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Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes - PubMed

  • ️Wed Jan 01 2014

Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes

Kazuaki Kajimoto et al. FEBS Open Bio. 2014.

Abstract

The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor α (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes.

Keywords: Adipocyte; Antioxidant; ER stress; ER, endoplasmic reticulum; Ern1, endoplasmic reticulum to nucleus signaling 1; FABP, fatty acid binding protein; FABP4; GSH, reduced glutathione; GSTA4, glutathione S-transferase A4; H2O2, hydrogen peroxide; Ormdl3, ORM1-like 3; Oxidative stress; ROS, reactive oxygen species; SOD, superoxide dismutase; Ssr1, signal sequence receptor α; UPR, unfolded protein response; VEGF, vascular endothelial growth factor; Xbp1, X-box binding protein 1.; siRNA, small interfering RNA.

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Figures

Fig. 1
Fig. 1

Increased oxidative stress mediated by the knockdown of FABP4 in differentiated 3T3-L1 adipocytes. (A and B) RT-PCR and Western blotting analyses for confirmation of FABP4 knockdown. At 48 h incubation of the differentiated 3T3-L1 adipocytes with 50 nM siFabp4 or siControl, the total RNA samples and cell lysates were subjected to RT-PCR (a) and Western blotting (b), respectively. Typical images obtained in three independent experiments are shown. NT: non-treatment. (C) Measurement of the intracellular ROS level. At 48 h transfection of siFabp4 or siControl, the 3T3-L1 adipocytes were fluorescently stained with CellROX Deep Red reagent and BODIPY493/503 for detection of cellular ROS and lipid droplets, and then subjected to flow cytometric analysis. The geometric mean values of CellROX fluorescence in the BODIPY-stained (fat accumulated) cells were acquired as the intracellular ROS levels. Data represent as mean ± SD (n = 6). †P < 0.005 (student’s t-test). (D) Immunocytochemical detection of 8-nitro-cGMP. At 48 h after transfection, the cells were fixed and immunostained with a pair of anti-nitroguanosine and Alexa568-labeled secondary antibodies (Red). Cell nuclei and lipid droplets were stained with Hoechst33342 (Green) and BODIPY (Blue). Typical CLSM images of 3 independent experiments were shown. Scale bars represent 50 μm. (E) Quantification of intracellular 8-nitro-cGMP contents. Mean fluorescent intensity (FI) (average intensity of pixels per cell) for 30 adipocytes per condition was measured. Open and closed circles represent the mean FI values in each cell, and black bars indicate the average values of mean FI in 30 cells. #P < 0.0001 (Student’s t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2
Fig. 2

No alteration in intracellular GSH content, SOD activity and GSTA4 expression in 3T3-L1 adipocytes was found as the result of FABP4 knockdown. (A and B) Intracellular GSH content and total SOD activity. At 48 h after siRNA transfection, cell lysates were prepared as described in Section 4, and GSH contents (a) and total SOD activity (b) were measured. The intracellular GSH contents and SOD activity per mg protein were calculated. Data represents mean ± SD (n = 3). (C) Western blotting of GSTA4. The protein expression of FABP4, GSTA4 and glyceraldehyde-3-dehydrogenase (GAPDH) was assessed. The chemiluminescent signals were detected using an Image Analyzer (LAS-4000mini).

Fig. 3
Fig. 3

Antioxidant activity of the recombinant FABP4 protein. (A) Measurement of superoxide anion. 1.5 μM of recombinant FABP4 protein and His6 peptide were subjected to quantification of the scavenger activity against superoxide anion. The percentages of the residual superoxide levels against the negative control are shown (n = 3). (B) Quantification of H2O2. 5 or 15 μM of recombinant FABP4 control peptide were incubated with H2O2, and then, the residual H2O2 level was measured. The relative H2O2 level in each sample against the negative control (buffer alone) was calculated. Data represent mean ± SD (n = 3). †P < 0.005, #P < 0.0001 (one-way ANOVA followed by Turkey-Kramer’s HSD test).

Fig. 4
Fig. 4

Reduced resistance to oxidative stress caused by FABP4 knockdown in the 3T3-L1 adipocytes and Raw264.7 macrophages. (A) Adipocyte viability. At 48 h after siRNA transfection, the cells were exposed with 300 mM H2O2 or vehicle for 1 h and then stained with 0.5% crystal violet. After extraction of the dye, the absorbance was measured at 540 nm to determine the cell viability. (B) Western blotting and macrophage viability. Raw264.7 cells were treated with 2 μM of Rosi or vehicle (DMSO) for 24 h. FABP4 expression was then assessed by Western blotting. After treatment with Rosi, the cells were incubated with 90 mM H2O2 or vehicle (water) for 1 h, and then cell viability was evaluated using crystal violet staining. Data represent mean ± SD (n = 3). ∗P < 0.05 (Student’s t-test).

Fig. 5
Fig. 5

Elevation of ER stress-related genes and intracellular Ca2+ level by FABP4 knockdown in the 3T3-L1 adipocytes. (A) RT-PCR analyses for ER stress-associated genes. At 48 h after siRNA transfection, the expression of several genes related to ER stress and/or UPR were analyzed by RT-PCR. Three independent samples transfected with siFabp4 or siControl were used in this evaluation. (B) Live cell calcium imaging. At 48 h after transfection of siFabp4 or siControl, the cells were stained with Fluo-8-AM (Green). Cell nuclei were counterstained with Hoechst33342 (Blue). Typical CLSM images of 3 independent experiments were shown. Scale bars represent 100 μm. (C) Quantification of intracellular Ca2+ level. Mean fluorescent intensity (FI) (average intensity of pixels per cell) for 67–73 adipocytes per condition was measured. Open and closed circles represent the mean FI values in each cell, and the black bars indicate the average values of the mean FI in each condition. #P < 0.0001 (Student’s t-test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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