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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

Comparison of LPS induced TNFalpha secretion by human peripheral blood mononuclear cells versus human mature adipocytes. Human mature adipocytes and peripheral blood mononuclear cells were treated with a LPS dose-response (5, 10 ng/mL and 1 μg/mL). TNFα concentrations in medium (pg/mL) were determined at 6 hours. Plating was 8 × 104 for macrophages and 3 × 104 for adipocytes. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition) for each cell type, representative of two experiments on two different patients (macrophages) and representative of three experiments on three different patients (adipocytes).
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Figure 6: Comparison of LPS induced TNFalpha secretion by human peripheral blood mononuclear cells versus human mature adipocytes. Human mature adipocytes and peripheral blood mononuclear cells were treated with a LPS dose-response (5, 10 ng/mL and 1 μg/mL). TNFα concentrations in medium (pg/mL) were determined at 6 hours. Plating was 8 × 104 for macrophages and 3 × 104 for adipocytes. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition) for each cell type, representative of two experiments on two different patients (macrophages) and representative of three experiments on three different patients (adipocytes).

Mentions: We evaluated the difference in the levels of TNFalpha synthesis between human macrophages and mature human adipocytes (both primary cultures). Figure 6 shows that macrophages were more sensitive to LPS stimulation than adipocytes, as these cells were able to respond to 5 ng/mL of LPS. Nevertheless, adipocytes secreted more TNFalpha than macrophages when they were treated with 1 μg/mL of LPS (around 4-fold more, following normalization of the cell number).


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)

Comparison of LPS induced TNFalpha secretion by human peripheral blood mononuclear cells versus human mature adipocytes. Human mature adipocytes and peripheral blood mononuclear cells were treated with a LPS dose-response (5, 10 ng/mL and 1 μg/mL). TNFα concentrations in medium (pg/mL) were determined at 6 hours. Plating was 8 × 104 for macrophages and 3 × 104 for adipocytes. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition) for each cell type, representative of two experiments on two different patients (macrophages) and representative of three experiments on three different patients (adipocytes).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Comparison of LPS induced TNFalpha secretion by human peripheral blood mononuclear cells versus human mature adipocytes. Human mature adipocytes and peripheral blood mononuclear cells were treated with a LPS dose-response (5, 10 ng/mL and 1 μg/mL). TNFα concentrations in medium (pg/mL) were determined at 6 hours. Plating was 8 × 104 for macrophages and 3 × 104 for adipocytes. The graph represents the mean ± SE of the results from one patient (n = 6 for each condition) for each cell type, representative of two experiments on two different patients (macrophages) and representative of three experiments on three different patients (adipocytes).
Mentions: We evaluated the difference in the levels of TNFalpha synthesis between human macrophages and mature human adipocytes (both primary cultures). Figure 6 shows that macrophages were more sensitive to LPS stimulation than adipocytes, as these cells were able to respond to 5 ng/mL of LPS. Nevertheless, adipocytes secreted more TNFalpha than macrophages when they were treated with 1 μg/mL of LPS (around 4-fold more, following normalization of the cell number).

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