Human PPAR Research Articles

Molecular docking and dynamic studies of Indian blackberry in contrast to X-ray structure of human PPAR gamma

Introduction and Aim: This study aims to elucidate the phytopharmacological and pharmacokinetic characteristics of Indian blackberries about human PPAR gamma receptors through in silico analyses. The protein receptor known as human PPAR gamma was retrieved, along with phytochemicals to perform molecular docking techniques to analyze the inhibitory effects of Indian blackberries.
 
 Methods: The human PPAR gamma receptor was obtained from the Protein Data Bank, and a set of phytochemicals was sourced from the IMPPAT database, and subsequently retrieved through the PubChem database. Molecular docking studies were conducted to assess the inhibitory interactions between Indian blackberry constituents and human PPAR gamma.
 
 Results: Among the various ligands investigated, ligand 12304985 exhibited the highest binding affinity towards the human PPAR gamma receptor. Subsequent analyses involved the assessment of ADMET profiling, secondary structure analysis, and Ramachandran plot analysis at the molecular level for human PPAR gamma.
 
 Conclusion: Our findings suggest that Indian blackberry, particularly ligand 12304985, exhibits promising inhibitory interactions with human PPAR gamma receptors. This in silico investigation provides valuable insights into the potential phytopharmacological and pharmacokinetic properties of Indian blackberries, paving the way for further in vitro and in vivo studies to validate these findings.

ANTI-DIABETIC POTENTENTAL OF Spatoglossum asperum (BROWN SEAWEED); INSILICO MODEL

This study used GC-MS analysis and computational biology to examine the diabetic effect of bioactive substances obtained from brown seaweed. Using AutoDock/Vina in Pymol 4.2, the molecular docking of Spatoglossum asperum on the human PPAR- protein was determined and compared to that of the well-known PPAR- agonist Gilbenclamide. The analysis the active components of seaweed extracts is the main focus of the current investigation. 12 substances were discovered using GC-MS analysis and evaluated using AutoDock/Vina against PPAR. Docking tests showed that the existing phytochemical 9-Octadecenoic Acid (Z)-, Octadecyl Ester, obtained from seaweed, has the highest fitness score of -398.9 kcal/mol and may therefore be a potent anti-diabetic drug. This study could result in the creation of brand-new marine nutraceuticals that use brown seaweed to treat a number of diseases.

Synthesis and anti-diabetic activity evaluation of phosphonates containing thiazolidinedione moiety

A sequence of substituted phosphonates containing the thiazolidinedione moiety was synthesized with good yields. The structures of all the synthesized compounds were confirmed by NMR (31P, 1H and 13C) and IR spectroscopy, mass spectrometry and C, H, N elemental analyses. In silico molecular docking study was also carried out to evaluate their interaction and binding modes on ligands against human PPAR γ protein for their anti-diabetic activity. From the docking results, it was determined that the compounds (Z)-dimethyl 5-(3-nitrobenzylidene)−2,4-dioxothiazolidin-3-ylphosphonate (7a), (Z)-dimethyl 5-(3-chloro-4-fluorobenzylidene)−2,4-dioxothiazolidin-3-ylphosphonate (7f), (Z)-dimethyl 5-(2,4-dichlorobenzylidene)−2,4-dioxothiazolidin-3-ylphosphonate (7e) and (Z)-dimethyl 5-((5-methoxypyridin-2-yl)methylene)−2,4-dioxothiazolidin-3-ylphosphonate (7j) have shown better binding energies (−7.8, −7.6, −7.5 and −7.6 Kcal/mol) with the target gene, PPAR γ than the reference drug, Rosiglitazone (−7.4 Kcal/mol). In vitro anti-diabetic activity of the title compounds was also screened by standard α-amylase inhibition assay. Some of the tested compounds proved to possess promising activity when compared with the reference drug.

Assessment of total, ligand-induced peroxisome proliferator activated receptor \u03b3 ligand activity in serum

BackgroundHumans are exposed to a complex mixture of environmental chemicals that impact bone and metabolic health, and traditional exposure assessments struggle to capture these exposure scenarios. Peroxisome proliferator activated receptor-gamma (PPARγ) is an essential regulator of metabolic and bone homeostasis, and its inappropriate activation by environmental chemicals can set the stage for adverse health effects. Here, we present the development of the Serum PPARγ Activity Assay (SPAA), a simple and cost-effective method to measure total ligand activity in small volumes of serum.MethodsFirst, we determined essential components of the bioassay. Cos-7 cells were transfected with combinations of expression vectors for human PPARγ and RXRα, the obligate DNA-binding partner of PPARγ, along with PPRE (DR1)-driven luciferase and control eGFP reporter constructs. Transfected cells were treated with rosiglitazone, a synthetic PPARγ ligand and/or LG100268, a synthetic RXR ligand, to characterize the dose response and determine the simplest and most efficacious format. Following optimization of the bioassay, we assessed the cumulative activation of PPARγ by ligands in serum from mice treated with a PPARγ ligand and commercial human serum samples.ResultsCos-7 cells endogenously express sufficient RXR to support efficacious activation of transfected PPARγ. Co-transfection of an RXR expression vector with the PPARγ expression vector did not increase PPRE transcriptional activity induced by rosiglitazone. Treatment with an RXR ligand marginally increased PPRE transcriptional activity in the presence of transfected PPARγ, and co-treatment with an RXR ligand reduced rosiglitazone-induced PPRE transcriptional activity. Therefore, the final bioassay protocol consists of transfecting Cos-7 cells with a PPARγ expression vector along with the reporter vectors, applying rosiglitazone standards and/or 10 μL of serum, and measuring luminescence and fluorescence after a 24 h incubation. Sera from mice dosed with rosiglitazone induced PPRE transcriptional activity in the SPAA in a dose-dependent and PPARγ-dependent manner. Additionally, human serum from commercial sources induced a range of PPRE transcriptional activities in a PPARγ-dependent manner, demonstrating the ability of the bioassay to detect potentially low levels of ligands.ConclusionsThe SPAA can reliably measure total PPRE transcriptional activity in small volumes of serum. This system provides a sensitive, straightforward assay that can be reproduced in any cell culture laboratory.

The Pioglitazone Trek via Human PPAR Gamma: From Discovery to a Medicine at the FDA and Beyond.

For almost two decades, pioglitazone has been prescribed primarily to prevent and treat insulin resistance in some type 2 diabetic patients. In this review, we trace the path to discovery of pioglitazone as a thiazolidinedione compound, the glitazone tracks through the regulatory agencies, the trek to molecular agonism in the nucleus and the binding of pioglitazone to the nuclear receptor PPAR gamma. Given the rise in consumption of pioglitazone in T2D patients worldwide and the increased number of clinical trials currently testing alternate medical uses for this drug, there is also merit to some reflection on the reported adverse effects. Going forward, it is imperative to continue investigations into the mechanisms of actions of pioglitazone, the potential of glitazone drugs to contribute to unmet needs in complex diseases associated with the dynamics of adaptive homeostasis, and also the routes to minimizing adverse effects in every-day patients throughout the world.

Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m2 (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

Synthesis, biological evaluation and molecular modeling studies of the PPARβ/δ antagonist CC618

Herein, we describe the synthesis, biological evaluation and molecular docking of the selective PPARβ/δ antagonist (4-methyl-2-(4-(trifluoromethyl)phenyl)-N-(2-(5-(trifluoromethyl)-pyridin-2-ylsulfonyl)ethyl)thiazole-5-carboxamide)), CC618. Results from in vitro luciferase reporter gene assays against the three known human PPAR subtypes revealed that CC618 selectively antagonizes agonist-induced PPARβ/δ activity with an IC50 = 10.0 μM. As observed by LC-MS/MS analysis of tryptic digests, the treatment of PPARβ/δ with CC618 leads to a covalent modification of Cys249, located centrally in the PPARβ/δ ligand binding pocket, corresponding to the conversion of its thiol moiety to a 5-trifluoromethyl-2-pyridylthioether. Finally, molecular docking is employed to shed light on the mode of action of the antagonist and its structural consequences for the PPARβ/δ ligand binding pocket.

Dihydrodehydrodiisoeugenol enhances adipocyte differentiation and decreases lipolysis in murine and human cells

Loss of subcutaneous fat is a hallmark of ageing usually starting in the face. Attempts to ameliorate cosmetically the appearance of subcutaneous fat loss have been of limited success as they fail to rebuild the missing subcutaneous tissue. Ageing-driven loss of subcutaneous fat results from (i) the reduced capacity of pre-adipocytes to differentiate into adipocytes and (ii) the fact that adipocytes of the elderly secrete increased amounts of TNFα, that in turn enhances lipolysis, inhibits pre-adipocyte differentiation and induces dedifferentiation of adipocytes. The neolignan dihydrodehydrodiisoeugenol (DDE) caused a 30% increase in lipid accumulation in murine 3T3-L1 cells. This effect was accompanied by an induction of the differentiation-associated transcription factors peroxisome proliferator-activated receptorγ (PPARγ2), CAAT/enhancer-binding protein α (C/EBPα), fatty acid binding protein 4 and adiponectin, and a loss of the pre-adipocyte marker Pref1. In addition, DDE diminished both basal and TNFα-induced lipolysis. Similar results were obtained in human subcutaneous (hsc) pre-adipocytes cultured in an age-adapted hormone mix with reduced levels of insulin and dexamethasone. In this system, DDE significantly increased lipid accumulation by 71% and 94% and was associated with an induction of PPARγ2 and adiponectin mRNA expression. DDE also reduced basal lipolysis in mature hsc adipocytes. DDE acted as a partial PPARγ agonist because (i) DDE displaced PPARγ ligand from the human PPAR ligand-binding site, (ii) DDE-induced lipid accumulation and (iii) DDE-induced adiponectin secretion could be overcome by the addition of PPARγ antagonists. Taken together, these studies identify DDE as a compound well suited to prevent and reverse loss of subcutaneous fat.

P50—A profile of human PPAR mRNA and protein expression during development

P50—A profile of human PPAR mRNA and protein expression during development

In silico design of potent agonists for human PPAR γ

AbstractPeroxisome proliferator-activated receptor (PPAR γ) acts as a key regulator on adipocyte differentiation and glucose homeostasis. PPAR γ has been implicated in the pathology of type 2 diabetes. As human PPAR γ activity is considered important in improving insulin sensitivity, in silico screening was carried out to find potent agonists for human PPAR γ protein. The co-crystal structure of PPAR γ, solved through X-Ray diffraction method was retrieved from the protein data bank. Four PPAR γ agonists selected from literature were submitted to subsequent 2D searching protocol using Ligand.Info, which yielded 1699 structural analogs. The PPAR γ co-crystal structure and ligand dataset were preprocessed using protein preparation wizard and LigPrep, respectively. Further, docking was performed by using three phased docking protocol of Maestro v9.2 that implements Glide v5.7. The obtained thirteen leads through docking were compared with the existing inhibitors and seven leads with good binding affinity with PPAR γ were proposed. The binding orientations of the seven leads were coinciding well with the native co-crystal structure of human PPAR γ. Thus, the proposed seven leads can be suggested as potential agonists for improving insulin sensitivity in the treatment of type 2 diabetes mellitus if synthesized and validated in animal models

In silico design of potent agonists for human PPAR γ

In silico design of potent agonists for human PPAR γ

Does Peroxisome Proliferator-activated Receptor-γ (PPARγ) Protect from Hypertension Directly through Effects in the Vasculature?

Peroxisome proliferator-activated receptor-gamma (PPAR gamma) is a ligand-activated transcription factor of the nuclear hormone receptor superfamily. Increasing evidence suggests that PPAR gamma is involved in the regulation of vascular function and blood pressure in addition to its well recognized role in metabolism. Thiazolidinediones, PPAR gamma agonists, lower blood pressure and have protective vascular effects through largely unknown mechanisms. In contrast, loss-of-function dominant-negative mutations in human PPAR gamma cause insulin resistance and severe early onset hypertension. Recent studies using genetically manipulated mouse models have begun to specifically address the importance of PPAR gamma in the vasculature. In this minireview, evidence for a protective role of PPAR gamma in the endothelium and vascular smooth muscle, derived largely from studies of genetically manipulated mice, will be discussed.

Mechanisms of Action and Medicinal Applications of Abscisic Acid

Since its discovery in the early 1960's, abscisic acid (ABA) has received considerable attention as an important phytohormone, and more recently, as a candidate medicinal in humans. In plants it has been shown to regulate important physiological processes such as response to drought stress, and dormancy. The discovery of ABA synthesis in animal cells has generated interest in the possible parallels between its role in plant and animal systems. The importance of this molecule has prompted the development of several methods for the chemical synthesis of ABA, which differ significantly from the biosynthesis of ABA in plants through the mevalonic acid pathway. ABA recognition in plants has been shown to occur at both the intra- and extracellularly but little is known about the perception of ABA by animal cells. A few ABA molecular targets have been identified in vitro (e.g., calcium signaling, G protein-coupled receptors) in both plant and animal systems. A unique finding in mammalian systems, however, is that the peroxisome proliferator-activated receptor, PPAR gamma, is upregulated by ABA in both in vitro and in vivo studies. Comparison of the human PPAR gamma gene network with Arabidopsis ABA-related genes reveal important orthologs between these groups. Also, ABA can ameliorate the symptoms of type II diabetes, targeting PPAR gamma in a similar manner as the thiazolidinediones class of anti-diabetic drugs. The use of ABA in the treatment of type II diabetes, offers encouragement for further studies concerning the biomedical applications of ABA.

Activation of PPAR γ and α by Punicic Acid Ameliorates Glucose Tolerance and Suppresses Obesity-Related Inflammation

Objective: Peroxisome proliferator-activated receptor γ (PPAR γ) is the molecular target for thiazolidinediones (TZDs), a class of synthetic antidiabetic agents. However, the naturally occurring agonists of PPARs remain largely unknown. Punicic acid (PUA) is a conjugated linolenic acid isomer found in pomegrante. The objective of this study was to test the hypothesis that PUA activates PPAR γ and thereby ameliorates glucose homeostasis and obesity-related inflammation.Methods: The ability of PUA to modulate PPAR reporter activity was determined in 3T3-L1 pre-adipocytes. A cell-free assay was used to measure PUA's binding to the ligand-binding domain (LBD) of human PPAR γ. The preventive actions of PUA were investigated using genetically obese db/db mice and a model of diet-induced obesity in PPAR γ-expressing and tissue-specific PPAR γ null mice. Expression of PPAR α, γ, PPAR-responsive genes and TNF-α was measured in tissues controlling glucose homeostasis.Results: PUA caused a dose-dependent increase PPAR α and γ reporter activity in 3T3-L1 cells and bound although weakly to the LBD of human PPAR γ. Dietary PUA decreased fasting plasma glucose concentrations, improved the glucose-normalizing ability, suppressed NF-κB activation, TNF-α expression and upregulated PPAR α- and γ-responsive genes in skeletal muscle and adipose tissue. Loss of PPAR γ impaired the ability of dietary PUA to improve glucose homeostasis and suppress inflammation.Conclusions: Our studies demonstrate that PUA binds and robustly activates PPAR γ, increases PPAR γ-responsive gene expression and the loss of PPAR γ in immune cells impairs its ability to ameliorate diabetes and inflammation.

Improvement of dyslipidemia, insulin sensitivity, and energy balance by a peroxisome proliferator–activated receptor α agonist

Peroxisome proliferator–activated receptor α (PPAR α) is a member of the nuclear receptor family of ligand-activated transcription factors. It plays an important role in the regulation of genes involved in lipid metabolism and transport. Compound A is a potent and orally active PPAR α agonist that activated both human and rat PPAR α receptors. The compound induced the expression of genes involved in fatty acid metabolism in a rodent hepatoma cell line and in the liver of db/db mouse. The ability of compound A to stimulate fatty acid β-oxidation was demonstrated in human hepatocytes and human skeletal muscle cells, which confirmed a functional activation of PPAR α-mediated activities. Compound A was shown to be a more potent and efficacious antidyslipidemic agent in atherogenic rat and db/db mouse models as compared with fenofibrate. The increase in high-density lipoprotein cholesterol levels by compound A was at least partially due to an increase in serum apolipoprotein A-I protein concentrations in human PPAR α transgenic mouse. The triglyceride-lowering effect was further confirmed in a higher species, obese dog models. In addition, compound A dose-dependently ameliorated hyperglycemia and hyperinsulinemia, and improved glucose tolerance in db/db mice. In a diet-induced obesity mouse model, compound A decreased body weight mainly by increasing energy expenditure and reducing fat deposition. In conclusion, the novel and potent PPAR α agonist improves lipid profile, insulin sensitivity, and energy balance in animal models.

Increasing alcohol-induced osteogenesis of human bone marrow-derived mesenchymal cells using siRNA transient suppression of peroxisome proliferator activated receptor gamma: an in vitro experiment study

To investigate the potential of small interfering RNA (siRNA) against human peroxisome proliferator activated receptor gamma (PPARgamma) to suppress the adipogenic effect of alcohol on human bone marrow-derived mesenchymal cells (hBMSCs) and increase osteogenesis. hBMSCs collected from joint replacement surgery were cultured, passaged, and transfected with PPARgamma-siRNA by using liposomal-based strategy. Then the cells were maintained in culture and treated with alcohol 50 mmol/L and dexamethasone, an osteogenic inducer, for 24 days. Oil red O staining was used to observe the fat drops in the cells so as to count the number of adipocytes. Histochemistry was performed to detect the protein expression of alkaline phosphatase, osteocalcium, and type 1 collagen on days 24 and 28 after treatment. Real-time PCR and Western blotting were used to detect the mRNA and protein expression of osteoblast-specific transcription factor-2 (Osf2)/core-binding factor alpha subunit 1 (Cbfa1) on day 10. The adipocyte number of the PPARgamma-siRNA group was significantly lower than those of the alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P < 0.05). The Calcium nodule formation rate of the PPARgamma-siRNA group was significantly higher than alcohol-induced group and the controls (adipogenic group, liposomal group, and negative siRNA group) (all P < 0.05). PCR and Western blotting showed significantly higher mRNA and protein expression of the human adipocyte-specific markers:Cbfa1, ALP, type 1 collagen, and osteocalcin, than the other groups. The adipogenic effect of alcohol on hBMSCs can be inhibited with a concomitant increase in osteogenesis by using siRNA technique to specific targeted human PPARgamma gene in vitro cell culture systems. PPARgamma-siRNA strategy may be a useful tool for studying the mechanisms of alcohol-induced osteoporosis and provides theoretical basis for its genetic therapy.

Exploring the human PPAR alpha dependent transcriptome in primary human hepatocytes

Regulation of hepatic gene expression by dietary fatty acids is primarilymediatedbyproxisomeproliferator activated receptor alpha (PPAR ). While the role of PPAR in mouse liver has been extensively studied, much less is known about its function in human liver. We used human primary hepatocytes whichwere isolated from 6 individual donors, including both male and female and ranging in age from 44 to 73 years. The cells were treated with PPAR agonist Wy14643 (50 M) for 6 and 24h. Gene expression profiles were assessed by using human genome U133 Plus 2.0 arrays. We observed that up to 204–174 genes (6 and 24h time point, respectively) were changed using a cutoff of P<0.05 and mean fold change ≥1.1. Significantly regulated genes were further analyzed using ingenuity pathway analyzer, which revealed fatty acid metabolism as the most regulated metabolic pathway. Therefore, we zoomed in to categorize the lipid metabolism connected genes based on their cellular localization and related lipidmetabolic process within the hepatocyte. A large number of genes implicated in lipid metabolism were found to be regulated by PPAR in primary human hepatocytes. This included classical PPAR targets involved in mitochondrial fatty acid oxidation, fatty acid binding and activation, glycerol metabolism, lipid transporting, and fatty acid desaturation. In addition, novel putative targets of PPAR were identified which are involved in lipid metabolism. Overall, we can conclude that the detailed functions of PPAR in lipid metabolism are extremely well conserved between mouse and human.

Structure-Based Design and Synthesis of Fluorescent PPAR.ALPHA./.DELTA. Co-agonist and Its Application as a Probe for Fluorescent Polarization Assay of PPAR.DELTA. Ligands

Based on the result of X-ray crystallographic analysis of our peroxisome proliferator-activated receptor alpha and delta (PPARalpha/delta) co-agonist complexed with human PPAR ligand binding domain (LBD), we designed and synthesized an optically active fluorescent PPARalpha/delta co-agonist, which has a pyrene unit incorporated directly at the hydrophobic tail part of the structure as a fluorophore. This fluorescent co-agonist was applied in a homogeneous fluorescent polarization assay format for the identification of PPARdelta ligands.

Structure–activity studies on 1,3-dioxane-2-carboxylic acid derivatives, a novel class of subtype-selective peroxisome proliferator-activated receptor α (PPARα) agonists

A series of 1,3-dioxane carboxylic acid derivatives was synthesized and evaluated for human PPAR transactivation activity. Structure–activity relationships on the phenyloxazole moiety of the lead compound 3 revealed that the introduction of small hydrophobic substituents at the 4-position of the terminal phenyl ring increased the PPARα agonist activity, and that the oxazole heterocycle was essential to the maintenance of both potency and PPARα subtype-selectivity. This investigation led to the identification of 14d (NS-220) and 14i as highly potent and selective human PPARα agonists. In KK-A y type 2 diabetic mice, these compounds significantly lowered plasma triglyceride and very-low-density plus low-density lipoprotein cholesterol levels while simultaneously raising HDL cholesterol levels. Our results suggest that highly potent and subtype-selective PPARα agonists will be promising drugs for the treatment of metabolic disorders in type 2 diabetes.

Ajulemic Acid, a Synthetic Nonpsychoactive Cannabinoid Acid, Bound to the Ligand Binding Domain of the Human Peroxisome Proliferator-activated Receptor γ

Ajulemic acid (AJA) is a synthetic analog of THC-11-oic acid, a metabolite of tetrahydrocannabinol (THC), the major active ingredient of the recreational drug marijuana derived from the plant Cannabis sativa. AJA has potent analgesic and anti-inflammatory activity in vivo, but without the psychotropic action of THC. However, its precise mechanism of action remains unknown. Biochemical studies indicate that AJA binds directly and selectively to the isotype gamma of the peroxisome proliferator-activated receptor (PPARgamma) suggesting that this may be a pharmacologically relevant receptor for this compound and a potential target for drug development in the treatment of pain and inflammation. Here, we report the crystal structure of the ligand binding domain of the gamma isotype of human PPAR in complex with ajulemic acid, determined at 2.8-A resolution. Our results show a binding mode that is compatible with other known partial agonists of PPAR, explaining their moderate activation of the receptor, as well as the structural basis for isotype selectivity, as observed previously in vitro. The structure also provides clues to the understanding of partial agonism itself, suggesting a rational approach to the design of molecules capable of activating the receptor at levels that avoid undesirable side effects.