Figure 5.

C. pneumoniae infection induces ER stress and the UPR in adipocytes, which leads to lipolysis and FABP4 secretion. A, relative levels of Chop, Bip, Atf4, or sXbp1 mRNA, as determined by real-time PCR, in 3T3-L1 adipocytes after mock or Cpn infection for 24 h and treatment with thapsigargin (1 μm) for 6 h. Gus mRNA served as the internal control. B, immunoblot analysis of CHOP, BIP, and p-eif2α in cell lysates of 3T3-L1 adipocytes after mock or Cpn infection at 4, 6, and 24 h. A 6-h incubation with thapsigargin (1 μm) served as the positive control for ER stress. β-Actin served as the standard. C, flow cytometry (left panel) and quantification (right panel) of MitoSOX-stained 3T3-L1 adipocytes at 24 h after Cpn infection in the presence or absence of azoramide (30 μm). D–G, glycerol (D and F) and FABP4 (E and G) levels in cultured medium of 3T3-L1 adipocytes at 24 h after Cpn infection at a MOI of 5 in the presence or absence of increasing doses of azoramide (D and E) or in the presence of GSK2606414) (PERK inhibitor) or STF-083010 (IRE1α RNase-specific inhibitor) (F and G). H, secretion of FABP4 in cultured medium of 3T3-L1 adipocytes at 6 h after treatment with thapsigardin (TG, 1 μm) and tunicamycin (TU, 5 μg/ml) was measured by ELISA. LDH assay using the supernatant was performed. As positive control, the cells were lysed with 2% Triton X-100 containing culture medium (n = 3/group; A and C–H). *, p < 0.05; **, p < 0.01, two-way ANOVA (A); one-way ANOVA (C–H). The data are shown as the means ± S.E. and are representative of at least three experiments.