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Cited by 38Peroxisome Proliferator-Activated ReceptorsLipid metabolism and biosynthesisCholesterol and Lipid Metabolism

The Peroxisome Proliferator–Activated Receptor (PPAR)-γ Antagonist 2-Chloro-5-Nitro-N-Phenylbenzamide (GW9662) Triggers Perilipin 2 Expression via PPARδ and Induces Lipogenesis and Triglyceride Accumulation in Human THP-1 Macrophages

Open access
Schubert, Martin, Becher, Stefanie, Wallert, Maria, Maeß, Marten B., Abhari, Masoumeh, Rennert, Knut, Mosig, Alexander S., Große, Silke +3 more·2020·Molecular Pharmacology
DOI 10.1124/mol.119.117887
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Abstract

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor family, playing pivotal roles in regulating glucose and lipid metabolism as well as inflammation. While characterizing potential PPARγ ligand activity of natural compounds in macrophages, we investigated their influence on the expression of adipophilin [perilipin 2 (PLIN2)], a well-known PPARγ target. To confirm that a compound regulates PLIN2 expression via PPARγ, we performed experiments using the widely used PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662). Surprisingly, instead of blocking upregulation of PLIN2 expression in THP-1 macrophages, expression was concentration-dependently induced by GW9662 at concentrations and under conditions commonly used. We found that this unexpected upregulation occurs in many human and murine macrophage cell models and also primary cells. Profiling expression of PPAR target genes showed upregulation of several genes involved in lipid uptake, transport, and storage as well as fatty acid synthesis by GW9662. In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels. Finally, we identified PPARδ as a mediator of the substantial unexpected effects of GW9662. Our findings show that: 1) the PPARγ antagonist GW9662 unexpectedly activates PPARδ-mediated signaling in macrophages, 2) GW9662 significantly affects lipid metabolism in macrophages, 3) careful validation of experimental conditions and results is required for experiments involving GW9662, and 4) published studies in a context comparable to this work may have reported erroneous results if PPARγ independence was demonstrated using GW9662 only. In light of our findings, certain existing studies might require reinterpretation regarding the role of PPARγ. SIGNIFICANCE STATEMENT Peroxisome proliferator–activated receptors (PPARs) are targets for the treatment of various diseases, as they are key regulators of inflammation as well as lipid and glucose metabolism. Hence, reliable tools to characterize the molecular effects of PPARs are indispensable. We describe profound and unexpected off-target effects of the PPARγ antagonist 2-chloro-5-nitro-N-phenylbenzamide (GW9662) involving PPARδ and in turn affecting macrophage lipid metabolism. Our results question certain existing studies using GW9662 and make better experimental design of future studies necessary.

AI snapshot

Written from the paper’s full text.

TL;DR

The widely used PPARγ antagonist GW9662 unexpectedly induces PLIN2 and other PPAR target genes in human and murine macrophages, rather than blocking PPARγ signaling. These effects are mediated at least in part by PPARδ activation and lead to increased lipogenesis and triglyceride accumulation in THP-1 macrophages.

Key findings

  • GW9662 (10 µM) significantly induced PLIN2 and CD36 mRNA in THP-1 macrophages by more than 2.1-fold and did not block rosiglitazone- or 15dPGJ2-induced expression; PLIN2 induction was concentration-dependent, from 2.2-fold at 1 µM up to 3.2-fold at 5–50 µM.
  • GW9662 increased PLIN2 protein about 1.9-fold at 24 hours and 1.4-fold at 48 hours, increased triglyceride content by 27% at 24 hours (no significant effect at 48 hours), and raised 14C-acetate incorporation by 34%, indicating increased lipogenesis.
  • In PPAR signaling qPCR arrays, 26.6% of genes were upregulated, 16.9% downregulated, 47.4% unchanged, and 9.1% not detectable; affected genes included those involved in lipid uptake, transport, storage, and lipogenesis.
  • PPARδ knockdown significantly reduced GW9662-induced PLIN2 and CD36 expression, and the PPARδ antagonist GSK3787 completely abolished GW9662 induction of PLIN2 and CD36, supporting PPARδ as a mediator.

Methods

  • Human THP-1 and U937 cells, murine RAW264.7 and J774A.1 cells, primary human PBMC-derived macrophages, and mouse peritoneal macrophages were treated with GW9662, PPAR agonists/antagonists, or siRNA.
  • Main analyses included RT-qPCR, Western blotting, immunofluorescence, Human PPAR Signaling 384 StellARray qPCR arrays, enzymatic triglyceride/cholesterol measurements, and 14C-acetate incorporation for lipogenesis.
  • Statistical comparisons used paired t tests, repeated-measures ANOVA with Tukey’s or Dunnett’s post hoc tests, and one-way ANOVA with Tukey’s post hoc test; P < 0.05 was considered significant.

Limitations

  • The paper states that the mechanism of PPARδ activation by GW9662 is not fully elucidated, that direct interaction was not proven experimentally, and that an indirect signaling cascade leading to PPARδ activation cannot be completely ruled out.
  • The paper notes that PPARδ knockdown did not completely abolish target gene induction, that assignment of array genes to specific PPAR subtypes is not unambiguous, and that most array genes cannot be finally proven to be regulated via PPARδ.
  • Evident from the text: some findings rely on pooled array samples, peritoneal macrophage experiments were performed only twice, CD36 responses were inconsistent across cell models, and GW9662 lot-to-lot variability produced fold changes ranging from about three- to sixfold.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1124/mol.119.117887

Cites

54 of 54 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

36 of 85 cited sources checked · 22 supported · 2 flagged · accuracy not yet measured

  • Interpretationcitation

    GW9662 influences genes in all classic PPAR-related metabolic pathways (lipid metabolism, glucose metabolism, immunity, and inflammation).

    “Taken together, GW9662 influences genes in all classic PPARrelated metabolic pathways (lipid metabolism, glucose metabolism, immunity, and inflammation) (Clark, 2002;Wang, 2010).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Clark, 2002 source
    • cites Wang, 2010 source

      The Introduction (page unavailable) describes PPARs as regulators of lipid metabolism and inflammation and discusses their relationship to insulin resistance. Our copy does not identify GW9662 or report that it influences genes in all the listed pathways, so it supports the PPAR-related background but not the claim about GW9662. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The Lonza Human PPAR Signaling 384 StellARray qPCR arrays measure expression of genes regulated by PPAR subtypes or PPAR-interacting partners.
  • Interpretationcitation

    The mediation of GW9662 effects by PPARδ is likely due to the abundant expression of PPARδ, a hallmark of THP-1 cell differentiation.

    “This is likely reasoned by the abundant expression of PPARd, a hallmark of THP-1 cell differentiation (Vosper et al., 2001) (Supplemental Fig. 16).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Vosper et al., 2001 source
  • Premisecitation

    GW9662 shows no effect in a cell-based full-length PPARγ luciferase assay.

    “Though GW9662 shows no effect in a cellbased full-length PPARg luciferase assay, the expression of the PPARg target genes FABP4 and CD36 tends to be increased by GW9662 in”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Brust et al., 2018 source
  • Premisecitation

    Malonyl-CoA suppresses beta-oxidation.

    “This enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which is not only a substrate for fatty acid biosynthesis but is also known to suppress b-oxidation (Tong and Harwood, 2006).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Tong and Harwood, 2006 source
  • Premisecitation

    PPARd activation promotes lipid accumulation in THP-1 macrophages in the presence of serum.

    “Consequently, the PPARd activation promotes lipid accumulation in THP-1 macrophages in the presence of serum (Vosper et al., 2001).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Vosper et al., 2001 source
  • Classified as probably not a substantial claim. 56
  • May combine two claims. 38
  • The quote only partly states this claim. 1

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A concentration of 10 mM GW9662 is commonly used in published scientific studies.

    “However, we used GW9662 at a concentration of 10 mM for further experiments because this concentration revealed reproducible results and is a commonly used concentration in published scientific studies (Lea et al., 2004;Kourtidis et al., 2009).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Lea et al., 2004
    • cites Kourtidis et al., 2009 source

      The Results (Figure 1; page not provided in our copy) report GW9662 at 10 μmol/l, not 10 mM—a 1,000-fold difference. The paper also does not establish that 10 mM is commonly used. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    A recent report supports the notion of different effects in artificial and native systems.

    “A recent report supports the notion of different effects in artificial and”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Brust et al., 2018 source

      The Results section, “T0070907 lowers basal activity of PPARγ” (page unavailable), reports that qPCR measurements of PPARγ target genes in 3T3-L1 cells confirmed the ligand activity profiles, showing similar activating, neutral, and repressive trends. Rather than supporting different effects in the reporter and native-gene systems it tested, the paper reports similar trends. (LLM, conf=0.86)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PPARα and PPARδ are related to lipid metabolism because of their key role in the regulation of fatty acid oxidation and lipoprotein metabolism.

    “PPARa and PPARd are related to lipid metabolism because of their key role in the regulation of fatty acid oxidation and lipoprotein metabolism (Luquet et al., 2005;Lefebvre et al., 2006).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Luquet et al., 2005 source
    • cites Lefebvre et al., 2006 source

      The ‘PPARα and intracellular lipid metabolism’ section links PPARα to fatty acid oxidation and lipoprotein assembly and transport; the ‘Lipoprotein metabolism’ section describes its effects on lipoproteins. The copy identifies PPARδ in the Introduction but does not establish the claimed roles for PPARδ. Page numbers are unavailable in our copy. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    GW9662 covalently and irreversibly binds to the PPARγ ligand binding pocket, preventing activating ligand binding and interrupting PPARγ signal transduction.

    “GW9662 has been shown to covalently and irreversibly bind to the PPARg ligand binding pocket (Leesnitzer et al., 2002;Brust et al., 2018).The binding of activating ligands is thereby prevented, and the signal transduction via PPARg is interrupted.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Leesnitzer et al., 2002 source
    • cites Brust et al., 2018 source

      The Introduction and Discussion state that GW9662 covalently binds the orthosteric PPARγ pocket and blocks other orthosteric ligands, preventing activation by those agonists; the Discussion also calls it nondissociative. But the Results report that GW9662 did not significantly affect basal PPARγ transcription, so the broader claim that PPARγ signal transduction is interrupted overstates the source. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Insulin-sensitizing effects of thiazolidinediones, a class of PPARγ ligands, are used for the treatment of type 2 diabetes mellitus.

    “the insulinsensitizing effects of thiazolidinediones, a class of PPARg ligands, are used for the treatment of type 2 diabetes mellitus (Tontonoz and Spiegelman, 2008;Hong et al., 2018).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Tontonoz and Spiegelman, 2008

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Hong et al., 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Chemical antagonists bind to the respective PPAR isoform, leading to maintenance of the corepressor complex and prevention of agonist binding.

    “Chemical antagonists bind to the respective PPAR isoform, leading to the maintenance of the corepressor complex and to the prevention of agonist binding (Harmon et al., 2011).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Harmon et al., 2011 source

      Section 4.4 (Coactivators and Corepressors; page unavailable) says corepressors bind in the presence of antagonists, whereas agonist binding displaces them. It does not state that chemical antagonists bind a particular PPAR isoform or prevent agonists from binding, so those parts of the mechanism are unsupported. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PPARγ is predominantly expressed in adipose tissue and plays a central role in adipogenesis.

    “PPARg is predominantly expressed in adipose tissue and plays a central role in adipogenesis (Wang, 2010).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Wang, 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Upon ligand binding, the corepressor complex is replaced by coactivators, resulting in transcription of PPAR target genes.

    “Upon ligand binding, the corepressor complex is replaced by coactivators, resulting in the transcription of PPAR target genes.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Varga et al., 2011 source

      In “Mechanisms of PPARγ mediated gene regulation” (page unavailable), the paper describes liganded PPARγ/RXR recruiting coactivators and usually upregulating target genes. It does not say that ligand binding replaces a corepressor complex with those coactivators; the corepressor-replacement step is unsupported. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PPARα is mainly found in liver and muscles.

    “PPARa is mainly found in liver and muscles (Lefebvre et al., 2006).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Lefebvre et al., 2006 source

      The “PPARα and intracellular lipid metabolism” section says PPARα is highly expressed in the liver and skeletal muscles. It also lists brown fat, heart, kidneys, and cells of atherosclerotic lesions, without saying expression is mainly in liver and muscle. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PPARs are activated by a variety of molecules and act mainly as sensors for fatty acids and fatty acid-derived metabolites.

    “PPARs are activated by a variety of molecules and act mainly as sensors for fatty acids and fatty acid-derived metabolites (Lefebvre et al., 2006;Tontonoz and Spiegelman, 2008;Neels and Grimaldi, 2014).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Lefebvre et al., 2006 source
    • cites Tontonoz and Spiegelman, 2008

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites Neels and Grimaldi, 2014 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The most common model for PPAR-dependent gene regulation is direct transcriptional regulation by heterodimerization of a PPAR isoform with retinoid X receptors.

    “The most common model for PPAR-dependent gene regulation is the direct transcriptional regulation by heterodimerization of a PPAR isoform with retinoid X receptors.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Varga et al., 2011 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    As transcription factors, PPARs regulate various genes implicated in lipid and glucose metabolism, inflammation, proliferation, and differentiation.

    “As transcription factors, PPARs regulate various genes implicated in lipid and glucose metabolism, inflammation, proliferation, and differentiation (Varga et al., 2011;Vrablík and Ce ska, 2015).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Varga et al., 2011 source
    • cites Vrablík and Ce ska, 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Anti-inflammatory effects and regulatory roles in glucose metabolism have been reported for both PPARα and PPARδ.

    “Furthermore, anti-inflammatory effects and regulatory roles in glucose metabolism have been reported for both isoforms (Lefebvre et al., 2006;Neels and Grimaldi, 2014;Magadum and Engel, 2018).”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Lefebvre et al., 2006 source

      The Introduction and Gene repression by PPARα sections report roles for PPARα in glucose homeostasis and anti-inflammatory effects. The Introduction links the three PPARs generally to diabetes and inflammation, but the passages do not establish both effects specifically for PPARδ. Page numbers were not provided in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Neels and Grimaldi, 2014 source
    • cites Magadum and Engel, 2018 source

      Section 3.3 (page not provided) describes anti-inflammatory properties of PPARβ/δ, and Section 3.1 reports that its overexpression increases glucose utilization in the liver and heart. The paper identifies PPARα as another isoform but does not establish both effects for PPARα, so it supports only the PPARδ portion of the claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    In the absence of ligands, the PPAR-RXR heterodimer binds to the PPAR response element in the promoter of target genes, binds a corepressor complex, and represses transcription.

    “In the absence of ligands, the heterodimer is bound to the so-called PPAR response element in the promoter of target genes.In this state, the heterodimer binds a corepressor complex and represses the transcription of target genes.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Varga et al., 2011 source

      The Introduction’s “DNA recognition by PPARs” section says PPAR–RXR heterodimers bind PPAR response elements, but describes binding in its simplified model as occurring upon ligand binding. The “Mechanisms of PPARγ mediated gene regulation” section discusses a repressor complex in a different mechanism involving ligand-bound PPARγ, not an unliganded PPAR–RXR heterodimer bound to a PPRE. Our copy therefore does not support the claimed ligand-free corepressor mechanism. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    T0070907 is a PPARγ antagonist with inverse agonist properties.

    “we were interested in the PPARg antagonist 2-chloro-5-nitro-N-4-pyridinyl-benzamide (T0070907) (Lee et al., 2002) with inverse agonist properties (Lee et al., 2002;Brust et al., 2018) as an alternative.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Lee et al., 2002 source
    • cites Brust et al., 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The expression of PPARγ target genes FABP4 and CD36 tends to be increased by GW9662 in native adipocyte-like cells.

    “Though GW9662 shows no effect in a cellbased full-length PPARg luciferase assay, the expression of the PPARg target genes FABP4 and CD36 tends to be increased by GW9662 in”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Brust et al., 2018 source

      The Methods section (‘3T3-L1 cell gene expression analysis’; page not specified) confirms that the study measured FABP4 and CD36 expression in 3T3-L1 cells after compound treatment. The Results section (‘T0070907 lowers basal activity of PPARγ’; page not specified) describes similar activating, neutral, and repressive trends in those cells, but characterizes GW9662 as having no significant effect in the transcription assay and does not state that it tended to increase either gene’s expression. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    The affinity of PPAR subtypes for ligands is thought to be mainly determined by the size of the ligand-binding pocket.

    “The affinity of the PPAR subtypes for ligands is thought to be mainly determined by the size of the ligand-binding pocket (Bugge and Mandrup, 2010).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Bugge and Mandrup, 2010 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetyl-coenzyme A carboxylase-a is the rate-limiting enzyme in fatty acid synthesis.

    “In particular, the upregulation of acetyl-coenzyme A carboxylase-a as the rate-limiting enzyme in fatty acid synthesis (Tong and Harwood, 2006) is noteworthy.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Tong and Harwood, 2006 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Prior studies report that PPARδ contributes to PLIN2 and CD36 expression.

    “These findings are in line with studies reporting the contribution of PPARd to PLIN2 (Chawla et al., 2003;Fan et al., 2009) and CD36 (Li et al., 2004;Bojic et al., 2012) expression.”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Chawla et al., 2003 source
    • cites Fan et al., 2009 source
    • cites Li et al., 2004

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    Differential tissue distribution is one main factor of subtype-specific effects of PPARs.

    “This assumption is supported by the fact that differential tissue distribution is one main factor of subtype-specific effects of PPARs (Bugge and Mandrup, 2010).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Bugge and Mandrup, 2010 source

      The Introduction reports that PPAR subtypes have different tissue distributions alongside distinct effects, but describes those expression patterns as a reflection of subtype-specific properties. Section 2 says the subtypes retain specificity when expressed at comparable levels in the same cell line and identifies intrinsic properties as key determinants. The paper therefore supports the difference in tissue distribution, but not its characterization as a main factor causing subtype-specific effects. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    T0070907 has similar effects to GW9662 on RNA but not necessarily on the protein level.

    “Furthermore, the structurally related antagonist or inverse agonist T0070907 (Lee et al., 2002;Hughes et al., 2014;Brust et al., 2018) might be a useful alternative depending on the experimental setup (Supplemental Fig. 13), as we observed similar effects to GW9662 on RNA but not necessarily on the protein level.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites Lee et al., 2002 source
    • cites Hughes et al., 2014 source
    • cites Brust et al., 2018 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA to malonyl-CoA.

    “This enzyme catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, which is not only a substrate for fatty acid biosynthesis but is also known to suppress b-oxidation (Tong and Harwood, 2006).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Tong and Harwood, 2006 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    The studies of Vosper et al. (2001) on PPARd and lipid accumulation in macrophages most accurately resemble our experimental setup because we did not use any lipoproteins as stimulus.

    “As we did not use any lipoproteins as stimulus, the studies of Vosper et al. (2001) on PPARd and lipid accumulation in macrophages most accurately resemble our experimental setup.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Vosper et al., 2001 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The higher lipid levels in BT474 cells are primarily caused by an increase in palmitic and stearic acid.

    “The higher lipid levels in BT474 cells are primarily caused by an increase in palmitic and stearic acid (Kourtidis et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kourtidis et al., 2009 source

      The Results section, “PPARγ inhibition results in increased levels of fats,” says the higher fat levels after PPARγ inhibition were primarily due to increases in palmitic and stearic acids. The citing sentence omits that treatment condition, so the passage does not establish that higher lipid levels in BT474 cells generally have the same explanation. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PPARd selectively responds to different stimuli of lipid metabolism; VLDL reliably activates PPARd, whereas HDL and LDL do not.

    “Furthermore, PPARd selectively responds to different”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Chawla et al., 2003 source
    • cites Lee et al., 2006 source
    • cites Bojic et al., 2012 source

  • May combine two claims.

    Findingreasoning

    GW9662 elevated lipogenesis and increased triglyceride levels in THP-1 macrophages.

    “In line with this and with upregulation of PLIN2 protein, GW9662 elevated lipogenesis and increased triglyceride levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The off-target action of GW9662 was confirmed in pharmacological antagonist experiments and in studies with siRNA-mediated knockdown of PPARδ.

    “The offtarget action of GW9662 was confirmed in pharmacological antagonist experiments as well as in studies with siRNA-mediated knockdown of PPARd.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In the Lonza Human PPAR Signaling 384 StellARray qPCR array, 26.6% of genes were upregulated by 24-hour treatment with 10 mM GW9662 in THP-1 macrophages.

    “As shown in Supplemental Fig. 7A, expression of most of the genes (47.4%) remained unchanged by GW9662, whereas expression was upregulated for 26.6% and downregulated for 16.9% of the genes.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The Lonza Human PPAR Signaling 384 StellARray qPCR arrays measure expression of genes regulated by PPAR subtypes or PPAR-interacting partners.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingnone given

    Results for the impact of GW9662 on CD36 expression were inconsistent.

    “However, as mentioned above, results for the impact of GW9662 on CD36 expression were inconsistent.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In the case of CD36, GW9662 was almost as potent as 15dPGJ2 to induce CD36 mRNA expression but was less potent than rosiglitazone.

    “In the case of CD36, GW9662 was almost as potent as 15dPGJ2 to induce CD36 mRNA expression but was less potent than rosiglitazone (Fig. 3A).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    In the Lonza Human PPAR Signaling 384 StellARray qPCR array, 16.9% of genes were downregulated by 24-hour treatment with 10 mM GW9662 in THP-1 macrophages.

    “As shown in Supplemental Fig. 7A, expression of most of the genes (47.4%) remained unchanged by GW9662, whereas expression was upregulated for 26.6% and downregulated for 16.9% of the genes.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The Lonza Human PPAR Signaling 384 StellARray qPCR arrays measure expression of genes regulated by PPAR subtypes or PPAR-interacting partners.

  • May combine two claims.

    Findingown data

    Pretreatment of THP-1 macrophages with GW9662 or simultaneous incubation with agonist and antagonist for 24 hours led to augmented levels of PLIN2 mRNA.

    “Pretreatment of THP-1 macrophages with GW9662 as well as incubating the cells in combination with agonist and antagonist at the same time for 24 hours also led to augmented levels of PLIN2 mRNA (Supplemental Fig. 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the panel of human and mouse macrophage cells tested, GW9662 (10 mM, 24 h) induced PLIN2/Plin2 mRNA expression in every cell type except RAW264.7, at least to 1.9-fold (P < 0.05 for THP-1, U937, and J774.A1).

    “Except for RAW264.7 cells, GW9662 induced expression of PLIN2/Plin2 mRNA in every cell type investigated at least to the 1.9-fold, as shown in Fig. 2 (top; P , 0.05 for THP-1, U937, and J774.A1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In PPARdelta-depleted THP-1 macrophages, GW9662 induced PLIN2 expression only to 2.5-fold, compared with 4.7-fold in negative control siRNA or PPARalpha siRNA cells and 4.2-fold in PPARgamma-depleted cells.

    “PLIN2 was merely induced to the 2.5fold compared with the respective control in PPARd-depleted cells, whereas an increase to the 4.7-fold in cells transfected with negative control siRNA or with PPARa siRNA and an increase to the 4.2-fold over the respective control was observed in PPARg-depleted cells (Fig. 7).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with GSK3787 or GW9662 alone significantly induced the expression of PLIN2 and CD36.

    “Treatment with GSK3787 or GW9662 alone significantly induced the expression of PLIN2 and CD36.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The genes with changed expression are involved in immunity and inflammation, signal transduction and transcription, and glucose metabolism and lipid metabolism.

    “These genes are involved in immunity and inflammation, signal transduction and transcription, and glucose metabolism and, particularly, lipid metabolism (Supplemental Table 5; Supplemental Fig. 7).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Findingown data

    The P value for GW9662 versus control was less than 0.001, calculated using a two-tailed paired t test.

    “***P , 0.001.The P value is provided for GW9662 vs. control, calculated using two-tailed paired t test.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GW9662 exhibits PPARg agonist-like activity and fails to block PPARg agonists in human THP-1 macrophages.

    “GW9662 exhibits PPARg agonist-like activity and fails to block PPARg agonists in human THP-1 macrophages.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GW9662 stimulated expression of PLIN2 and CD36 significantly.

    “GW9662 stimulated expression of PLIN2 and CD36 significantly.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In GW9662-treated THP-1 cells, hormone-sensitive lipase was upregulated in expression profiling but downregulated in confirmatory RT-qPCR experiments.

    “However, patatin-like phospholipase domain containing 2 remained unaffected, whereas hormone-sensitive lipase was upregulated in our expression profiling (Supplemental Table 3) but was downregulated in confirmatory RT-qPCR experiments (Supplemental Fig. 12A) in GW9662-treated THP-1 cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    GW9662 induces expression of PLIN2/Plin2 and CD36/Cd36 in different human and murine macrophage cell models.

    “GW9662 induces expression of PLIN2/ Plin2 and CD36/Cd36 in different human and murine macrophage cell models.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GW9662 itself induces expression of target genes linked to PPARg, such as PLIN2 and CD36, rather than blocking it.

    “Moreover, GW9662 itself induces expression of target genes that have been linked to PPARg, such as PLIN2 and CD36, rather than blocking it.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Transfection efficiency was determined after the 24-hour incubation period by RT-qPCR.

    “Transfection efficiency was determined after the 24-hour incubation period by RT-qPCR (Supplemental Fig. 14).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    PPARδ is ubiquitously expressed, most abundantly in tissues with high fatty acid metabolism.

    “While PPARd is ubiquitously expressed (most abundantly in tissues with high fatty acid metabolism) (Neels and Grimaldi, 2014)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Neels and Grimaldi, 2014 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PPARγ exerts considerable effects on lipid and glucose metabolism and insulin sensitivity.

    “In addition, PPARg exerts considerable effects on lipid and glucose metabolism and insulin sensitivity (Tontonoz and Spiegelman, 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Tontonoz and Spiegelman, 2008 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Mononuclear cells were isolated from buffy coats of healthy volunteers with written informed consent and ethics committee approval.

    “Blood donors were informed about the aim of the study and gave written informed consent.The procedure was approved by the ethics committee of the University Hospital Jena (2446-12/08) and conducted according to the ethical principles defined by the declaration of Helsinki.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All cell lines were routinely tested for mycoplasma contamination.

    “All cell lines were routinely tested for mycoplasma contamination using the mycoplasma detection kit PP-401 (Jena Bioscience, Jena, Germany) based on a PCR reaction.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisereasoning

    Chemical antagonists are considered useful tools in PPAR research, especially for verification of possible PPAR ligands.

    “Thus, chemical antagonists are considered as useful tools in PPAR research, especially for the verification of possible PPAR ligands.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Animal procedures were approved by the Animal Care and Use Committee of Thuringia.

    “Animal procedures, including isolation of peritoneal macrophages, were approved by the Animal Care and Use Committee of Thuringia (permit number 02-003/09) and were in line with the National Institutes of Health guidelines for the care and use of laboratory animals.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    GW9662 is known as a selective PPARγ antagonist and is widely accepted to demonstrate participation of PPARγ in signaling cascades.

    “GW9662, known as a selective peroxisome proliferator (PPAR)g antagonist, is widely accepted to demonstrate participation of PPARg in signaling cascades (Leesnitzer et al., 2002;Lea et al., 2004;Nielsen et al., 2006).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Leesnitzer et al., 2002 source
    • cites Lea et al., 2004
    • cites Nielsen et al., 2006 source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Samples were prepared in biologic replicates, and analyses were performed as technical duplicates.

    “Samples were prepared in biologic replicates, and analyses were performed as technical duplicates as indicated.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Experiments were performed at least three times.

    “Experiments were performed at least three times.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PPARδ is regarded as a promising target for the treatment of metabolic syndrome because of its positive effects on serum cholesterol and lipid profiles.

    “PPARd is regarded as a promising target for the treatment of metabolic syndrome because of its positive effects on serum cholesterol and lipid profiles (Neels and Grimaldi, 2014)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Neels and Grimaldi, 2014 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Key findings were confirmed using different lots of GW9662 obtained either from Sigma-Aldrich or Enzo Life Sciences.

    “Key findings were confirmed using different lots of GW9662 obtained either from Sigma-Aldrich or Enzo Life Sciences.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Fibrates, agonists of PPARα, have been used as hypolipidemic drugs to treat hypertriglyceridemia.

    “In brief, fibrates, agonists of PPARa, have been used as hypolipidemic drugs to treat hypertriglyceridemia (Vrablík and Ce ska, 2015)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Vrablík and Ce ska, 2015

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    The expression of alpha-tubulin is not affected by GW9662 in THP-1 macrophages.

    “The expression of a-tubulin was not affected by GW9662 (Fig. 4A).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Expression of the reference genes RPL37A and Ppib remained unchanged in all experiments.

    “Expression of the reference genes RPL37A and Ppib remained unchanged in all experiments (Fig. 2, bottom).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Transfection efficiency was verified for siRNA knockdown of each PPAR subtype in THP-1 macrophages.

    “Resulting THP-1 macrophages were transfected with either negative control siRNA or siRNA for each PPAR subtype and transfection efficiency was verified (Supplemental Fig. 14).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data

    Expression of the reference gene RPL37A was not affected and was used for normalization.

    “Expression of the reference gene RPL37A was not affected and was used for normalization (Fig. 1, bottom).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    GW9662 reliably induces PPARγ target genes, a robust effect in different experimental setups.

    “These findings indicate that upregulation of PPARg target genes is a robust effect in different experimental setups.”
    • Also stated in: § Concentration-Dependent Activation of PLIN2 Expression by GW9662. · boosted
      “As GW9662 reliably induces PPARg target genes”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    Most PPAR target genes are regulated by all three PPAR subtypes.

    “thus, most PPAR target genes are regulated by all three subtypes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    GW9662 directly activates PPARδ in macrophages, although this interaction was not proven experimentally.

    “Summarizing the above delineated results, we propose that GW9662 directly activates PPARd in macrophages but do not prove this interaction experimentally.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    Expression of the reference gene remained unchanged in the experimental setup and was used for normalization.

    “Expression of the reference gene remained unchanged in the experimental setup and was used for normalization.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The profiling analysis allows the attribution of these genes to PPAR subtypes.

    “This profiling analysis allows the attribution of these genes to PPAR subtypes (Supplemental Table 4).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Supplemental Table 4

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    PPARγ has a reported regulatory function on PLIN2 and CD36 expression in macrophages.

    “the reported regulatory function of PPARg on PLIN2 (Buechler et al., 2001;Fan et al., 2009) and CD36 (Chawla et al., 2001;Moore et al., 2001;Tontonoz et al., 1998) expression in macrophages.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Buechler et al., 2001 source
    • cites Fan et al., 2009 source
    • cites Chawla et al., 2001 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GW9662 covalently modifies PPARα and PPARδ at higher concentrations.

    “Although GW9662 was identified as a potent antagonist of PPARg, it also covalently modifies PPARa and PPARd at higher concentrations (Leesnitzer et al., 2002).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Leesnitzer et al., 2002 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GW9662 was initially characterized as an antagonist of PPARδ using an assay based on a fusion protein containing the ligand binding domain of PPARδ and the galactose-responsive transcription factor 4.

    “Contradictory to this, GW9662 has initially been characterized as an antagonist of PPARd using an assay based on a fusion protein containing the ligand binding domain of PPARd and the galactose-responsive transcription factor 4 (Leesnitzer et al., 2002)”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Leesnitzer et al., 2002 source

  • Probably not a substantial claim: may not pass S2 (checkable).

    Interpretationreasoning

    The awareness of the PPARδ-activating properties of GW9662 requires a reinterpretation of results obtained using GW9662 in previous studies.

    “The awareness of the PPARdactivating properties of GW9662 requires a reinterpretation of results obtained using GW9662 in previous studies.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: GW9662 is a PPARγ antagonist.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    The expression of PPAR target genes is dependent on the differential expression of the PPAR subtypes in cells as well as the setting of the target sites, such as chromatin modification and the combination of other transcription factors, which is highly cell-type specific.

    “The expression of PPAR target genes is undoubtedly dependent on the differential expression of the PPAR subtypes in cells as well as the”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Nielsen et al., 2006 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    The initial characterization of GW9662 as a PPARδ antagonist was later confirmed with a similar approach.

    “and was later confirmed with a similar approach (Leesnitzer et al.,”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Leesnitzer et al.,

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisecitation

    GW9662 was identified as a potent antagonist of PPARγ.

    “Although GW9662 was identified as a potent antagonist of PPARg, it also covalently modifies PPARa and PPARd at higher concentrations (Leesnitzer et al., 2002).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Leesnitzer et al., 2002 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PPARd is known for stimulation of fatty acid oxidation in different tissues such as adipose tissue and skeletal muscles.

    “In contrast to this assumption, PPARd is known for the stimulation of fatty acid oxidation in different tissues such as adipose tissue and skeletal muscles (Reilly and Lee, 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Reilly and Lee, 2008 source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Pretreatment with PPARd agonists attenuates VLDL-induced triglyceride accumulation by indirect inhibition of lipoprotein lipase.

    “Interestingly, pretreatment with PPARd agonists attenuates VLDL-induced triglyceride accumulation by indirect inhibition of lipoprotein lipase (Bojic et al., 2012).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Bojic et al., 2012 source

  • May combine two claims.

    Interpretationcitation

    PPARd apparently exerts different effects on lipid metabolism and is not invariably a stimulator of lipid catabolism.

    “PPARd apparently exerts different effects on lipid metabolism and thus is not invariably a stimulator of lipid catabolism.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    PPARd possibly acts as a switch: moderate activation favors lipid storage, whereas excessive activation shifts metabolism to lipid catabolism, likely to prevent lipid overload.

    “Thus, PPARd possibly acts as a switch; moderate activation of PPARd (e.g., by VLDL or a PPARd agonist) might favor lipid storage, whereas excessive PPARd activation could shift the metabolism to lipid catabolism, likely to prevent lipid overload.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GW9662 causes triglyceride accumulation in T47D cells.

    “GW9662 causes triglyceride accumulation in T47D cells (Lea et al., 2004) as well as BT474 and MCF-7 cells (Kourtidis et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lea et al., 2004

  • May combine two claims.

    Interpretationreasoning

    GW9662 induces PLIN2 and CD36 only moderately and no additional stimuli were used, so moderate activation of PPARd is expected.

    “As GW9662 induces PLIN2 and CD36 only moderately and no additional stimuli were used, we expect moderate activation of PPARd.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Study design and conductown data

    Expression of the reference gene RPL37A remained unchanged in both experimental setups and was used for normalization.

    “Expression of the reference gene remained unchanged in both experimental setups and was used for normalization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    The sparse data available on PPARd and lipid accumulation in macrophages are in agreement with our finding that GW9662 promotes lipid accumulation via activation of PPARd and consequent induction of genes involved in lipid metabolism, such as PLIN2 and CD36.

    “Taken together, the sparse data available on PPARd and lipid accumulation in macrophages are in agreement with our finding that GW9662 promotes lipid accumulation via activation of PPARd and consequent induction of genes involved in lipid metabolism, such as PLIN2 and CD36.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: Most of the genes in the expression array are regulated via activation of PPARd by GW9662.
    • The authors state this as an assumption: The observed effects on lipogenesis and triglyceride accumulation in response to GW9662 are a global consequence of PPARd activation.
79 of 79

  • The paper relies on this without support.

    CD36 is a PPARγ target gene.

    “the PPARg target genes perilipin 2 (PLIN2) and cluster of differentiation 36 (CD36)”

  • The paper relies on this without support.

    GW9662 is widely used in scientific studies that do not necessarily resemble the initial experimental setup.

    “This is of interest, as GW9662 is widely used in scientific studies that do not necessarily resemble our initial setup.”

  • The paper relies on this without support.

    GW9662 is often used to confirm PPARγ-mediated effects.

    “Because GW9662 is often used to confirm PPARg-mediated effects”

  • The authors state this as an assumption.

    No comparable effects to the authors' observations have been reported.

    “and no comparable effects to our observations have been reported (to the best of our knowledge)”

  • The paper relies on this without support.

    The Lonza Human PPAR Signaling 384 StellARray qPCR arrays measure expression of genes regulated by PPAR subtypes or PPAR-interacting partners.

    “These arrays allow the measurement of the expression of 384 genes that are either regulated by the PPAR subtypes (PPARa, PPARb/d, and PPARg) themselves, are PPAR-interacting partners, or serve as controls (HsGenomic and Hs18s) and reference genes (nine most stably expressed genes).”

  • The paper relies on this without support.

    GW9662 is a PPARγ antagonist.

    “the well-known PPARg antagonist GW9662”
    “the accepted and frequently used PPARg antagonist GW9662”

  • The authors state this as an assumption.

    The predominance of PPARδ in THP-1-derived macrophages (and other macrophage cell lines in this study) likely favors the effects of GW9662 via PPARδ over PPARγ and PPARα.

    “The resulting predominance of PPARd in THP-1-derived macrophages (as well as in the other macrophage cell lines in this study; Supplemental Table 6) likely favors the effects of GW9662 via PPARd over PPARg and PPARa.”

  • The authors state this as an assumption.

    Most of the genes in the expression array are regulated via activation of PPARd by GW9662.

    “Given the identification of PPARd as mediator of PLIN2 and CD36 regulation, most of the genes in the array are likely also regulated via activation of PPARd by GW9662.”
    “Although the predominance of PPARd in THP-1 macrophages supports this assumption, we cannot give final proof.”

  • The authors state this as an assumption.

    The observed effects on lipogenesis and triglyceride accumulation in response to GW9662 are a global consequence of PPARd activation.

    “Given that GW9662 regulates PLIN2 and CD36 via PPARd, the observed effects on lipogenesis and triglyceride accumulation in response to GW9662 (Figs. 5 and 6) might be a global consequence of the activation of PPARd.”

  • The paper relies on this without support.

    The role of PPARd in lipid metabolism of macrophages is insufficiently characterized, and results of studies on this topic are inconsistent.

    “However, the role of PPARd in lipid metabolism of macrophages is insufficiently characterized, and results of studies on this topic are inconsistent.”
10 of 10

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Authors
Schubert, M., Becher, S., Wallert, M., Maeß, M. B., Abhari, M., Rennert, K., Mosig, A. S., Große, S., Heller, R., Grün, M., & Lorkowski, S.
Affiliations
Jena University Hospital (DE)
ORCID
Venue
Molecular Pharmacology
Publisher
Elsevier BV
Published
2020-03
Volume
97
Issue
3
Pages
212-225
ISSN
0026-895X (ISSN-L)
DOI
10.1124/mol.119.117887
OpenAlex
W2997062013
Semantic Scholar
89a983538846b5576a7920f3550fa89e29336017
Cited by
35
References
54
Citations by year
2026: 2 · 2025: 7 · 2024: 7 · 2023: 4 · 2022: 7 · 2021: 8
FWCI
1.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Pharmacology (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft (RTG1715, DFG RTG 1715)
  • Ministerul Educaţiei Naţionale
  • Thüringer Ministerium für Bildung, Wissenschaft und Kultur
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Peroxisome Proliferator-Activated Receptors
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Lipid metabolism and biosynthesis
  • Health Sciences › Medicine › Surgery › Cholesterol and Lipid Metabolism
Keywords
GW9662, PPARγ antagonist, PPARδ, PLIN2, adipophilin, THP-1 macrophages, lipogenesis, triglyceride accumulation, lipid metabolism, PPARγ target genes, nuclear receptors, fatty acid synthesis
MeSH terms
RAW 264.7 Cells, Perilipin-2, Anilides, Animals, Cells, Cultured, Female, Humans, Macrophages, Male, Mice, Inbred C57BL, Triglycerides, Gene Expression, U937 Cells, PPAR delta, PPAR gamma, Lipogenesis, Mice

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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