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Chlamydia pneumoniae infection-induced endoplasmic reticulum stress causes fatty acid-binding protein 4 secretion in murine adipocytes - PubMed

  • ️Wed Jan 01 2020

Chlamydia pneumoniae infection-induced endoplasmic reticulum stress causes fatty acid-binding protein 4 secretion in murine adipocytes

Nirwana Fitriani Walenna et al. J Biol Chem. 2020.

Abstract

Fatty acid-binding protein 4 (FABP4) is predominantly expressed in adipocytes and macrophages and regulates metabolic and inflammatory pathways. FABP4 is secreted from adipocytes during lipolysis, and elevated circulating FABP4 levels are associated with obesity, metabolic disease, and cardiac dysfunction. We previously reported that the bacterial respiratory pathogen Chlamydia pneumoniae infects murine adipocytes and exploits host FABP4 to mobilize fat and replicate within adipocytes. However, whether C. pneumoniae induces FABP4 secretion from adipocytes has not been determined. Here, we show that FABP4 is actively secreted by murine adipocytes upon C. pneumoniae infection. Chemical inhibition of lipase activity and genetic deficiency of hormone-sensitive lipase blocked FABP4 secretion from C. pneumoniae-infected adipocytes. Mechanistically, C. pneumoniae infection induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in elevated levels of mitochondrial reactive oxygen species and cytosolic Ca2+ Of note, exposure to a mitochondrial reactive oxygen species-specific scavenger, MitoTEMPO, reduced FABP4 release from C. pneumoniae-infected adipocytes. Furthermore, treatment with azoramide, which protects cells against ER stress, decreased FABP4 release from C. pneumoniae-infected adipocytes. Using gene silencing of CHOP (C/EBP homologous protein), a central regulator of ER stress, we further validated the role of C. pneumoniae infection-induced ER stress/UPR in promoting FABP4 secretion. Overall, these results indicate that C. pneumoniae infection robustly induces FABP4 secretion from adipocytes by stimulating ER stress/UPR. Our findings shed additional light on the etiological link between C. pneumoniae infection and metabolic syndrome.

Keywords: Chlamydia; adipocyte; bacteria; endoplasmic reticulum stress (ER stress); fatty acid binding protein; infection; lipolysis; metabolic disorder; oxidative stress; unfolded protein response (UPR).

© 2020 Walenna et al.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.

C. pneumoniae infection induces the secretion of FABP4 from murine adipocytes. A, immunoblot analysis of FABP4 in cultured medium (CM) and cell lysates (CL) of 3T3-L1 adipocytes after mock or C. pneumoniae (Cpn) infection for 2–24 h. β-Actin served as the standard. B, secretion of FABP4 was measured in the cultured medium of 3T3-L1 adipocytes after mock or Cpn infection for 2–24 h. A 4-h incubation with forskolin (20 μ

m

) served as the positive control for lipolysis. C, relative levels of Fabp4 mRNA in 3T3-L1 adipocytes after mock or Cpn infection for 4–24 h, as determined by real-time PCR. Gus mRNA served as the internal control. D, LDH assay using the supernatant of 3T3-L1 adipocytes at 2–24 h after mock or Cpn infection (n = 3/group; B–D). **, p < 0.01 by two-way ANOVA (B and C). The data are shown as the means ± S.E. and are representative of at least three experiments.

Figure 2.
Figure 2.

C. pneumoniae infection–induced FABP4 secretion is regulated by cAMP–PKA–HSL pathway. A, the lipolytic pathway inhibitors used in this experiment. B–E, FABP4 levels were measured in the cultured medium of 3T3-L1 adipocytes at 24 h after Cpn infection at a MOI of 5 in the presence or absence of atglistatin (50 μ

m

), CAY10499 (50 μ

m

) or JZL184 (1 μ

m

) (B), KH7 (50 μ

m

) (C), H89 (50 μ

m

) (D), or DMSO 1% solvent control (E). F and G, 3T3-L1 adipocytes differentiated from 3T3-L1 preadipocyte lines each stably expressing a short hairpin RNA (shRNA) against mRNAs encoding either murine EGFP (control) or HSL were infected with Cpn MOI 5 for 24 h. F, immunoblot analysis of FABP4 in the cultured medium (CM) and cell lysates (CL) of 3T3-L1 adipocytes. β-Actin served as the standard. G, secretion of FABP4 in cultured medium of these 3T3-L1 adipocytes was examined by ELISA (n = 3/group; B–E and G). **, p < 0.01, one-way ANOVA (B–E); two-way ANOVA (G). The data are shown as the means ± S.E. and are representative of at least three experiments. AC, adenylyl cyclase; PKA, protein kinase A; MAGL, monoacylglycerol lipase; TAG, triacylglycerol; DAG, diacylglycerol; MAG, monoacylglycerol; FFA, free fatty acid.

Figure 3.
Figure 3.

C. pneumoniae infection–induced FABP4 secretion depends on mitochondrial ROS. A, flow cytometry (left panel) and quantification (right panel) of MitoSOX-stained 3T3-L1 adipocytes at 24 h after Cpn infection at a MOI of 5 in the presence or absence of MitoTEMPO (100 μ

m

). B and E, immunoblot analysis of p-HSL and HSL in cell lysates of 3T3-L1 adipocytes at 24 h after Cpn infection in the presence or absence of MitoTEMPO (100 μ

m

) (B) or increasing doses of MitoQ (E). A 2-h incubation with forskolin (20 μ

m

) served as the positive control for lipolysis. β-Actin served as the standard. C, glycerol levels were measured in cultured medium of 3T3-L1 adipocytes at 24 h after Cpn infection in the presence or absence of MitoTEMPO. D and F, FABP4 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 MitoTEMPO (100 μ

m

) (D), increasing doses of MitoQ (Mitoquinone) (F). In A, C, D, and F, n = 3/group. *, p < 0.05; **, p < 0.01, one-way ANOVA (A, C, D, and F). The data are shown as the means ± S.E. and are representative of at least three experiments.

Figure 4.
Figure 4.

C. pneumoniae infection–induced FABP4 secretion is regulated by cytoplasmic calcium elevation. A, intracellular Ca2+ was assessed by Fluo-4 AM fluorescence in 3T3-L1 adipocytes after mock or Cpn infection for 2, 4, 12, and 24 h. Thapsigargin (10 μ

m

) for 6 h served as a positive control. B and C, FABP4 (B) and glycerol (C) 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 BAPTA-AM (20 μ

m

). D, the number of infectious EB progeny of 3T3-L1 adipocytes at 24 h after Cpn infection in the presence or absence of BAPTA-AM (20 μ

m

) or BAPTA (50 μ

m

) was determined using an IFU assay (n = 3/group; B–D). *, p < 0.05; **, p < 0.01, one-way ANOVA (B–D). The data are shown as the means ± S.E. and are representative of at least three experiments.

Figure 5.
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 μ

m

) 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 μ

m

) 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 μ

m

). 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 μ

m

) 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.

Figure 6.
Figure 6.

Gene silencing of CHOP or PERK abolishes C. pneumoniae infection–induced FABP4 secretion. A, at 48 h after transfection (siCHOP, siPERK, or siControl), 3T3-L1 adipocytes were infected with Cpn MOI 5 for 24 h, and immunoblot analysis was done to confirm the effectiveness of CHOP or PERK gene silencing. B, FABP4 levels were measured in the cultured medium of 3T3-L1 siControl, siCHOP, or siPERK adipocytes at 24 h after Cpn infection at a MOI of 5 (n = 3/group). **, p < 0.01, two-way ANOVA (B). The data are shown as the means ± S.E. and are representative of at least three experiments.

Figure 7.
Figure 7.

C. pneumoniae usurps infection-induced ER stress/UPR and subsequent elevation of mitochondrial ROS for its replication. A–D, the number of infectious EB progeny of 3T3-L1 adipocytes at 24 h after Cpn infection at a MOI of 5 in the presence or absence of MitoTEMPO (A, 100 μ

m

), MitoQ (B, 1 or 5 μ

m

), azoramide (C, 20 μ

m

to 50 μ

m

), GSK2606414 (D, PERK inhibitor, 2 μ

m

), or STF-083010 (IRE1α RNase-specific inhibitor, 50 μ

m

) was determined using an IFU assay. E, at 48 h after transfection (siCHOP, siPERK, or siControl), 3T3-L1 adipocytes were infected with Cpn MOI 5 for 24 h, and infectious EB progeny was determined using an IFU assay (n = 3/group). *, p < 0.05; **, p < 0.01, Student's t test (A); one-way ANOVA (B–E). The data are shown as the means ± S.E. and are representative of at least three experiments.

Figure 8.
Figure 8.

The possible model of FABP4 secretion in C. pneumoniae–infected adipocytes. C. pneumoniae infection–induced ER stress/UPR in murine adipocytes causes the elevation of mitochondrial ROS and cytoplasmic calcium levels, followed by HSL-mediated lipolysis and FABP4 secretion. The right part of this illustration (italics) is based on our previous report (20). CPT-1, carnitine palmitoyltransferase 1; FFA, free fatty acid.

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