Activity Relationships of Novel Template, Truncated 1'-Homologated Adenosine Derivatives as Pure Dual PPARγ/δ Modulators.

Following our report that A3 adenosine receptor (AR) antagonist 1 exhibited a polypharmacological profile as a dual modulator of peroxisome proliferator-activated receptor (PPAR)γ/δ, we discovered a new template, 1'-homologated adenosine analogues 4a-4t, as dual PPARγ/δ modulators without AR binding. Removal of binding affinity to A3AR was achieved by 1'-homologation, and PPARγ/δ dual modulation was derived from the structural similarity between the target nucleosides and PPAR modulator drug, rosiglitazone. All the final nucleosides were devoid of AR-binding affinity and exhibited high binding affinities to PPARγ/δ but lacked PPARα binding. 2-Cl derivatives exhibited dual receptor-binding affinity to PPARγ/δ, which was absent for the corresponding 2-H derivatives. 2-Propynyl substitution prevented PPARδ-binding affinity but preserved PPARγ affinity, indicating that the C2 position defines a pharmacophore for selective PPARγ ligand designs. PPARγ/δ dual modulators functioning as both PPARγ partial agonists and PPARδ antagonists promoted adiponectin production, suggesting their therapeutic potential against hypoadiponectinemia-associated cancer and metabolic diseases.

Silencing of peroxisome proliferator-activated receptor-alpha alleviates myocardial injury in diabetic cardiomyopathy by downregulating 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 expression.

Diabetic cardiomyopathy (DCM) is a cardiac disorder, which affects around 12% diabetic patients, resulting in overt heart death. Our initial bioinformatic analysis identified the differentially expressed gene 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2 (HMGCS2) in DCM, which may be activated by peroxisome proliferator-activated receptor-alpha (PPARα) based on previous evidence. Therefore, the present study aims to explore the effect of PPARα on the development of DCM through regulating HMGCS2. The expression of PPARα and HMGCS2 was detected by reverse transcription quantitative polymerase chain reaction in cardiomyocytes and high-glucose-cultured cardiomyocytes. The proliferation and apoptosis of cardiomyocytes were examined by 5-ethynyl-2'-deoxyuridine assay and flow cytometry, separately. Mitoehondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels were determined. Then, the protein levels of B-cell lymphoma 2, Bcl-2-associated X protein, and cleaved Caspase-3 were detected by Western blot analysis. The myocardial apoptosis index, heart weight, and serum lipids of rats were examined. At last, the expressions of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), transforming growth factor ß1 (TGFß1), peroxisome proliferator activator receptor gamma coactivator-1 alpha (PGC1α), nuclear respiratory factor (NRF)-1, NRF-2, NAD(P)H oxidase 1, and superoxide dismutase-1 were examined. HMGCS2 was the most differentially expressed gene in DCM. The levels of HMGCS2 and PPARα were upregulated in patients with DCM. HMGCS2 silencing was shown to inhibit HMGCS2 expression to suppress the apoptosis of high-glucose-induced cardiomyocytes and the loss of MMP, reduce the accumulation of ROS, and promote cardiomyocyte proliferation. Silencing of HMGCS2 and PPARα alleviated myocardial injury, decreased blood glucose, and lipid in DCM rats, downregulated the expression of ANP, BNP, and TGFß1 to reduce myocardial injury, and elevated PGC1α, NRF-1, and NRF-2 levels to enhance oxidative stress levels. Our results demonstrated that silencing of PPARα could alleviate cardiomyocyte injury and oxidative stress via a mechanism related to the downregulation of HMGCS2, which could provide a novel target for DCM treatment.

Regulation of Hox and ParaHox genes by perfluorochemicals in mouse liver.

Homeobox (Hox) genes encode homeodomain proteins, which play important roles in the development and morphological diversification of organisms including plants and animals. Perfluorinated chemicals (PFCs), which are well recognized industrial pollutants and universally detected in human and wildlife, interfere with animal development. In addition, PFCs produce a number of hepatic adverse effects, such as hepatomegaly and dyslipidemia. Homeodomain proteins profoundly contribute to liver regeneration. Hox genes serve as either oncogenes or tumor suppressor genes during target organ carcinogenesis. However, to date, no study investigated whether PFCs regulate expression of Hox genes. This study was designed to determine the regulation of Hox (including Hox-a to -d subfamily members) and paraHox [including GS homeobox (Gsx), pancreatic and duodenal homeobox (Pdx), and caudal-related homeobox (Cdx) family members] genes by PFCs including perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) in mouse liver. 46.4 mg/kg PFNA induced mRNA expression of Hoxa5, b7, c5, d10 and Pdx1 in wild-type and CAR-null mouse livers, but not in PPARα-null mouse livers, indicating a PPARα-dependent manner. PFOA, PFNA, and PFDA all induced mRNA expression of Hoxa5, b7, c5, d10, Pdx1 and Zeb2 in wild-type but not PPARα-null mouse livers. In addition, in Nrf2-null mouse livers, PFNA continued to increase mRNA expression of Hoxa5 and Pdx1, but not Hoxb7, c5 or d10. Furthermore, Wy14643, a classical PPARα agonist, induced mRNA expression of Hoxb7 and c5 in wild-type but not PPARα-null mouse livers. However, Wy14643 did not induce mRNA expression of Hoxa5, d10 or Pdx1 in either wild-type or PPARα-null mouse livers. TCPOBOP, a classical mouse CAR agonist, increased mRNA expression of Hoxb7, c5 and d10 but not Hoxa5 or Pdx1 in mouse livers. Moreover, PFNA decreased cytoplasmic and nuclear Hoxb7 protein levels in mouse livers. However, PFNA increased cytoplasmic Hoxc5 protein level but decreased nuclear Hoxc5 protein level in mouse livers. In conclusion, PFCs induced mRNA expression of several Hox genes such as Hoxb7, c5 and d10, mostly through the activation of PPARα and/or Nrf2 signaling.

Anti-Inflammatory Effects of Fucoxanthinol in LPS-Induced RAW264.7 Cells through the NAAA-PEA Pathway.

Palmitoylethanolamide (PEA) is an endogenous lipid mediator with powerful anti-inflammatory and analgesic functions. PEA can be hydrolyzed by a lysosomal enzyme N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages and other immune cells. The pharmacological inhibition of NAAA activity is a potential therapeutic strategy for inflammation-related diseases. Fucoxanthinol (FXOH) is a marine carotenoid from brown seaweeds with various beneficial effects. However, the anti-inflammatory effects and mechanism of action of FXOH in lipopolysaccharide (LPS)-stimulated macrophages remain unclear. This study aimed to explore the role of FXOH in the NAAA-PEA pathway and the anti-inflammatory effects based on this mechanism. In vitro results showed that FXOH can directly bind to the active site of NAAA protein and specifically inhibit the activity of NAAA enzyme. In an LPS-induced inflammatory model in macrophages, FXOH pretreatment significantly reversed the LPS-induced downregulation of PEA levels. FXOH also substantially attenuated the mRNA expression of inflammatory factors, including inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), and markedly reduced the production of TNF-α, IL-6, IL-1ß, and nitric oxide (NO). Moreover, the inhibitory effect of FXOH on NO induction was significantly abolished by the peroxisome proliferator-activated receptor α (PPAR-α) inhibitor GW6471. All these findings demonstrated that FXOH can prevent LPS-induced inflammation in macrophages, and its mechanisms may be associated with the regulation of the NAAA-PEA-PPAR-α pathway.

Keratinocyte aquaporin-3 expression induced by histone deacetylase inhibitors is mediated in part by peroxisome proliferator-activated receptors (PPARs).

The water and glycerol channel, aquaporin-3 (AQP3), plays an important role in the skin epidermis, with effects on hydration, permeability barrier repair and wound healing; therefore, information about the mechanisms regulating its expression is important for a complete understanding of skin function physiologically and in disease conditions. We previously demonstrated that histone deacetylase inhibitors (HDACi) induce the mRNA and protein expression of AQP3, in part through the p53 family, transcription factors for which acetylation is known to affect their regulatory activity. Another set of transcription factors previously shown to induce AQP3 expression and/or regulate skin function are the peroxisome proliferator-activated receptors (PPARs). Since there are reports that PPARs are also acetylated, we examined the involvement of these nuclear hormone receptors in HDACi-induced AQP3 expression. We first verified that a PPARγ agonist upregulated AQP3 mRNA and protein levels and that this increase was blocked by a PPARγ antagonist. We then showed that the PPARγ antagonist also inhibited AQP3 expression induced both by a broad-spectrum HDACi and an HDAC3-selective inhibitor. Interestingly, a PPARα antagonist also inhibited HDACi-induced AQP3 expression. These antagonist effects were observed in both primary mouse and normal human keratinocytes. Furthermore, PPARγ overexpression enhanced HDACi-stimulated AQP3 mRNA levels. Thus, our results suggest that PPARγ and/or PPARα may play a role in regulating AQP3 levels in the skin; based on the ability of PPAR agonists to promote epidermal differentiation and/or inhibit proliferation, topical PPAR agonists might be considered as a therapy for hyperproliferative skin disorders, such as psoriasis.

Functional analysis of molecular and pharmacological modulators of mitochondrial fatty acid oxidation.

Fatty acid oxidation (FAO) is a key bioenergetic pathway often dysregulated in diseases. The current knowledge on FAO regulators in mammalian cells is limited and sometimes controversial. Previous FAO analyses involve nonphysiological culture conditions or lack adequate quantification. We herein described a convenient and quantitative assay to monitor dynamic FAO activities of mammalian cells in physiologically relevant settings. The method enabled us to assess various molecular and pharmacological modulators of the FAO pathway in established cell lines, primary cells and mice. Surprisingly, many previously proposed FAO inhibitors such as ranolazine and trimetazidine lacked FAO-interfering activity. In comparison, etomoxir at low micromolar concentrations was sufficient to saturate its target proteins and to block cellular FAO function. Oxfenicine, on the other hand, acted as a partial inhibitor of FAO. As another class of FAO inhibitors that transcriptionally repress FAO genes, antagonists of peroxisome proliferator-activated receptors (PPARs), particularly that of PPARα, significantly decreased cellular FAO activity. Our assay also had sufficient sensitivity to monitor upregulation of FAO in response to environmental glucose depletion and other energy-demanding cues. Altogether this study provided a reliable FAO assay and a clear picture of biological properties of potential FAO modulators in the mammalian system.

The marine-derived furanone reduces intracellular lipid accumulation in vitro by targeting LXRα and PPARα.

Recent studies have demonstrated that commercially available lipid-lowering drugs cause various side effects; therefore, searching for anti-hyperlipidaemic compounds with lower toxicity is a research hotspot. This study was designed to investigate whether the marine-derived compound, 5-hydroxy-3-methoxy-5-methyl-4-butylfuran-2(5H)-one, has an anti-hyperlipidaemic activity, and the potential underlying mechanism in vitro. Results showed that the furanone had weaker cytotoxicity compared to positive control drugs. In RAW 264.7 cells, the furanone significantly lowered ox-LDL-induced lipid accumulation (~50%), and its triglyceride (TG)-lowering effect was greater than that of liver X receptor (LXR) agonist T0901317. In addition, it significantly elevated the protein levels of peroxisome proliferator-activated receptors (PPARα) and ATP-binding cassette (ABC) transporters, which could be partially inhibited by LXR antagonists, GSK2033 and SR9243. In HepG2 cells, it significantly decreased oleic acid-induced lipid accumulation, enhanced the protein levels of low-density lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPARα, and reduced the expression of sterol regulatory element-binding protein 2 (~32%). PPARα antagonists, GW6471 and MK886, could significantly inhibit the furanone-induced lipid-lowering effect. Furthermore, the furanone showed a significantly lower activity on the activation of the expression of lipogenic genes compared to T0901317. Taken together, the furanone exhibited a weak cytotoxicity but had powerful TC- and TG-lowering effects most likely through targeting LXRα and PPARα, respectively. These findings indicate that the furanone has a potential application for the treatment of dyslipidaemia.

Peroxisome proliferator-activated receptor agonists and antagonists: a patent review (2014-present).

Introduction: Peroxisome proliferator-activated receptors (PPARs), PPARα, PPARδ, and PPARγ, play an important role in the regulation of various physiological processes, specifically lipid and energy metabolism and immunity. PPARα agonists (fibrates) and PPARγ agonists (thiazolidinediones) are used for the treatment of hypertriglyceridemia and type 2 diabetes, respectively. PPARδ activation enhances mitochondrial and energy metabolism but PPARδ-acting drugs are not yet available. Many synthetic ligands for PPARs have been developed to expand their therapeutic applications.Areas covered: The authors searched recent patent activity regarding PPAR ligands. Novel PPARα agonists, PPARδ agonists, PPARγ agonists, PPARα/γ dual agonists, and PPARγ antagonists have been claimed for the treatment of metabolic disease and inflammatory disease. Methods for the combination of PPAR ligands with other drugs and expanded application of PPAR agonists for bone and neurological disease have been also claimed.Expert opinion: Novel PPAR ligands and the combination of PPAR ligands with other drugs have been claimed for the treatment of mitochondrial disease, inflammatory/autoimmune disease, neurological disease, and cancer in addition to metabolic diseases including dyslipidemia and type 2 diabetes. Selective therapeutic actions of PPAR ligands should be exploited to avoid adverse effects. More basic studies are needed to elucidate the molecular mechanisms of selective actions.

Peroxisome proliferator-activated receptor alpha regulates the expression of the immune response mediators in the porcine endometrium during the estrous cycle and early pregnancy.

PROBLEM: Cytokines are immune response mediators that play an important role in the regulation of reproductive functions. An association between cytokines and peroxisome proliferator receptors (PPARs) has been reported in various tissues, including the endometrium. The present study aimed to evaluate the impact of PPARα ligands on the expression of nuclear factor kappa B (NF-κB) and cytokines (interleukin [IL]-1ß, IL-4, IL-6, IL-8, IL-10, and LIF) in the porcine endometrium in different reproductive stages. METHODS OF STUDY: Endometrial slices were collected from gilts on days 10-12 or 14-16 of the estrous cycle and pregnancy. Endometrial tissue explants were incubated in vitro in the presence or absence of PPARα agonist WY-14643 and antagonist MK886. Expression of mRNA and protein for NF-ĸB and selected cytokines was evaluated by real-time PCR and immunoblot. RESULTS: PPARα agonist WY-14643 decreased the mRNA expression of NF-κB in most of the analyzed stages (excluding days 10-12 of the estrous cycle), but increased the expression of NF-κB protein (excluding days 14-16 of pregnancy). The WY-14643 increased expression of IL-1ß and IL-6 proteins, and the mRNA expression of IL-8 and LIF, decreased IL-4 expression, and did not affect the mRNA and protein expression of IL-10. CONCLUSION: The obtained results demonstrate that PPARα is involved in the regulation of NF-κB and cytokine expression in the porcine endometrium. PPARα ligands exert a varied influence on immune system components, which could be attributed to differences in the receptivity of porcine endometrial tissue during the reproductive cycle.

Oleic acid-induced perilipin 5 expression and lipid droplets formation are regulated by the PI3K/PPARα pathway in HepG2 cells.

Perilipin 5 (Plin5), a member of the PAT (Perilipin, ADRP, and Tip47) protein family, has been implicated in the regulation of cellular neutral lipid accumulation in nonalcoholic fatty liver diseases. However, the underlying regulatory mechanisms of Plin5 are not clear. The goal of the present study was to explore the mechanism of oleic acid (OA)-induced Plin5 expression in HepG2 cells. We found that the expression of Plin5 was increased during OA-induced lipid droplets formation in a dose- and time-dependent manner. During this process of OA-stimulated lipid droplets formation, peroxisome proliferator-activated receptor alpha (PPARα) was also upregulated. When PPARα activation was blocked by GW6471, OA-induced Plin5 expression and lipid droplets formation were effectively ablated. We further found that the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was able to downregulate both PPARα and Plin5 expression and lipid droplets formation. Thus, we concluded that PI3K may, at least in part, act upstream of PPARα to regulate Plin5 expression and lipid droplets formation in HepG2 cells.

PPARα-Mediated Positive-Feedback Loop Contributes to Cold Exposure Memory.

Fluctuations in food availability and shifts in temperature are typical environmental changes experienced by animals. These environmental shifts sometimes portend more severe changes; e.g., chilly north winds precede the onset of winter. Such telltale signs may be indicators for animals to prepare for such a shift. Here we show that HEK293A cells, cultured under starvation conditions, can "memorize" a short exposure to cold temperature (15 °C), which was evidenced by their higher survival rate compared to cells continuously grown at 37 °C. We refer to this phenomenon as "cold adaptation". The cold-exposed cells retained high ATP levels, and addition of etomoxir, a fatty acid oxidation inhibitor, abrogated the enhanced cell survival. In our standard protocol, cold adaptation required linoleic acid (LA) supplementation along with the activity of Δ-6-desaturase (D6D), a key enzyme in LA metabolism. Moreover, supplementation with the LA metabolite arachidonic acid (AA), which is a high-affinity agonist of peroxisome proliferator-activated receptor-alpha (PPARα), was able to underpin the cold adaptation, even in the presence of a D6D inhibitor. Cold exposure with added LA or AA prompted a surge in PPARα levels, followed by the induction of D6D expression; addition of a PPARα antagonist or a D6D inhibitor abrogated both their expression, and reduced cell survival to control levels. We also found that the brief cold exposure transiently prevents PPARα degradation by inhibiting the ubiquitin proteasome system, and starvation contributes to the enhancement of PPARα activity by inhibiting mTORC1. Our results reveal an innate adaptive positive-feedback mechanism with a PPARα-D6D-AA axis that is triggered by a brief cold exposure in cells. "Cold adaptation" could have evolved to increase strength and resilience against imminent extreme cold temperatures.

Discovery of potent and selective PPARα/δ dual antagonists and initial biological studies.

We previously published on the design and synthesis of novel, potent and selective PPARα antagonists suitable for either i.p. or oral in vivo administration for the potential treatment of cancer. Described herein is SAR for a subsequent program, where we set out to identify selective and potent PPARα/δ dual antagonist molecules. Emerging literature indicates that both PPARα and PPARδ antagonism may be helpful in curbing the proliferation of certain types of cancer. This dual antagonism could also be used to study PPARs in other settings. After testing for selective and dual potency, off-target counter screening, metabolic stability, oral bioavailability and associated toxicity, compound 11, the first reported PPARα/δ dual antagonist was chosen for more advanced preclinical evaluation.

N-Oleoyl-glycine reduces nicotine reward and withdrawal in mice.

Cigarette smokers with brain damage involving the insular cortex display cessation of tobacco smoking, suggesting that this region may contribute to nicotine addiction. In the present study, we speculated that molecules in the insular cortex that are sensitive to experimental traumatic brain injury (TBI) in mice might provide leads to ameliorate nicotine addiction. Using targeted lipidomics, we found that TBI elicited substantial increases of a largely uncharacterized lipid, N-acyl-glycine, N-oleoyl-glycine (OlGly), in the insular cortex of mice. We then evaluated whether intraperitoneal administration of OlGly would alter withdrawal responses in nicotine-dependent mice as well as the rewarding effects of nicotine, as assessed in the conditioned place preference paradigm (CPP). Systemic administration of OlGly reduced mecamylamine-precipitated withdrawal responses in nicotine-dependent mice and prevented nicotine CPP. However, OlGly did not affect morphine CPP, demonstrating a degree of selectivity. Our respective in vitro and in vivo observations that OlGly activated peroxisome proliferator-activated receptor alpha (PPAR-α) and the PPAR-α antagonist GW6471 prevented the OlGly-induced reduction of nicotine CPP in mice suggests that this lipid acts as a functional PPAR-α agonist to attenuate nicotine reward. These findings raise the possibility that the long chain fatty acid amide OlGly may possess efficacy in treating nicotine addiction.

Identification of the First PPARα/γ Dual Agonist Able To Bind to Canonical and Alternative Sites of PPARγ and To Inhibit Its Cdk5-Mediated Phosphorylation.

A new series of derivatives of the PPARα/γ dual agonist 1 allowed us to identify the ligand ( S)-6 as a potent partial agonist of both PPARα and γ subtypes. X-ray studies in PPARγ revealed two different binding modes of ( S)-6 to the canonical site. However, ( S)-6 was also able to bind an alternative site as demonstrated by transactivation assay in the presence of a canonical PPARγ antagonist and supported from docking experiments. This compound did not activate the PPARγ-dependent program of adipocyte differentiation inducing a very less severe lipid accumulation compared to rosiglitazone but increased the insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Finally, ( S)-6 inhibited the Cdk5-mediated phosphorylation of PPARγ at serine 273 that is currently considered the mechanism by which some PPARγ partial agonists exert antidiabetic effects similar to thiazolidinediones, without showing their typical side effects. This is the first PPARα/γ dual agonist reported to show this inhibitory effect representing the potential lead of a new class of drugs for treatment of dyslipidemic type 2 diabetes.

Selective peroxisome proliferator-activated receptor-α modulator K-877 regulates the expression of ATP-binding cassette transporter A1 in pancreatic beta cells.

ATP-binding cassette transporter A1 (ABCA1) protein is a pivotal regulator of cholesterol and phospholipid efflux from cells to high-density lipoprotein (HDL) particles. Pancreatic ABCA1 functions in beta cell cholesterol homeostasis and affects insulin secretion. We investigated the effect of pemafibrate (K-877), a novel selective PPARα modulator (SPPARMα), on pancreatic ABCA1 expression. In vivo experiment, mice were divided into four treatment groups, namely, normal food plus placebo, high fat diet (HFD) plus placebo, normal food plus K-877 (0.3 mg/kg/day), or HFD plus K-877 (0.3 mg/kg/day), and treated for eight weeks. The results in vitro experiment indicate that K-877 treatment increased levels of ABCA1 mRNA, as well as protein, subsequently reduced the cellular cholesterol content in INS-1 cells. PPARα specific antagonist GW6471 attenuate K-877 induced ABCA1 expression in INS-1 cells. ABCA1 promoter activity increased with K-877 treatment at concentration 1 µM and 10 µM. Glucose-stimulated insulin secretion was ameliorated by K-877 treatment in INS-1 cells and isolated mouse islets. Although the expression of ABCA1 was reduced in mice with HFD treatment, both ABCA1 protein and mRNA levels were increased in mice with K-877 treatment. K-877 treatment improved glucose intolerance induced by HFD in mice. These findings raise the possibility that K-877 may affect insulin secretion by controlling ABCA1 expression in pancreatic beta cells.

Regulation of steroidogenic function of mouse Leydig cells: G-coupled membrane estrogen receptor and peroxisome proliferator-activated receptor partnership.

We tested whether G-coupled membrane estrogen receptor (GPER) and peroxisome proliferator activated receptor (PPAR) partnership exists and whether this interaction regulates mouse Leydig cell function. Mature and aged mice were treated with the antagonist of GPER (G-15; 50 µg/kg b.w). Leydig cells (MA-10) were treated with G-15 (10 nM) alone or in combination with peroxisome proliferator-activated receptor α or γ antagonists, respectively (PPARα, 10 µM; PPARγ, 10 µM). GPER blockage affected testis steroidogenic status via changes in lutropin and cholesterol levels as well as protein expression alterations of the lutropin receptor, acute steroidogenesis activating protein, translocator protein, and protein kinase A in mouse Leydig cells both in vivo and in vitro. Inactivation of both GPER and PPAR in vitro revealed expressional modulation of other steroidogenesis-controlling molecules acting on various steps of lipid homeostasis e.g. cytochrome P450scc, perilipin, hormone sensitive lipase, and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. Concomitantly, microscopic analysis of cells treated with antagonists showed changes in morphology, migration competences and cytoskeleton structure. In the above processes, the action of GPER and PPARα was regulated through the PI3K/Akt pathway, while PPARγ was mediated by the Ras/Raf pathway. In addition, GPER and PPARs specifically controlled individual signaling proteins. For the first time, we report here the importance of GPER-PPARα and -PPARγ 'neopartnership' in maintenance of Leydig cell morpho-functional status.