Figure 1.
SAA and saaHDL induce pro-inflammatory adipokine secretion by hMADS adipocytes
. hMADS adipocytes were cultured in presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL), humans HDL and saaHDL (12.5, 25, 50 and 100 µg/mL) for 24 h. Upon treatment, the supernatants were recovered and secreted concentrations of MCP-1 (Panels A and B), IL-6 (Panels C and D) and IL-8 (Panels E and F) were measured by ELISA. Data are expressed as mean ± SD from n = 3–4 independent experiments. Statistical significance: * p<0.05; ** p<0.01, *** p<0.001 vs. control.
Figure 2.
SAA pro-inflammatory effects in hMADS adipocytes are not due to endotoxin contamination.
Fully differentiated hMADS adipocytes were preincubated with or without 2 µM TAK-242 inhibitor for 1 h. Upon preincubation, cells were treated in the presence or absence of recombinant human SAA (1, 3 and 10 µg/mL) or LPS (1, 3 and 10 ng/mL) for 24 h. At the end of the treatment period, the supernatants were recovered and secreted MCP-1 (Panels A and B) and IL-6 (Panels C and D) were measured by ELISA. Data are expressed as mean ± SD from 3 independent experiments.
Figure 3.
SAA and saaHDL decrease adiponectin secretion by hMADS adipocytes.
hMADS adipocytes were cultured in the presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL), humans HDL and saaHDL (12.5, 25, 50 and 100 µg/mL) for 3 days following which secreted adiponectin (Panels A and B) and leptin (Panels C and D) were measured by ELISA. Data are expressed as mean ± SD from 3 independent experiments. Statistical significance: * p<0.05; *** p<0.001 vs. control.
Figure 4.
SAA decreases the expression of adipocyte markers.
hMADS adipocytes were cultured in the presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL) for 24 h. Upon treatment, total RNA was extracted and expression levels of various genes were analyzed by RTqPCR. Panel A: three important transcription factor (PPARγ2, C/EBPα and SREBP-1c). Panel B: lipogenic genes and adiponectin. Panel C: inflammation related proteins. The mRNA levels, normalized to LRP10 RNA expression, were determined relative to untreated control cells. Data are expressed as mean ± SD from n = 3–5 independent experiments. Statistical significance: * p<0.05; ** p<0.01; *** p<0.001 vs. control.
Figure 5.
activates NF-κB and MAPK pathways and NF-κB inhibition suppresses inflammation. hMDAS adipocytes were stimulated with 2 µg/mL recombinant human SAA or 5 ng/mL TNFα (positive control) for different periods of time. Nuclear proteins were extracted and EMSA performed as described in “Materials and Methods”. The NF-κB/DNA complex was detected by [γ-32P]-labeled NF-κB probe (Panel A). Total proteins were also extracted to determine total and phosphorylated forms of MAP kinases, p44/42, p38 and SAPK/JNK (Panel B). Following a 48 h insulin deprivation, hMADS adipocytes were incubated for 1 h with various inhibitors (10 µM) and subsequently incubated for 6 h and 24 h with SAA (10 µg/mL). SB203580, a p38 inhibitor; SP600125, a JNK inhibitor; PD98059, a MEK1/2 inhibitor; BAY11-7082, an inhibitor of IκB-alpha phosphorylation. At the end of the treatment period, the supernatants were recovered. Secreted IL-8 (Panel C) was measured by ELISA. MCP-1 gene expression was analyzed by RT-qPCR (Panel D). Glycerol was measured in the cell supernatant using a colorimetric assay (Panel E). * p<0.05; ** p<0.01; *** p<0.001 vs. control.
Figure 6.
SAA exerts pro-inflammatory effects in adipocytes isolated from human subcutaneous adipose tissue.
Isolated human adipocytes were prepared as described in “Methods” and were cultured for 24 h in presence or absence of human recombinant SAA (1, 3, 10 and 30 µg/mL). Secreted IL-6 (Panel A) was measured by ELISA. Gene expression levels of PPARγ2, C/EBPα and SREBP-1c were analyzed by RT-qPCR (Panel B). The mRNA levels normalized to LRP10 RNA expression were determined relative to untreated control cells. Statistical significance: * p<0.05; ** p<0.01; *** p<0.001 vs. control for three preparations.