Development of a Novel PPARγ Ligand Screening System Using Pinpoint Fluorescence-Probed Protein

Nagai, Hirokazu; Ebisu, Shogo; Abe, Ryoji; Goto, Tsuyoshi; Takahashi, Nobuyuki; Hosaka, Takahiro; Kawada, Teruo

doi:10.1271/bbb.100810

Cited by 10 publications

(8 citation statements)

References 22 publications

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“…The HL diet might induce up-regulated COX-2 mRNA in the coronary artery as well, similar to liver, and cause coronary atherosclerosis or cardiovascular disease [16]. In contrast, the products generated by COX-2 might serve as ligands for differentiation [55] and proliferation of adipocytes [56] through up-regulation of Pparg mRNA [57][58][59][60]. Indeed, significant up-regulation of the mRNA level of Pparg in the HL group was observed ( Table 6).…”

Section: Discussionmentioning

confidence: 97%

Three Dissimilar High Fat Diets Differentially Regulate Lipid and Glucose Metabolism in Obesity‐Resistant Slc:Wistar/ST Rats

Hashimoto

Yamada

Tsushima

et al. 2013

Lipids

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Epidemiologic and ecologic studies suggest that dietary fat plays an important role in the development of obesity. Certain Wistar rat strains do not become obese when fed high-fat diets unlike others. In a preliminary study, we confirmed that Slc:Wistar/ST rats did not become obese when fed high-fat diets. The mechanisms governing the response of hepatic lipid-metabolizing enzymes to large quantities of dietary lipids consumed by obesity-resistant animals are unknown. The aim of the present study is to examine how obesity-resistant animals metabolize various types of high-fat diets and why they do not become obese. For this purpose, male Slc:Wistar/ST rats were fed a control low-fat diet (LS) or a high-fat diet containing fish oil (HF), soybean oil (HS), or lard (HL) for 4 weeks. We observed their phenotypes and determined lipid profiles in plasma and liver as well as mRNA expression levels in liver of genes related to lipid and glucose metabolism using DNA microarray and quantitative reverse transcriptase polymerase chain analyses. The body weights of all dietary groups were similar due to isocaloric intakes, whereas the weight of white adipose tissues in the LS group was significantly lower. The HF diet lowered plasma lipid levels by accelerated lipolysis in the peroxisomes and suppressed levels of very-low-density lipoprotein (VLDL) secretion. The HS diet promoted hepatic lipid accumulation by suppressed lipolysis in the peroxisomes and normal levels of VLDL secretion. The lipid profiles of rats fed the LS or HL diet were similar. The HL diet accelerated lipid and glucose metabolism.

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Section: Discussionmentioning

confidence: 97%

Three Dissimilar High Fat Diets Differentially Regulate Lipid and Glucose Metabolism in Obesity‐Resistant Slc:Wistar/ST Rats

Hashimoto

Yamada

Tsushima

et al. 2013

Lipids

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“…The PPARbinding site (amino acids 623–770) of human SRC-1 was cloned into pROX-FL92 (Protein Express, Choshi, Japan). This expression vector enabled the addition of the FL tag of 12 amino acids [22] and the His tag to upstream and downstream of SRC-1 respectively. With an umber codon inserted during the addition of the FL tag and CloveDirect TAMRA (6-carboxytetramethylrhodamine; Protein Express), the pinpoint fluorescent labelling protein to which TAMRA-aminophenylalanine was introduced into the ninth amino acid was produced.…”

Section: Methodsmentioning

confidence: 99%

Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist

Goto

Nagai

Egawa

et al. 2011

Biochemical Journal

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The cholesterol biosynthetic pathway produces not only sterols but also non-sterol mevalonate metabolites involved in isoprenoid synthesis. Mevalonate metabolites affect transcriptional and post-transcriptional events that in turn affect various biological processes including energy metabolism. In the present study, we examine whether mevalonate metabolites activate PPARγ (peroxisome-proliferator-activated receptor γ), a ligand-dependent transcription factor playing a central role in adipocyte differentiation. In the luciferase reporter assay using both GAL4 chimaera and full-length PPARγ systems, a mevalonate metabolite, FPP (farnesyl pyrophosphate), which is the precursor of almost all isoprenoids and is positioned at branch points leading to the synthesis of other longer-chain isoprenoids, activated PPARγ in a dose-dependent manner. FPP induced the in vitro binding of a co-activator, SRC-1 (steroid receptor co-activator-1), to GST (glutathione transferase)–PPARγ. Direct binding of FPP to PPARγ was also indicated by docking simulation studies. Moreover, the addition of FPP up-regulated the mRNA expression levels of PPARγ target genes during adipocyte differentiation induction. In the presence of lovastatin, an HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitor, both intracellular FPP levels and PPARγ-target gene expressions were decreased. In contrast, the increase in intracellular FPP level after the addition of zaragozic acid, a squalene synthase inhibitor, induced PPARγ-target gene expression. The addition of FPP and zaragozic acid promotes lipid accumulation during adipocyte differentiation. These findings indicated that FPP might function as an endogenous PPARγ agonist and regulate gene expression in adipocytes.

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“…Several nonsterol isoprenoids are known agonists for nuclear receptors, including thyroid hormone receptor (FPP), estrogen receptor (FPP), PPARa (FOH, geranylgeraniol), and PPARg (FPP, FOH, geranylgeraniol) (Takahashi et al, 2002;Das et al, 2007;Goto et al, 2011b;Nagai et al, 2011). In the current study, FOH also induced CYP2B6-PBREM/XREM reporter activity in the presence of either mCAR or hCAR1; however, SQ1 did not increase the interaction of hCAR1 with the hSRC1-RID, suggesting that SQ1's mechanism of CAR activation differs from that of the ligand agonist CITCO.…”

Section: Discussionmentioning

confidence: 99%

Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes

Rondini

Duniec-Dmuchowski

Kocarek

2016

Drug Metabolism and Disposition

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Our laboratory previously reported that accumulation of nonsterol isoprenoids following treatment with the squalene synthase inhibitor, squalestatin 1 (SQ1) markedly induced cytochrome P450 (CYP)2B1 mRNA and reporter activity in primary cultured rat hepatocytes, which was dependent on activation of the constitutive androstane receptor (CAR). The objective of the current study was to evaluate whether isoprenoids likewise activate murine CAR (mCAR) or one or more isoforms of human CAR (hCAR) produced by alternative splicing (SPTV, hCAR2; APYLT, hCAR3). We found that SQ1 significantly induced Cyp2b10 mRNA (∼3.5-fold) in primary hepatocytes isolated from both CAR-wild-type and humanized CAR transgenic mice, whereas the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor pravastatin had no effect. In the absence of CAR, basal Cyp2b10 mRNA levels were reduced by 28-fold and the effect of SQ1 on Cyp2b10 induction was attenuated. Cotransfection with an expression plasmid for hCAR1, but not hCAR2 or hCAR3, mediated SQ1-induced CYP2B1 and CYP2B6 reporter activation in hepatocytes isolated from CAR-knockout mice. This effect was also observed following treatment with the isoprenoid trans,trans-farnesol. The direct agonist CITCO increased interaction of hCAR1, hCAR2, and hCAR3 with steroid receptor coactivator-1. However, no significant effect on coactivator recruitment was observed with SQ1, suggesting an indirect activation mechanism. Further results from an in vitro ligand binding assay demonstrated that neither farnesol nor other isoprenoids are direct ligands for hCAR1. Collectively, our findings demonstrate that SQ1 activates CYP2B transcriptional responses through farnesol metabolism in an hCAR1-dependent manner. Further, this effect probably occurs through an indirect mechanism.

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Development of a Novel PPARγ Ligand Screening System Using Pinpoint Fluorescence-Probed Protein

Cited by 10 publications

References 22 publications

Three Dissimilar High Fat Diets Differentially Regulate Lipid and Glucose Metabolism in Obesity‐Resistant Slc:Wistar/ST Rats

Three Dissimilar High Fat Diets Differentially Regulate Lipid and Glucose Metabolism in Obesity‐Resistant Slc:Wistar/ST Rats

Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist

Nonsterol Isoprenoids Activate Human Constitutive Androstane Receptor in an Isoform-Selective Manner in Primary Cultured Mouse Hepatocytes

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