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Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus

p38 MAP Kinase phosphorylation by LPS. Mature adipocyte cells were treated with LPS at 1 μg/mL for 5, 10 and 20 min, or with LPS + p38 MAP Kinase inhibitor (SB, 1 μM) for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-p38 MAP Kinase protein antibody (Thr180/Tyr182, panel B). Loading equality was controlled using antibody against the unphosphorylated isoform of p38 (panel A). The data represent a typical result from two independent experiments.
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Figure 2: p38 MAP Kinase phosphorylation by LPS. Mature adipocyte cells were treated with LPS at 1 μg/mL for 5, 10 and 20 min, or with LPS + p38 MAP Kinase inhibitor (SB, 1 μM) for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-p38 MAP Kinase protein antibody (Thr180/Tyr182, panel B). Loading equality was controlled using antibody against the unphosphorylated isoform of p38 (panel A). The data represent a typical result from two independent experiments.

Mentions: Figure 2 confirms that the action of LPS on mature adipocytes results in p38 protein phosphorylation with a peak obtained 5 minutes after stimulation. The quantity of phosphorylated p38 protein subsequently decreases and is no longer detectible 20 minutes after treatment with LPS. The use of SB202190 (1 μM) greatly decreases the LPS induced phosphorylation of the p38 protein, resulting in a level that is near identical to the control.


Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

Hoareau L, Bencharif K, Rondeau P, Murumalla R, Ravanan P, Tallet F, Delarue P, Cesari M, Roche R, Festy F - J Inflamm (Lond) (2010)

p38 MAP Kinase phosphorylation by LPS. Mature adipocyte cells were treated with LPS at 1 μg/mL for 5, 10 and 20 min, or with LPS + p38 MAP Kinase inhibitor (SB, 1 μM) for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-p38 MAP Kinase protein antibody (Thr180/Tyr182, panel B). Loading equality was controlled using antibody against the unphosphorylated isoform of p38 (panel A). The data represent a typical result from two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2819999&req=5

Figure 2: p38 MAP Kinase phosphorylation by LPS. Mature adipocyte cells were treated with LPS at 1 μg/mL for 5, 10 and 20 min, or with LPS + p38 MAP Kinase inhibitor (SB, 1 μM) for 5 min. Proteins (50 μg per lane) were separated by SDS-PAGE and analyzed by Western blotting using an anti-phospho-p38 MAP Kinase protein antibody (Thr180/Tyr182, panel B). Loading equality was controlled using antibody against the unphosphorylated isoform of p38 (panel A). The data represent a typical result from two independent experiments.
Mentions: Figure 2 confirms that the action of LPS on mature adipocytes results in p38 protein phosphorylation with a peak obtained 5 minutes after stimulation. The quantity of phosphorylated p38 protein subsequently decreases and is no longer detectible 20 minutes after treatment with LPS. The use of SB202190 (1 μM) greatly decreases the LPS induced phosphorylation of the p38 protein, resulting in a level that is near identical to the control.

Bottom Line: Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors.Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway.Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

View Article: PubMed Central - HTML - PubMed

Affiliation: LBGM-GEICO, Laboratoire de Biochimie et de Génétique Moléculaire - Groupe d'Etude sur l'Inflammation Chronique et l'Obésité, Université de l'île de la Réunion, 15 avenue René Cassin 97715 Saint Denis Messag Cedex, France.

ABSTRACT

Background: The development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.

Methods: Primary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.

Results: WE SHOW FOR THE FIRST TIME THAT THE PRODUCTION OF TNFALPHA IN MATURE HUMAN ADIPOCYTES IS MAINLY DEPENDENT UPON TWO PATHWAYS: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.

Conclusion: This study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

No MeSH data available.


Related in: MedlinePlus