Facilitative glucose transporter type 1 is differentially regulated by progesterone and estrogen in murine and human endometrial stromal cells

Abstract

Embryo implantation is a highly synchronized event between an activated blastocyst and a receptive endometrium. The success of this process relies on the dynamic interplay of estrogen (E(2)) and progesterone (P(4)), however, the details of this interaction are not entirely clear. Recent data implicate E(2) and P(4) in the regulation of glucose utilization by affecting facilitative glucose transporter (GLUT) expression. In this study we examine GLUT1 expression in murine and human endometrial stromal cells (ESCs) using a primary culture system. We show that expression of GLUT1 is increased during ESC decidualization in vitro. P(4) up-regulates, whereas E(2) down-regulates, GLUT1 expression. In addition, P(4) increases and E(2) decreases glucose uptake in ESCs, suggesting that GLUT1 may be a major player in glucose utilization in these cells. Moreover, GLUT1 expression is increased in human ESCs when decidualized in vitro with P(4) and dibutyryl cAMP, suggesting a similar role for P(4) in human endometrium. In conclusion, an imbalance between P(4) and E(2) seen in patients with polycystic ovary syndrome, luteal phase defect, and recurrent pregnancy loss may have a critical impact on glucose utilization in the endometrial stroma, and, thus, may be responsible for endometrial dysfunction and failure of embryo implantation in these patient populations.

Figure 1

Expression of facilitative GLUTs in murine ESCs decidualized in vitro. A, ESCs were isolated from mice on d 4 pregnancy and cultured for 72 h in the presence of 1 μm P₄ and 10 nm E₂ to induce decidualization. Control cells were cultured without hormones. GLUT1 protein expression was quantitated by Western blot and normalized to the internal control, β-actin, for loading variability. Fold change compared with the “Control” value is represented in the graph. Values are the mean ± sem of three independent experiments. *, P < 0.001. B, Representative Western blot demonstrates GLUT1 expression and the unchanged loading control, β-actin. C, ESCs cultured in vitro with hormones (“D”) or without (“C”). GLUT1 protein expression was analyzed after 24, 48, and 72 h culture. A representative Western blot is shown. GLUT1 begins to increase after the initial 24 h E₂ and P₄ treatment in decidualized ESCs from pregnant mice, and the increase is sustained at 48 and 72 h. D, Protein levels of GLUT4, GLUT8, GLUT9b, and GLUT12 were analyzed by Western blot in decidualized ESCs compared with control. GLUT proteins have a high degree of N-linked glycosylation and, thus, appear as multiple bands or heterogeneous bands when subjected to SDS-PAGE analysis (35). E, qPCR was used to monitor levels of Prp mRNA, a decidualization marker, in ESCs after 72 h culture.

Figure 2

GLUT1 protein expression is differentially regulated by P₄ and E₂ in murine ESCs. ESCs were isolated from uteri of nonpregnant mice and cultured in phenol red-free media for 4 d before hormone treatment. A, Cells were then plated at 5 × 10⁵ cells per well in six-well cell culture plates and cultured for 72 h in the presence of 10 nm E₂, 1 μm P₄, or both 10 nm E₂/1 μm P₄. GLUT1 protein expression was analyzed by Western blot and normalized to the internal control, β-actin, for loading variability. Fold change compared with the “Control” value is represented in the graph. C, ESCs were also treated with 1 μm RU486, a PR antagonist. B and D, Representative Western blots show relative expression of GLUT1 and the loading control β-actin. Values are the mean ± sem of three independent experiments. *, P < 0.001 compared with control by ANOVA with Fisher’s test. E, ESCs were isolated from uteri of nonpregnant mice, maintained in phenol red-free media for 4 d, and then treated with 1 μm P₄ for the durations indicated. mRNA expression was analyzed by qPCR, and samples were normalized to a housekeeping gene, Gapdh, for a loading control. Values are a mean ± sem of four independent experiments. *, P < 0.005 compared with 0 h by ANOVA with Fisher’s test. F, GLUT1 protein expression was analyzed by Western blot. Values are a mean ± sem of three independent experiments. *, P < 0.005; **, P < 0.05 compared with the 0 h sample by ANOVA with Fisher’s test. G, A representative Western blot showing GLUT1 expression.

Figure 3

GLUT1 localizes to the plasma membrane. A, Cell surface GLUTs were labeled by incubating ESCs with Bio-ATB-BMPA and exposing the mixture to UV light. Cells were then solubilized for immunoprecipitation with streptavidin-agarose beads. Cell surface GLUT1 expression was analyzed by Western blot. To determine whether differences in cell surface expression levels of GLUTs among the treatment groups were attributable to differences in GLUT expression levels or in GLUT localization, the total expression levels of GLUTs in each sample was also analyzed by Western blotting and normalized to β-actin for loading variability. B, A representative Western blot of GLUT1 cell surface expression is shown. C, A representative Western blot of total GLUT1 expression is shown. D, ESCs isolated from the uteri of pregnant mice were decidualized in vitro. Cell surface GLUTs were labeled by incubating ESCs with Bio-ATB-BMPA and then solubilized for immunoprecipitation with streptavidin-agarose beads. Cell surface GLUT1 expression and total GLUT1 expression were analyzed by Western blot. E, A representative Western blot of cell surface GLUT1 expression in ESCs isolated from mice at 4 dpc is shown. F, A representative Western blot of total GLUT1 in ESCs isolated from mice at 4 dpc is shown. Values are the mean ± sem of six samples analyzed in each treatment group. *, P < 0.001 compared with control by ANOVA with Fisher’s test. G, GLUT1 localization in vivo in the uterine decidual cells at 7 dpc. Uteri were collected from five mice, and a representative section is shown.

Figure 4

P₄ increases glucose uptake in ESCs. DG uptake in ESCs cultured in the presence of P₄, E₂, or P₄+E₂ for 72 h. Uptake was measured after 60 min incubation with 0.1 mm DG, and each sample was normalized to total protein. Values are mean ± sem of six samples analyzed in each treatment group. *, P < 0.001 compared with vehicle control by ANOVA with Fisher’s test.

Figure 5

GLUT1 is expressed in vivo in ESCs at pregnancy. A, ESCs were isolated from mice on the indicated day of pregnancy, and protein expression of GLUT1 was analyzed by Western blot. Values are a mean of five mice. *, P < 0.05 by ANOVA with Fisher’s test. B, Western blot showing GLUT1 expression and the loading control, β-actin. C, GLUT1 expression at 5 dpc by immunohistochemistry in the implantation site (IS) and interimplantation site (Inter-IS). Five mice were used, and a representative section is shown. The protein can be seen in the decidualizing stroma (s) close to the embryo (em), as well as the glandular and luminal epithelium (ge and le, respectively). D, By 7 dpc, GLUT1 expression can be seen primarily in the growing decidualizing stroma in the implantation site surrounding the embryo (em) and no longer in the epithelial tissues. Five mice were used, and a representative section is shown.

Figure 6

GLUT1 expression increases in hESCs during decidualization in vitro. A, GLUT1 protein expression increases significantly in hESCs after 3, 6, and 9 d 1 μm MPA and 0.5 mm db-cAMP treatment in vitro. Fold changes are calculated compared with the 3 d untreated control sample. Values are a mean of three independent experiments ± sem. *, P < 0.05; **, P < 0.001 by ANOVA with Fisher’s test. B, Representative Western blot of GLUT1 and the loading control β-actin showing protein lysates from control (“C”) and decidualized (“D”) hESCs collected after the indicated length of treatment time. C, qPCR was used to monitor levels of Igfbp1 mRNA, a decidualization marker in hESCs after 3, 6, and 9 d culture. Samples are normalized to the untreated-control sample on the appropriate days. D, qPCR was used to monitor levels of Prl mRNA, a decidualization marker in hESCs after 3, 6, and 9 d culture. Samples are normalized to the untreated-control sample on the appropriate days.