Suppression of adipocyte differentiation by aldo-keto reductase 1B3 acting as prostaglandin F2alpha synthase

Abstract

Prostaglandin (PG) F(2alpha) suppresses adipocyte differentiation by inhibiting the function of peroxisome proliferator-activated receptor gamma. However, PGF(2alpha) synthase (PGFS) in adipocytes remains to be identified. Here, we studied the expression of members of the aldo-keto reductase (AKR) 1B family acting as PGFS during adipogenesis of mouse 3T3-L1 cells. AKR1B3 mRNA was expressed in preadipocytes, and its level increased about 4-fold at day 1 after initiation of adipocyte differentiation, and then quickly decreased the following day to a level lower than that in the preadipocytes. In contrast, the mRNA levels of Akr1b8 and 1b10 were clearly lower than that level of Akr1b3 in preadipocytes and remained unchanged during adipogenesis. The transient increase in Akr1b3 during adipogenesis was also observed by Western blot analysis. The mRNA for the FP receptor, which is selective for PGF(2alpha), was also expressed in preadipocytes. Its level increased about 2-fold within 1 h after the initiation of adipocyte differentiation and was maintained at almost the same level throughout adipocyte differentiation. The small interfering RNA for Akr1b3, but not for Akr1b8 or 1b10, suppressed PGF(2alpha) production and enhanced the expression of adipogenic genes such as peroxisome proliferator-activated receptor gamma, fatty acid-binding protein 4 (aP2), and stearoyl-CoA desaturase. Moreover, an FP receptor agonist, Fluprostenol, suppressed the expression of those adipogenic genes in 3T3-L1 cells; whereas an FP receptor antagonist, AL-8810, efficiently inhibited the suppression of adipogenesis caused by the endogenous PGF(2alpha). These results indicate that AKR1B3 acts as the PGFS in adipocytes and that AKR1B3-produced PGF(2alpha) suppressed adipocyte differentiation by acting through FP receptors.

FIGURE 1.

Expression of Akr family and Pparγ genes in 3T3-L1 cells. A, Oil Red O staining of 3T3-L1 cells. 3T3-L1 cells were cultured with dexamethasone, 3-isobutyl-1-methylxanthine, and insulin for 2 days and then in medium with insulin alone for 6 more days. Undifferentiated (U) and differentiated (D) 3T3-L1 cells were stained with Oil Red O to visualize the accumulation of lipid droplet in the cells. Bar, 0.5 μm (inset, 0.1 μm). B, measurement of Oil Red O dye extracted from lipid droplet-laden cells. U, undifferentiated cells; D, differentiated cells. *, p < 0.01 as compared with undifferentiated cells. C, expression level of Akr1b3, Cox-2, and Pparγ genes during differentiation of 3T3-L1 cells. Expression levels of each gene were measured by quantitative PCR. The data are presented as the mean ± S.D. from three independent experiments. Protein levels of AKR1B3, COX-2, and PPARγ were detected during the adipogenesis of 3T3-L1 cells. *, p < 0.01 as compared with undifferentiated cells. Crude cell extracts (20 μg) were loaded in each lane in Western blot analysis. The PGF_2α level was measured by enzyme immunoassay. Cells were cultured with A23187 (5 μm) for 10 min at 37 °C, after which the medium was collected to measure the PGF_2α level. *, p < 0.01 as compared with undifferentiated cells.

FIGURE 2.

Suppression of Akr1b family gene expression by siRNA. A, siRNA-mediated suppression of Akr1b family gene expression. Preadipocytic 3T3-L1 cells were transfected with either of three siRNAs for each Akr1b family genes or NC (N.C.) siRNA (Invitrogen). Transfection was performed every 2 days. Eight days after the initial transfection, cells were harvested for extraction of RNA. Each gene expression was then measured by quantitative PCR. The data are presented as the mean ± S.D. from three independent experiments. *, p < 0.01 as compared with NC siRNA. B, suppression of the AKR1B3 protein level by Akr1b3 siRNAs. 3T3-L1 cells were knocked down by each of three Akr1b3 siRNAs as described in A. Crude cell extracts (30 μg) were loaded in each lane, and protein levels of AKR1B3 and actin as an internal control were detected in Western blot analysis. C, PGF_2α production was measured by enzyme immunoassay. Cells were transfected with each siRNA as described in A. At 8 days after the initial transfection, the medium was removed and fresh medium with A23187 (5 μm) was added. Then the cells were incubated for 10 min at 37 °C, after which the medium was collected to measure the PGF_2α level. *, p < 0.01 as compared with NC siRNA. D, Oil Red O staining of Akr1b3-knockdown 3T3-L1 cells. 3T3-L1 cells were transfected with Akr1b3 or NC siRNA, and differentiated for 8 days. Cells were stained with Oil Red O to visualize the accumulation of lipid droplet in the cells. Bar, 0.5 μm (inset, 0.1 μm). E, measurement of Oil Red O dye extracted from lipid droplet-laden cells. *, p < 0.01 as compared with NC siRNA.

FIGURE 3.

Expression of adipogenic genes in Akr1b-knockdown cells. Measurement of the mRNA level of adipogenic genes such as Pparγ, aP2, and Scd in Akr1b siRNA-transfected cells. Cells were transfected with each siRNA as described in the legend to Fig. 2A. At 8 days after the initial transfection, RNA was extracted and further utilized for cDNA synthesis. Messenger RNA levels were measured by quantitative PCR. Data are the mean ± S.D. from at least three independent experiments. *, p < 0.01 as compared with NC (N.C.) siRNA. The protein level of PPARγ was detected by Western blot analysis using crude cell extracts (30 μg/lane, inset).

FIGURE 4.

Expression of FP receptor and suppression of adipogenic gene expression by FP receptor agonist. A, the expression level of the FP receptor gene during differentiation of 3T3-L1 cells was measured by quantitative PCR. The data are presented as the mean ± S.D. from three independent experiments. *, p < 0.01 as compared with undifferentiated cells. B, Oil Red O staining of 3T3-L1 cells treated with the FP receptor agonist Fluprostenol. 3T3-L1 cells were caused to differentiate for 8 days in the presence of Fluprostenol (5 nm). Lipid droplets in the cells were visualized by staining with Oil Red O. Bar, 0.5 μm (inset, 0.1 μm). C, Oil Red O was extracted from lipid droplet-laden cells and quantified by colorimetric intensity. *, p < 0.01 as compared with vehicle control. D, down-regulation of adipogenic gene expression in Fluprostenol-treated 3T3-L1 cells. 3T3-L1 cells were transfected with the Akr1b3 siRNA together with or without various concentrations of Fluprostenol. Transfection was carried out every 2 days and Fluprostenol was added for every transfection. At 8 days after the initial transfection, RNA was extracted, and the transcription level of adipogenic genes Pparγ, aP2, and Scd were measured by quantitative PCR. The data are presented as the mean ± S.D. from three independent experiments. #, p < 0.01 as compared with vehicle control; *, p < 0.01 and **, p < 0.05 as compared with vehicle control. The protein level of PPARγ was detected by the use of crude cell extracts (30 μg/lane, inset).

FIGURE 5.

Antagonistic effect of FP receptor in 3T3-L1 cells. A, Oil Red O staining of 3T3-L1 cells. 3T3-L1 cells were caused to differentiate for 4 days in the presence or absence of AL-8810 (10 nm), fixed, and then stained with Oil Red O. Lipid droplet in the cells was visualized by staining with Oil Red O. Bar, 0.2 μm (inset, 0.1 μm). B, Oil Red O was extracted from lipid droplet-laden cells and quantified by colorimetric intensity. *, p < 0.01 as compared with vehicle control. C, enhancement of adipogenic gene expression in AL-8810-treated 3T3-L1 cells. The cells were incubated with AL-8810 in the differentiation medium for 4 days. The expression level of the adipogenic genes was quantified by PCR. Data are the mean ± S.D. from three independent experiments. Crude cell extracts (30 μg) were loaded in each lane, and the protein level of PPARγ was detected in Western blot analysis (inset).

FIGURE 6.

Proposed model of AKR1B3-mediated suppression of adipocyte differentiation in 3T3-L1 cells.