Table 1.
List of primers used in this study for RT-PCR on SW872 RNA.
Figure 1.
HMGB1 expression and localization in the human preadipocyte cell line SW872.
A) Expression of HMGB1 by RT-PCR from total RNA obtained from SW872 cell after 12h of culture, with (+) and without (−) Reverse Transcriptase (RT). B) Detection of HMGB1 protein in different cell fraction by Western blot analysis. β-actin is a cytoplasmic protein (Cy) and lamin B a nuclear protein (Nu). C) Immunofluorescence of HMGB1 in SW872 cells (X 200). Cells were incubated with mouse anti-HMGB1 primary antibody (1:200), Alexa Fluor 594 linked secondary antibody (1∶1000) and DAPI. Scale bar indicated 50 µm.
Figure 2.
Secreted HMGB1 levels correlate with IL-6 secretion in SW872 cells.
A) Expression of IL-6 by RT-PCR from total RNA obtained from SW872 cell after 12 h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) IL-6 secretion by ELISA assay from SW872 supernatant culture 6h after media renewal (expressed in mean ± SD) (n = 3). D) Correlation between IL-6 secretion and HMGB1 release in SW872 cell culture media after renewal of media (n = 3). Pearson's correlation coefficient (r = 0.9108, p = 0.0116) and Spearman's rank correlation coefficient (r = 0.9429, p = 0.0167). All values are expressed as means +/− SD.
Figure 3.
Secreted HMGB1 controls IL-6 production in SW872 cells.
IL-6 levels were determined by ELISA from SW872 cells A) treated with increasing concentrations of recombinant HMGB1 for 5 h (n = 4) or B) treated or not with rHMGB1 with a dose of 1 µg.mL−1 for 5 h in presence of either an irrelevant control rabbit IgG (CTL) or a rabbit monoclonal anti-HMGB1 (abHMGB1) (n = 4). Detection of IL-6 secretion in SW872 cells treated for 5 h with C) ethyl-pyruvate (n = 3) or D) glycyrrhizin (n = 3). All values are expressed as means +/− SD. Degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001.
Figure 4.
HMGB1 shRNA dowregulates IL6 secretion.
HMGB1 mRNA expression or protein expression were assessed respectively by RT-PCR (A) or Western blot (B) after 12h in culture of stably SW872 infected with scrumble shRNA (Sh CTL) or HMGB1 shRNA (sh HMGB1) constructs. C) Detection of IL-6 secretion in stably SW872 infected cells with scramble shRNA (Sh CTL) or HMGB1 shRNA (sh HMGB1) constructs after 5h of media renewal (n = 3). All values are expressed as means +/− SD. Degrees of significance are indicated in the figure captions as follow *** p<0.001.
Figure 5.
HMGB1 mediates IL-6 release through RAGE.
A) TLR2, TLR4, RAGE and HMGB1 mRNA expression was assessed by RT-PCR from total RNA obtained from SW872 cell after 12h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) Cell surface expression of TLR2, TLR4 and RAGE were analyzed by FACS C) SW872 cells were treated with rHMGB1 1 µg.mL−1 for 5 h with control mouse IgG (CTL), TLR2 or TLR4 blocking antibody (n = 4). D) SW872 cells were treated as in (C) except that control goat IgG (CTL) or goat polyclonal anti-RAGE were used (n = 4). All values are expressed as means +/− SD and degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001 and ns = not significant.
Figure 6.
HMGB1 controls MCP1 (CCL2) secretion.
A) MCP1 and TNFα mRNA expression were assessed by RT-PCR from total RNA obtained from SW872 cell after 12h in culture, with (+) and without (−) Reverse Transcriptase (RT). B) MCP1 and TNFα secretion by ELISA assay from SW872 supernatant culture 6h after media renewal (n = 6). C) IL-6 levels were determined by ELISA from SW872 cells treated with increasing concentrations of recombinant HMGB1 for 5 h (n = 3). D) Detection of MCP1 secretion in stably infected SW872 with scramble shRNA (Sh CTL) or HMGB1 shRNA (Sh HMGB1) constructs after 5h of media renewal (n = 3). All values are expressed as means +/− SD and degrees of significance are indicated in the figure captions as follow, * p<0.05, ** p<0.01, *** p<0.001.
Figure 7.
Central role of HMGB1 adipokine in LGI of fat tissue.
Model depicting the novel role of HMGB1 as an adipokine involved in fat tissue inflammation, obesity and associated-chronic disorders such as diabetes type II. We found that HMGB1 is secreted and interacts via an autocrine and/or paracrine manner with RAGE on preadipocytes. This could contribute to low grade inflammation (LGI) of the fat tissue with the elevated expression of major pro-inflammatory cytokines such as IL-6 and MCP1. We could further hypothesize that HMGB1 released by preadipocytes can attract, via MCP1, macrophages and leading to activation through TLR2, TLR4 and RAGE. Finally, the pool of proinflammatory factors and recruited immune cells could instigate the LGI and the insulin resistance in liver and muscle cells.