Upregulation of PGC-1α expression by pioglitazone mediates prevention of sepsis-induced acute lung injury.

The imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages plays a critical role in the pathogenesis of sepsis-induced acute lung injury (ALI). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) may modulate macrophage polarization toward the M2 phenotype by altering mitochondrial activity. This study aimed to investigate the role of the PGC-1α agonist pioglitazone (PGZ) in modulating sepsis-induced ALI. A mouse model of sepsis-induced ALI was established using cecal ligation and puncture (CLP). An in vitro model was created by stimulating MH-S cells with lipopolysaccharide (LPS). qRT-PCR was used to measure mRNA levels of M1 markers iNOS and MHC-II and M2 markers Arg1 and CD206 to evaluate macrophage polarization. Western blotting detected expression of peroxisome proliferator-activated receptor gamma (PPARγ) PGC-1α, and mitochondrial biogenesis proteins NRF1, NRF2, and mtTFA. To assess mitochondrial content and function, reactive oxygen species levels were detected by dihydroethidium staining, and mitochondrial DNA copy number was measured by qRT-PCR. In the CLP-induced ALI mouse model, lung tissues exhibited reduced PGC-1α expression. PGZ treatment rescued PGC-1α expression and alleviated lung injury, as evidenced by decreased lung wet-to-dry weight ratio, pro-inflammatory cytokine secretion (tumor necrosis factor-α, interleukin-1ß, interleukin-6), and enhanced M2 macrophage polarization. Mechanistic investigations revealed that PGZ activated the PPARγ/PGC-1α/mitochondrial protection pathway to prevent sepsis-induced ALI by inhibiting M1 macrophage polarization. These results may provide new insights and evidence for developing PGZ as a potential ALI therapy.

Cold acclimation and pioglitazone combined increase thermogenic capacity of brown and white adipose tissues but this does not translate into higher energy expenditure in mice.

Cold acclimation and pharmacological peroxisome proliferator-activated receptor γ (PPARγ) activation have each earlier been shown to recruit brown adipose tissue (BAT) and beige adipocytes thermogenic machinery, enhancing uncoupling protein 1 (UCP1)-mediated thermogenic capacity. We here investigated whether cold acclimation and PPARγ agonism combined have additive effects in inducing brown and beige adipocytes UCP1 content and whether this translates into a higher thermogenic capacity and energy expenditure. C57BL/6J mice treated or not with pioglitazone (30 mg/kg/day) were maintained at 21°C or exposed to cold (7°C) for 15 days and evaluated for thermogenic capacity, energy expenditure and interscapular BAT (iBAT) and inguinal white adipose tissue (iWAT) mass, morphology, UCP1 content and gene expression, glucose uptake and oxygen consumption. Cold acclimation and PPARγ agonism combined synergistically increased iBAT and iWAT total UCP1 content and mRNA levels of the thermogenesis-related proteins PGC1a, CIDEA, FABP4, GYK, PPARa, LPL, GLUTs (GLUT1 in iBAT and GLUT4 in iWAT), and ATG when compared to cold and pioglitazone individually. This translated into a stronger increase in body temperature in response to the ß3-adrenergic agonist CL316,243 and iBAT and iWAT respiration induced by succinate and pyruvate in comparison to that seen in either cold-acclimated or pioglitazone-treated mice. However, basal energy expenditure, BAT glucose uptake and glucose tolerance were not increased above that seen in cold-acclimated untreated mice. In conclusion, cold acclimation and PPARγ agonism combined induced a robust increase in brown and beige adipocytes UCP1 content and thermogenic capacity, much higher than each treatment individually. However, our findings enforce the concept that increases in total UCP1 do not innately lead to higher energy expenditure.NEW & NOTEWORTHY Cold acclimation and PPARγ agonism combined markedly increase brown and white adipose tissue total UCP1 content and mRNA levels of thermogenesis-related proteins. Higher UCP1 protein levels did not result in higher energy expenditure. The high thermogenic capacity induced by PPARγ agonism in cold-exposed animals markedly increases animals' body temperature in response to the ß3-adrenergic agonist CL316,243.

Assessment of lipid metabolism-disrupting effects of non-phthalate plasticizer diisobutyl adipate through in silico and in vitro approaches.

Diisobutyl adipate (DIBA), as a novel non-phthalate plasticizer, is widely used in various products. However, little effort has been made to investigate whether DIBA might have adverse effects on human health. In this study, we integrated an in silico and in vitro strategy to assess the impact of DIBA on cellular homeostasis. Since numerous plasticizers could activate peroxisome proliferator-activated receptor γ (PPARγ) pathway to interrupt metabolism systems, we first utilized molecular docking to analyze interaction between DIBA and PPARγ. Results indicated that DIBA had strong affinity with the ligand-binding domain of PPARγ (PPARγ-LBD) at Histidine 499. Afterwards, we used cellular models to investigate in vitro effects of DIBA. Results demonstrated that DIBA exposure increased intracellular lipid content in murine and human hepatocytes, and altered transcriptional expression of genes related to PPARγ signaling and lipid metabolism pathways. At last, target genes regulated by DIBA were predicted and enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Protein-protein interaction (PPI) network and transcriptional factors (TFs)-genes network were established accordingly. Target genes were enriched in Phospholipase D signaling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and Epidermal growth factor receptor (EGFR) signaling pathway which were related to lipid metabolism. These findings suggested that DIBA exposure might disturb intracellular lipid metabolism homeostasis via targeting PPARγ. This study also demonstrated that this integrated in silico and in vitro methodology could be utilized as a high throughput, cost-saving and effective tool to assess the potential risk of various environmental chemicals on human health.

The Potential Roles of Post-Translational Modifications of PPARγ in Treating Diabetes.

The number of patients with type 2 diabetes mellitus (T2DM), which is mainly characterized by insulin resistance and insulin secretion deficiency, has been soaring in recent years. Accompanied by many other metabolic syndromes, such as cardiovascular diseases, T2DM represents a big challenge to public health and economic development. Peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated nuclear receptor that is critical in regulating glucose and lipid metabolism, has been developed as a powerful drug target for T2DM, such as thiazolidinediones (TZDs). Despite thiazolidinediones (TZDs), a class of PPARγ agonists, having been proven to be potent insulin sensitizers, their use is restricted in the treatment of diabetes for their adverse effects. Post-translational modifications (PTMs) have shed light on the selective activation of PPARγ, which shows great potential to circumvent TZDs' side effects while maintaining insulin sensitization. In this review, we will focus on the potential effects of PTMs of PPARγ on treating T2DM in terms of phosphorylation, acetylation, ubiquitination, SUMOylation, O-GlcNAcylation, and S-nitrosylation. A better understanding of PTMs of PPARγ will help to design a new generation of safer compounds targeting PPARγ to treat type 2 diabetes.

Protection against influenza-induced Acute Lung Injury (ALI) by enhanced induction of M2a macrophages: possible role of PPARγ/RXR ligands in IL-4-induced M2a macrophage differentiation.

Many respiratory viruses cause lung damage that may evolve into acute lung injury (ALI), a cytokine storm, acute respiratory distress syndrome, and ultimately, death. Peroxisome proliferator activated receptor gamma (PPARγ), a member of the nuclear hormone receptor (NHR) family of transcription factors, regulates transcription by forming heterodimers with another NHR family member, Retinoid X Receptor (RXR). Each component of the heterodimer binds specific ligands that modify transcriptional capacity of the entire heterodimer by recruiting different co-activators/co-repressors. However, the role of PPARγ/RXR ligands in the context of influenza infection is not well understood. PPARγ is associated with macrophage differentiation to an anti-inflammatory M2 state. We show that mice lacking the IL-4Rα receptor, required for M2a macrophage differentiation, are more susceptible to mouse-adapted influenza (A/PR/8/34; "PR8")-induced lethality. Mice lacking Ptgs2, that encodes COX-2, a key proinflammatory M1 macrophage mediator, are more resistant. Blocking the receptor for COX-2-induced Prostaglandin E2 (PGE2) was also protective. Treatment with pioglitazone (PGZ), a PPARγ ligand, increased survival from PR8 infection, decreased M1 macrophage gene expression, and increased PPARγ mRNA in lungs. Conversely, conditional knockout mice expressing PPARγ-deficient macrophages were significantly more sensitive to PR8-induced lethality. These findings were extended in cotton rats: PGZ blunted lung inflammation and M1 cytokine gene expression after challenge with non-adapted human influenza. To study mechanisms by which PPARγ/RXR transcription factors induce canonical M2a genes, WT mouse macrophages were treated with IL-4 in the absence or presence of rosiglitazone (RGZ; PPARγ ligand), LG100754 (LG; RXR ligand), or both. IL-4 dose-dependently induced M2a genes Arg1, Mrc1, Chil3, and Retnla. Treatment of macrophages with IL-4 and RGZ and/or LG differentially affected induction of Arg1 and Mrc1 vs. Chil3 and Retnla gene expression. In PPARγ-deficient macrophages, IL-4 alone failed to induce Arg1 and Mrc1 gene expression; however, concurrent treatment with LG or RGZ + LG enhanced IL-4-induced Arg1 and Mrc1 expression, but to a lower level than in WT macrophages, findings confirmed in the murine alveolar macrophage cell line, MH-S. These findings support a model in which PPARγ/RXR heterodimers control IL-4-induced M2a differentiation, and suggest that PPARγ/RXR agonists should be considered as important tools for clinical intervention against influenza-induced ALI.

A preliminary cumulative risk assessment of Diethylhexyl phthalate and Dibutyl phthalate based on the inhibition of embryonic development via the PPARγ pathway.

BACKGROUND: Diethylhexyl phthalate (DEHP) and dibutyl phthalate (DBP) are the two most widely used plasticizers (PAEs) that may act as endocrine disruptors and cause developmental toxicity. METHODS: We measured intrauterine exposure to DEHP and DBP which are the two most widely used phthalates (PAEs) in the cord blood of 50 postpartum women using ultra-HPLC-tandem mass spectrometry and solid-phase extraction. The embryotoxicity of DEHP and DBP was evaluated using the human embryonic stem cell test (hEST). Based on the intrauterine exposure concentration of DEHP and DBP in pregnant women and the reference point of toxic effects in hEST, we used the reference point index (RPI) method to assess the cumulative risk of DBP and DEHP. RESULTS: The mean concentrations of DEHP and DBP were 99.9 µg/L and 24.7 µg/L, respectively. DEHP and DBP were weakly embryotoxic, and the benchmark dose lower confidence intervals were 29.99 and 0.99 µg/mL, respectively, as determined using hEST. Both DEHP and DBP inhibited embryonic development via PPAR/PTEN/Akt signal pathway. CONCLUSION: The findings suggest that the cumulative risk in pregnant women with a high level of exposure should receive more attention in the future.

Insulin-stimulated endoproteolytic TUG cleavage links energy expenditure with glucose uptake.

TUG tethering proteins bind and sequester GLUT4 glucose transporters intracellularly, and insulin stimulates TUG cleavage to translocate GLUT4 to the cell surface and increase glucose uptake. This effect of insulin is independent of phosphatidylinositol 3-kinase, and its physiological relevance remains uncertain. Here we show that this TUG cleavage pathway regulates both insulin-stimulated glucose uptake in muscle and organism-level energy expenditure. Using mice with muscle-specific Tug (Aspscr1)-knockout and muscle-specific constitutive TUG cleavage, we show that, after GLUT4 release, the TUG C-terminal cleavage product enters the nucleus, binds peroxisome proliferator-activated receptor (PPAR)γ and its coactivator PGC-1α and regulates gene expression to promote lipid oxidation and thermogenesis. This pathway acts in muscle and adipose cells to upregulate sarcolipin and uncoupling protein 1 (UCP1), respectively. The PPARγ2 Pro12Ala polymorphism, which reduces diabetes risk, enhances TUG binding. The ATE1 arginyltransferase, which mediates a specific protein degradation pathway and controls thermogenesis, regulates the stability of the TUG product. We conclude that insulin-stimulated TUG cleavage coordinates whole-body energy expenditure with glucose uptake, that this mechanism might contribute to the thermic effect of food and that its attenuation could promote obesity.

Assessment of the hypoglycemic effect of Bixin in alloxan-induced diabetic rats: in vivo and in silico studies.

The objectives of this study were to extract and purify Bixin from the seeds of Bixa orellana and to evaluate its hypoglycemic activity in vivo, as well as, to conduct an in silico study of selectivity on peroxisome proliferator-activated receptors via molecular docking and molecular dynamics simulations. Oral administration of Bixin (10 mg/kg) significantly reduced their glucose level that was alloxan-induced diabetic rats. Bixin showed in silico selectivity on peroxisome proliferator-activated receptors (PPARs), particularly by the peroxisome proliferator-activated receptor gamma (PPARγ), which supports the hypoglycemic activity of Bixin. From the results obtained, it can be inferred that Bixin presents hypoglycemic characteristics, which was confirmed by the results obtained from the in vivo and in silico tests. Bixin may act by other pathways to control blood glucose and thus it is possible that it presents a different toxicity profile than troglitazone, rosiglitazone and pioglitazone. However, more studies on the activity and toxicity of Bixin are needed to evaluate for further clinical use. Communicated by Ramaswamy H. Sarma.

Exploring conformational changes of PPAR-Ɣ complexed with novel kaempferol, quercetin, and resveratrol derivatives to understand binding mode assessment: a small-molecule checkmate to cancer therapy.

Peroxisome proliferator-activated receptors-γ (PPAR-γ), a ligand-activated transcription factor, activated by several ligands like fatty acids (linoleic acid being the most common) or their metabolites, can function as potential therapeutic target for various cancers. Although various synthetic ligands, thiazolidinediones (TZDs), serves as full agonist for PPAR-γ, application of these molecules has been discontinued due to adverse toxicity profile. Hence, with a dire need to identify novel PPAR-γ-agonists, the present in silico study aimed to determine the effectiveness of potent flavonoids, kaempferol (CID: 5280863), quercetin (CID: 5280343), and stilbenoid resveratrol (CID: 445154) and their 806 derivatives towards PPAR-γ that could combat the deleterious effect of TZDs. The molecular docking experiment performed by FlexX elucidated the efficacy of derivatives; Kem204, Qur8, and Res183 of kaempferol, quercetin, and resveratrol respectively to be more effective against PPAR-γ as compared with other derivatives. The physicochemical and pharmacokinetic parameters of Kem204, Qur8, and Res183 follow the drug-likeness and thus comprise a pharmacologically active model to be considered for advancing further potential hits. Further molecular dynamics (MD) simulation study revealed the Qur8 compound to have favorable dynamic interactions within the PPAR-γ which certainly paves away in developing futuristic potential anticancer drugs. Graphical abstract.

Experimental Exposure Assessment of Ionizable Organic Chemicals in In Vitro Cell-Based Bioassays.

Exposure assessment in in vitro cell-based bioassays is challenging for ionizable organic chemicals (IOCs), because they are present as more than one chemical species in the bioassay medium. Furthermore, compared to neutral organic chemicals, their binding to medium proteins and lipids is driven by more complex molecular interactions. Total medium concentrations (Ctotal,medium) and/or freely dissolved medium concentrations (Cfree,medium) were determined for one neutral chemical and 14 IOCs (acids, bases, multifunctional) at concentrations relevant for determination of cytotoxicity and effect. Cfree,medium was measured in two in vitro bioassays at the time of dosing and after 24 h of incubation using solid-phase microextraction. Cfree,medium was maximally 1.7 times lower than the nominal concentrations (Cnom) for the hydrophilic chemicals (caffeine and lamotrigine). For the organic acids (naproxen, ibuprofen, warfarin, and diclofenac), Cfree,medium was by a factor of 4 lower than Cnom at high concentrations, but the ratio was much higher at low concentrations, indicating a nonlinear binding behavior. The experimental Cfree,medium was also compared with Cfree,medium predicted with a mass balance model accounting for binding to medium proteins and lipids. The mass balance model performed well for five of the test chemicals (within a factor of 10), but it underestimated Cfree,medium by up to a factor of 1200 for chemicals that showed nonlinear binding to medium components. These findings emphasize that experimental exposure assessment is required for improved understanding of in vitro toxicity data.

Metabolomic-based assessment reveals dysregulation of lipid profiles in human liver cells exposed to environmental obesogens.

Significant attention has been given to the potential of environmental chemicals to disrupt lipid homeostasis at the cellular level. These chemicals, classified as obesogens, are abundantly used in a wide variety of consumer products. However, there is a significant lack of information regarding the mechanisms by which environmental exposure can contribute to the onset of obesity and non-alcoholic fatty liver disease (NAFLD). Several studies have described the interaction of potential obesogens with lipid-related peroxisome proliferator-activated receptors (PPAR). However, no studies have quantified the degree of modification to lipidomic profiles in relevant human models, making it difficult to directly link PPAR agonists to the onset of lipid-related diseases. A quantitative metabolomic approach was used to examine the dysregulation of lipid metabolism in human liver cells upon exposure to potential obesogenic compounds. The chemicals rosiglitazone, perfluorooctanoic acid, di-2-ethylexylphthalate, and tributyltin significantly increased total lipids in liver cells, being diglycerides, triglycerides and phosphatidylcholines the most prominent. Contrarily, perfluorooctane sulfonic acid and the pharmaceutical fenofibrate appeared to lower total lipid concentrations, especially those belonging to the acylcarnitine, ceramide, triglyceride, and phosphatidylcholine groups. Fluorescence microscopy analysis for cellular neutral lipids revealed significant lipid bioaccumulation upon exposure to obesogens at environmentally relevant concentrations. This integrated omics analysis provides unique mechanistic insight into the potential of these environmental pollutants to promote diseases like obesity and NAFLD. Furthermore, this study provides a significant contribution to advance the understanding of molecular signatures related to obesogenic chemicals and to the development of alternatives to in vivo experimentation.

α-Ketoglutarate Modulates Macrophage Polarization Through Regulation of PPARγ Transcription and mTORC1/p70S6K Pathway to Ameliorate ALI/ARDS.

As tissue-resident cells in the lung, alveolar macrophages display remarkable heterogeneity and play a crucial role in the development and control of septic acute lung injury/acute respiratory distress syndrome (ALI/ARDS). Recent evidence suggests that α-ketoglutarate (α-KG) plays an important role in alternative activation of macrophage (M2) through metabolic and epigenetic reprogramming, and thus possesses anti-inflammatory properties. However, the underlying mechanisms of α-KG's effect on alveolar macrophage polarization and the potential effects of α-KG in ALI/ARDS remain unclear. Here, we examined the effects and mechanisms of α-KG on alveolar macrophage polarization, and investigated the possible effects of α-KG on lipopolysaccharide (LPS)-induced ALI/ARDS in a mouse model. We found that α-KG inhibited M1 macrophage polarization and promoted IL-4-induced M2 macrophage polarization in MH-S cells (a murine alveolar macrophage cell line). Further experiments showed that α-KG down-regulated the expression of M1-polarized marker genes and inhibited the activities of mammalian target of rapamycin complex 1 (mTORC1)/p70 ribosomal protein S6 kinase (p70S6K) signaling pathway in M1-polarized MH-S cells. Moreover, our results showed that α-KG promoted IL-4-induced M2 polarization of MH-S cells by augmenting nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ) and increasing expression of relevant fatty acid metabolic genes. Finally, using an LPS-induced ALI/ARDS mouse model, we found that α-KG ameliorated the LPS-induced inflammation and lung pathological damage, as well as α-KG pretreated mice had better clinical scores compared with the LPS group. These findings reveal new mechanisms of α-KG in regulating macrophage polarization which may provide novel strategies for the prevention and treatment of inflammatory diseases, including sepsis and septic ALI/ARDS.

Quantitative structural assessment of graded receptor agonism.

Ligand-receptor interactions, which are ubiquitous in physiology, are described by theoretical models of receptor pharmacology. Structural evidence for graded efficacy receptor conformations predicted by receptor theory has been limited but is critical to fully validate theoretical models. We applied quantitative structure-function approaches to characterize the effects of structurally similar and structurally diverse agonists on the conformational ensemble of nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). For all ligands, agonist functional efficacy is correlated to a shift in the conformational ensemble equilibrium from a ground state toward an active state, which is detected by NMR spectroscopy but not observed in crystal structures. For the structurally similar ligands, ligand potency and affinity are also correlated to efficacy and conformation, indicating ligand residence times among related analogs may influence receptor conformation and function. Our results derived from quantitative graded activity-conformation correlations provide experimental evidence and a platform with which to extend and test theoretical models of receptor pharmacology to more accurately describe and predict ligand-dependent receptor activity.

Assessment of total, ligand-induced peroxisome proliferator activated receptor γ ligand activity in serum.

BACKGROUND: Humans 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. METHODS: First, 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. RESULTS: Cos-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. CONCLUSIONS: The 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 Suv420h histone methyltransferases regulate PPAR-γ and energy expenditure in response to environmental stimuli.

Obesity and its associated metabolic abnormalities have become a global emergency with considerable morbidity and mortality. Epidemiologic and animal model data suggest an epigenetic contribution to obesity. Nevertheless, the cellular and molecular mechanisms through which epigenetics contributes to the development of obesity remain to be elucidated. Suv420h1 and Suv420h2 are histone methyltransferases responsible for chromatin compaction and gene repression. Through in vivo, ex vivo, and in vitro studies, we found that Suv420h1 and Suv420h2 respond to environmental stimuli and regulate metabolism by down-regulating peroxisome proliferator-activated receptor gamma (PPAR-γ), a master transcriptional regulator of lipid storage and glucose metabolism. Accordingly, mice lacking Suv420h proteins activate PPAR-γ target genes in brown adipose tissue to increase mitochondria respiration, improve glucose tolerance, and reduce adipose tissue to fight obesity. We conclude that Suv420h proteins are key epigenetic regulators of PPAR-γ and the pathways controlling metabolism and weight balance in response to environmental stimuli.

Decision tree learning to predict overweight/obesity based on body mass index and gene polymporphisms.

The new technologies for data analysis, such as decision tree learning, may help to predict the risk of developing diseases. The aim of the present work was to develop a pilot decision tree learning to predict overweight/obesity based on the combination of six single nucleotide polymorphisms (SNP) located in feeding-associated genes. Genotype study was performed in 151 healthy individuals, who were anonymized and randomly selected from the TALAVERA study. The decision tree analysis was performed using the R package rpart. The learning process was stopped when 15 or less observation was found in a node. The participant group consisted of 78 men and 73 women, who 100 individuals showed body mass index (BMI) ≥ 25 kg/m2 and 51 BMI < 25 kg/m2. Chi-square analysis revealed that individuals with BMI ≥ 25 kg/m2 showed higher frequency of the allelic variation Ala67Ala in AgRP rs5030980 with respect to those with BMI <25 kg/m2. However, the variant Thr67Ala in AgRP rs5030980 was the most frequently found in individuals with BMI <25 kg/m2. There were no statistical differences in the other analyzed SNPs. Decision tree learning revealed that carriers of the allelic variants AgRP (rs5030980) Ala67Ala, ADRB2 (rs1042714) Gln27Glu or Glu27Glu, INSIG2 (rs7566605) 73 + 9802 with CC or GG genotypes and PPARG (rs1801282) with the allelic variants of Ala12Ala or Pro12Pro, will most likely develop overweight/obesity (BMI ≥ 25 kg/m2). Moreover, the decision tree learning indicated that age and gender may change the developed three decision learning associated with overweight/obesity development. The present work should be considered as a pilot demonstrative study to reinforce the broad field of application of new data analysis technologies, such as decision tree learning, as useful tools for diseases prediction. This technology may achieve a potential applicability in the design of early strategies to prevent overweight/obesity.

Role of peroxisome proliferator-activated receptor gamma Pro12Ala polymorphism in human adipose tissue: assessment of adipogenesis and adipocyte glucose and lipid turnover.

The protective mechanisms of peroxisome proliferator-activated receptor gamma (PPARγ) Pro12Ala polymorphism in type 2 diabetes (T2D) are unclear. We obtained subcutaneous adipose tissue (AT) before and 3 h after oral glucose (OGTT) in carriers and non-carriers of the Ala allele (12 Pro/Pro, 15 Pro/Ala, and 13 Ala/Ala). Adipogenesis, adipocyte glucose uptake and lipolysis as well as PPARγ target gene expression were investigated and compared between the genotype groups. During fasting and post-OGTT, neither basal nor insulin-stimulated adipocyte glucose uptake differed between genotypes. Compared to fasting, a decreased hormone-sensitive lipase gene expression in Pro/Pro (p < 0.05) was accompanied with a higher antilipolytic effect of insulin post-OGTT (p < 0.01). The adipocyte size was similar across groups. Preadipocyte differentiation rates between Pro/Pro and Ala/Ala were unchanged. In conclusion, no major differences in AT differentiation, glucose uptake, lipolysis or expression of PPARγ target genes were observed between different PPARγ Pro12Ala genotypes. Albeit small, our study may suggest that other pathways in AT or effects exerted in other tissues might contribute to the Pro12Ala-mediated protection against T2D.

Norbixin, an apocarotenoid derivative activates PPARγ in cardiometabolic syndrome: Validation by in silico and in vivo experimental assessment.

The increased prevalence of cardio-metabolic disorders worldwide prompted the exploration of new strategies for its treatment. Peroxisome Proliferator activated receptor (PPAR) play major role in regulation of lipid as well as glucose metabolism and thus, natural PPARγ activators seem to be drug of choice. AIMS: In the present work, we studied norbixin which is a natural apocarotenoid derivative for its agonistic activity for PPAR γ followed by in vivo studies for amelioration of cardio-metabolic syndrome (CMetS). MAIN METHODS: The methods include computational studies, TR-FRET binding analysis and in vivo studies on high fat diet induced rats. KEY FINDINGS: Molecular docking and molecular dynamics (MD) simulation studies showed that norbixin could be embedded into hydrophobic pocket of PPARγ and stable hydrogen bonding interactions were found with residues Glu273, Tyr327, Ser289, His323, His449 and Tyr473 of PPARγ. These results were substantiated by significant in vitro PPAR agonistic activity of norbixin in TR-FRET binding assay studies. The experimental results of norbixin in high fat diet induced CMetS in rats further confirmed that norbixin decreased insulin resistance (IR), hyperglycemia and dyslipidemia. These results were accompanied by reduced inflammatory marker hs-CRP as well as decreased oxidative stress and arterial pressure. The histopathology of heart sections also showed that norbixin could prevent the abnormal fibrotic changes in heart. Furthermore, PPARγ protein expressions were increased, whereas NF-κB expression was decreased by norbixin treatment in western blot studies. SIGNIFICANCE: These results validate norbixin as a novel PPARγ agonist and prove therapeutic potential of norbixin in treatment of CMetS.

Assessment of potential biomarkers of atherosclerosis in Indian patients with type 2 diabetes mellitus.

BACKGROUND & OBJECTIVES: : Various biological markers of subclinical atherosclerosis have been proposed to predict cardiovascular events in patients with diabetes mellitus (DM). However, there are only a few clinical studies assessing the role of invasive biomarkers [CD-36, peroxisome proliferator-activated receptor gamma (PPAR-γ) and YKL-40] in Indian patients with type 2 DM (T2DM). Hence, the present study was conducted to assess protein levels and gene expression of CD-36, PPAR-γ and YKL-40 in patients with T2DM and compare that with hypertensive and healthy controls. METHODS: : All the participants were subjected to medical history, anthropometric measurements and biochemical and biomarker (ELISA and real-time polymerase chain reaction) estimations. The study groups consisted of patients with T2DM (>5 yr) with hypertension (n=55), patients with T2DM (<2 yr) without hypertension (n=28), hypertensive controls (n=31) and healthy controls (n=30). RESULTS: : Gene expressions of YKL-40 and CD36 were significantly higher in patients with T2DM (>5 yr) with hypertension compared to healthy controls (P=0.006). In addition, a significant increase in serum levels of sCD36, PPAR-γ and YKL-40 was observed in patients with T2DM (>5 yr) with hypertension compared to healthy controls (P< 0.05). Serum levels as well as gene expression of CD36 showed significant correlation with serum levels as well as gene expression of PPAR-γ (ρ=0.45 and ρ=0.51; P< 0.001), respectively. INTERPRETATION & CONCLUSIONS: : CD36 and YKL-40 may be potential inflammatory biomarkers for early onset of atherosclerosis in patients with T2DM.

Deciphering PPARγ activation in cardiometabolic syndrome: studies by in silico and in vivo experimental assessment.

Cardiometabolic syndrome (CMetS) is a consolidation of metabolic disorders characterized by insulin resistance, dyslipidemia and hypertension. Curcumin, a natural bioactive compound, has been shown to possess notable anti-oxidant activity and it has also been included as a super natural herb in the super natural herbs database. Most of the beneficial effects of Curcumin are possibly due to activation of the nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ). The present study investigates molecular interactions of curcumin with PPARγ protein through molecular docking and molecular dynamics (MD) simulation studies. Further, effect of curcumin on high fat diet induced CMetS was studied in rats along with western blot for PPARγ and nuclear factor-κB (NF-κB) expressions and histopathological studies. Computational studies presented several significant molecular interactions of curcumin including Ser289, His323, His449 and Tyr473 of PPARγ. The in vivo results further confirmed that curcumin was able to ameliorate the abnormal changes and also, increased PPARγ expressions. The results confirm our hypothesis that activation of PPARγ by curcumin possesses the therapeutic potential to ameliorate the altered levels of metabolic changes in rats in the treatment of CMetS. This is the first report of CMetS treatment by curcumin and study of its underlying mechanism through in silico as well as in vivo experiments.