Fig 2: GPR120 protected against mice from LPS induced abortion by promoting decidualization. WT female mice were mated with WT male mice to induce pregnancy. LPS (1 mg/kg) was injected i.p. on GD7.5. In treated groups, TUG891 (10 mg/kg) or GW9508 (10 mg/kg) was injected i.p. from GD4.5 to GD7.5. Mice were sacrificed on GD8.5. (a) Uterus and fetus were dissected. Scale bar: 5 mm. (b) Litter size was counted and litters were weighed (n = 6). (c) Uterine tissues were collected and protein level of IGFBP1 was detected by western blot. (d) Working model of the regulation of GPR120 on decidualization. Results are presented as mean ± SEM. *P < .05, **P < .01 (one-way ANOVA).

Fig 3: Regulatory function of GPR120 in decidualization was mediated by ERK1/2 signaling. (a) Decidualization was induced in HESCs for 6 days. Expression of FOXO1 and IGFBP1 during decidualization were detected by western blot. (b) HESCs were treated with DMSO (D), TUG891 (T) or AH7614 (A) during decidualization. Expression of FOXO1 and IGFBP1 were detected by western blot. (c) shScramble or shGPR120 HESCs were incubated in induction medium for 3 days and western blot was performed. (d) Decidualization was induced in HESCs for 3 days and FOXO1 inhibitor AS1842856 (AS, 10/20 μM) was supplemented. Protein level of FOXO1, GLUT1 and IGFBP1 were examined by western blot. (e) HESCs were pretreated with 0% FBS medium for 24 h. Then cells were stimulated with TUG891 (10 μM) with or without 1 h of pretreatment of 10 μM AH7614 for 5, 15, 30 and 60 min. AMPK and ERK1/2 signaling pathways were determined by western blot. Decidualization was induced in HESCs for 3 days, supplemented with TUG891 (10 μM) and (f) AMPK inhibitor Compound C (C·C, 5/10 μM) or (g) ERK1/2 inhibitor SCH772984 (SCH, 1/5/10 μM). Western blot was performed. (h) HESCs were treated with DMSO (D), TUG891 (T, 10 μM) plus ERK1/2 inhibitor (SCH, 5 μM) or AMPK inhibitor (C·C, 5 μM) during decidualization for 3 days. Cells were collected and western blot was performed.

Fig 4: GPR120 was downregulated in human spontaneous abortion decidua. Decidual tissues from women of normal pregnancies (n = 20) and women of spontaneous abortion (n = 20) were collected and the expression of GPR120 was determined. (a) Western blot was performed to detect the protein level of GPR120 and IGFBP1. (b) mRNA expression level of GPR120, IGFBP1 and PRL were determined by real-time PCR. (c) Parraffin section of human decidual tissues were prepared and GPR120 was deteceted by IHC. (d, e) Normal and abortion DSCs were isolated from decidual tissues of normal pregnancy and abortion with collagnease and DNase. Protein level of GPR120 and IGFBP1 were measured by western blot (d) and real-time PCR was performed to detect mRNA expression level of GPR120, IGFBP1 and PRL (e). (f) GPR120 (green) and the marker of DSCs vimentin (red) were detected in decidual tissues by IFH to confim the expression of GPR120 in vimentin+ DSCs. Results are presented as mean ± SEM. *P < .05, **P < .01 (two-tailed Student's t-test).

Fig 5: GPR120 upregulated GLUT1 mediated glucose uptake and G6PD mediated PPP. (a) HESCs were incubated in culture or induction medium plus DMSO, TUG891 or AH7614 for 3 days. 2-NBDG was added and glucose uptake was determined by FACS analysis. MFI of 2-NBDG was shown (n = 6). (b) HESCs transfected with shScramble or shGPR120 were incubated in induction or culture medium for 3 days. Glucose uptake was detected by 2-NBDG assay and MFI was shown (n = 5). (c) mRNA expression of SLC2As were determined in HESCs and DSCs by real-time PCR. HESCs incubated in induction medium for D1-D6 were collected and mRNA expression level of SLC2A1 was measured by real-time PCR (d) and protein level detected by western blot during decidualization (e). (f) HESCs were transfected with shScramble or shSLC2A1 to knock down GLUT1. Cells were cultured in induction medium for 3 days and western blot was performed to detect the expression of GLUT1 and IGFBP1. (g) Glucose uptake was detected by 2-NBDG assay in shScramble or shSLC2A1 HESCs. MFI was shown (n = 5). HESCs were incubated in induction medium plus DMSO, TUG891 or AH7614 for 3 days. (h) GLUT1, IGFBP1 and PRL were detected by western blot. (i) Expression level of key enzymes involved in glucose metabolism were determined by western blot. (j) mRNA expression of G6PD was examined by real-time PCR. (k) HESCs were transfected with shScramble or shG6PD to knock down G6PD and incubated in induction medium for 3 days. G6PD and IGFBP1 were detected by western blot. (l) HESCs were incubated in induction medium with or without TUG891 (TUG, 10 μM) plus PPP inhibitor Glucosamine hydrochloride (GlcN, 1/5/10 mM). Decidualization was determined by protein level of IGFBP1. Results are presented as mean ± SEM. *P < .05, **P < .01, ***P < .001 (one-way ANOVA in Fig. 4a-d and 4j, two-tailed Student's t-test in Fig. 4g).