scholarly journals Branched-Chain Fatty Acids as Mediators of the Activation of Hepatic Peroxisome Proliferator-Activated Receptor Alpha by a Fungal Lipid Extract

Biomolecules ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1259 ◽  
Author(s):  
Garima Maheshwari ◽  
Robert Ringseis ◽  
Gaiping Wen ◽  
Denise K. Gessner ◽  
Johanna Rost ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Reporter Gene ◽  
Target Genes ◽  
Reporter Gene Assay ◽  
Lipid Extract ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Branched Chain Fatty Acids ◽  
Gene Assay ◽  
Branched Chain

The study aimed to test the hypothesis that monomethyl branched-chain fatty acids (BCFAs) and a lipid extract of Conidiobolus heterosporus (CHLE), rich in monomethyl BCFAs, are able to activate the nuclear transcription factor peroxisome proliferator-activated receptor alpha (PPARalpha). Rat Fao cells were incubated with the monomethyl BCFAs 12-methyltridecanoic acid (MTriA), 12-methyltetradecanoic acid (MTA), isopalmitic acid (IPA) and 14-methylhexadecanoic acid (MHD), and the direct activation of PPARalpha was evaluated by reporter gene assay using a PPARalpha responsive reporter gene. Furthermore, Fao cells were incubated with different concentrations of the CHLE and PPARalpha activation was also evaluated by using the reporter gene assay, and by determining the mRNA concentrations of selected PPARalpha target genes by real-time RT-PCR. The reporter gene assay revealed that IPA and the CHLE, but not MTriA, MHD and MTA, activate the PPARalpha responsive reporter gene. CHLE dose-dependently increased mRNA concentrations of the PPARalpha target genes acyl-CoA oxidase (ACOX1), cytochrome P450 4A1 (CYP4A1), carnitine palmitoyltransferase 1A (CPT1A) and solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5). In conclusion, the monomethyl BCFA IPA is a potent PPARalpha activator. CHLE activates PPARalpha-dependent gene expression in Fao cells, an effect that is possibly mediated by IPA.

scholarly journals Multiplexing Nuclear Receptors for Agonist Identification in a Cell-Based Reporter Gene High-Throughput Screen

2003 ◽  
Vol 8 (3) ◽  
pp. 239-246 ◽  
Author(s):  
G. Scott Grover ◽  
Benjamin A. Turner ◽  
Christian N. Parker ◽  
Jannika Meier ◽  
Deepak S. Lala ◽  
...  
Keyword(s):  
Data Quality ◽  
Nuclear Receptors ◽  
Reporter Gene ◽  
High Throughput ◽  
High Throughput Screening ◽  
Peroxisome Proliferator ◽  
Human Hepatoma Cells ◽  
Peroxisome Proliferator Activated Receptor ◽  
Screening Cost ◽  
Gene Assay

High-throughput screening (HTS) has become an essential part of the drug discovery process. Due to the rising requirements for both data quality and quantity, along with increased screening cost and the demand to shorten the time for lead identification, increasing throughput and cost-effectiveness has become a necessity in the hit identification process. The authors present a multiplexed HTS for 2 nuclear receptors, the farnesoid X-activated receptor and the peroxisome proliferator-activated receptor delta in a viable cell-based reporter gene assay. The 2 nuclear receptors were individually transfected into human hepatoma cells, and the transient transfected cell lines were pooled for the multiplexed screen. Hits identified by the multiplexed screen are similar to those identified by the individual receptor screens. Furthermore, the multiplexed screen provides selectivity information if ligands selective for one and not the other receptor are one of the hit criteria. The data demonstrate that multiplexing nuclear receptors can be a simple, efficient, cost-effective, and reliable alternative to traditional HTS of individual targets without compromising data quality. ( Journal of Biomolecular Screening 2003:239-246)

Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPARγ in 3T3-L1 cells

2012 ◽  
Vol 302 (12) ◽  
pp. E1461-E1471 ◽  
Author(s):  
Takeshi Kobayashi ◽  
Ko Fujimori
Keyword(s):  
Fatty Acids ◽  
Target Genes ◽  
Mrna Level ◽  
Peroxisome Proliferator ◽  
Long Chain Fatty Acids ◽  
Peroxisome Proliferator Activated Receptor ◽  
Lipogenic Genes ◽  
Pparγ Agonist ◽  
Long Chain ◽  
Chain Fatty Acids

Here, we show that Elovl3 (elongation of very long-chain fatty acids 3) was involved in the regulation of the progression of adipogenesis through activation of peroxisome proliferator-activated receptor (PPAR)γ in mouse adipocytic 3T3-L1 cells. The expression of the Elovl3 gene increased during adipogenesis, the expression pattern of which was similar to that of the PPARγ gene. Troglitazone, a PPARγ agonist, enhanced Elovl3 expression in adipocytes, as it did that of other PPARγ target genes. Promoter-reporter analysis demonstrated that three PPAR-responsive elements in the Elovl3 gene promoter had the potential to activate its expression in 3T3-L1 cells. Moreover, a chromatin immunoprecipitation assay revealed that PPARγ bound these PPAR-responsive elements of the Elovl3 promoter. When the Elovl3 mRNA level was suppressed by its siRNAs, the level of intracellular triglycerides was significantly decreased, and the expression levels of adipogenic, lipolytic, and lipogenic genes were also repressed. In a mammalian two-hybrid assay, C18:1 and C20:1 very long-chain fatty acids (VLCFAs), which are the products of Elovl3 and activated PPARγ function. In addition, these same VLCFAs could prevent the Elovl3 siRNA-mediated suppression of adipogenesis by enhancing the expression of adipogenic, lipolytic, and lipogenic genes in adipocytes. Moreover, this VLCFAs-mediated activation was repressed by a PPARγ antagonist. These results indicate that the expression of the Elovl3 gene was activated by PPARγ during adipogenesis. Elovl3-produced C18:1 and C20:1 VLCFAs acted as agonists of PPARγ in 3T3-L1 cells. Thus, the Elovl3-PPARγ cascade is a novel regulatory circuit for the regulation of adipogenesis through improvement of PPARγ function in adipocytes.

scholarly journals In vitro bioanalytical assessment of toxicity of wetland samples from Spanish Mediterranean coastline

2021 ◽  
Vol 33 (1) ◽  
Author(s):  
Alberto Celma ◽  
Geeta Mandava ◽  
Agneta Oskarsson ◽  
Juan Vicente Sancho ◽  
Lubertus Bijlsma ◽  
...  
Keyword(s):  
Water Quality ◽  
Reporter Gene ◽  
Biological Activities ◽  
Reporter Gene Assay ◽  
Water Bodies ◽  
Good Tool ◽  
Adverse Health Effects ◽  
Aryl Hydrocarbon ◽  
Gene Assay

Abstract Background Fresh water bodies represent less than 1% of overall amount of water on earth and ensuring their quality and sustainability is pivotal. Although several campaigns have been performed to monitor the occurrence of micropollutants by means of chemical analysis, this might not cover the whole set of chemicals present in the sample nor the potential toxic effects of mixtures of natural and anthropogenic chemicals. In this sense, by selecting relevant toxicity endpoints when performing in vitro bioanalysis, effect-based methodologies can be of help to perform a comprehensive assessment of water quality and reveal biological activities relevant to adverse health effects. However, no prior bioanalytical study was performed in wetland water samples from the Spanish Mediterranean coastline. Methods Eleven samples from relevant water bodies from the Spanish Mediterranean coastline were collected to monitor water quality on 8 toxicity endpoints. Aryl hydrocarbon receptor (AhR), androgenicity (AR+ and AR−), estrogenicity (ER+ and ER−), oxidative stress response (Nrf2) and vitamin D receptor (VDR+ and VDR−) reporter gene assays were evaluated. Results AhR was the reporter gene assay showing a more frequent response over the set of samples (activated by 9 out of 11 samples), with TCDD-eq in the range 7.7–22.2 pM. For AR, ER and VDR assays sporadic activations were observed. Moreover, no activity was observed on the Nrf2 reporter gene assay. Wastewater and street runaway streams from Valencia could be responsible for enhanced activities in one of the water inputs in the Natural Park ‘L’Albufera’. Conclusions Water quality of relevant wetlands from the Spanish Mediterranean coastline has been evaluated. The utilization of a panel of 5 different bioassays to cover for different toxicity endpoints has demonstrated to be a good tool to assess water quality.

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