Glycogen metabolism in mink uterine epithelial cells and its regulation by estradiol, progesterone and insulin

Abstract

Glycogen content in mink uterine glandular and luminal epithelia (GE and LE) is maximal during estrus and is depleted before implantation while embryos are in diapause. Uterine glycogen synthesis in vivo is stimulated by estradiol (E₂) while its mobilization is induced by progesterone (P₄). Nevertheless, treatment of an immortalized mink uterine epithelial cell line (GMMe) with E₂ did not affect glycogen production. Interestingly, insulin alone significantly increased synthesis of the nutrient and glycogen content in response to insulin + E₂ was greater than for insulin alone. Our objectives were to determine: 1) If insulin receptor protein (INSR) is expressed by mink uterine GE and LE in vivo and if the amount differs between estrus, diapause and pregnancy; 2) if E₂, P₄ or insulin regulate insulin receptor gene (Insr) expression by GMMe cells, and 3) if E₂ and P₄ act independently to regulate glycogen metabolism by GMMe cells and/or if their effects are mediated in part through the actions of insulin. The mean (±S.E.) percent INSR content of uterine epithelia was greatest during diapause (GE: 15.65 ± 0.06, LE:16.56 ± 1.25), much less during pregnancy (GE: 2.53 ± 0.60, LE:2.25 ± 0.32) and barely detectable in estrus (GE: 0.03 ± 0.01, LE:0.02 ± 0.01). Glycogen concentrations in GMMe cells increased 10-fold in response to insulin and 20-fold with insulin + E₂ when compared to controls. Expression of Insr was increased 2-fold by insulin and insulin + E₂ when compared to controls and there was no difference between the two hormone treatments, indicating that E₂ does not increase Insr expression in insulin-treated cells. To simulate E₂-priming, cells were treated with Insulin + E₂ for 24 h, followed by the same hormones + P₄ for the second 24 h (Insulin + E₂ → P₄) which resulted in Insr and glycogen levels not different from controls. Similarly, cells treated with Insulin + P₄ resulted in glycogen concentrations not different from controls. We conclude that the glycogenic actions of E₂ on GMMe cells are due to increased responsiveness of the cells to insulin, but not as a result of up-regulation of the insulin receptor. Glycogen mobilization in response to P₄ was the result of decreased glycogenesis and increased glycogenolysis occurring concomitantly with reduced Insr expression. Mink uterine glycogen metabolism appears to be regulated in a reproductive cycle-dependent manner in part as a result of the actions of E₂ and P₄ on cellular responsiveness to insulin.

Keywords: Estradiol; Glycogen; Insulin; Mink; Progesterone; Uterus.

Fig. 1

GMMe cells were treated with E₂ (10nM) or P₄ (10µM; Groups 2, 3) for 48 h, with a wash being conducted at the end of the first 24 h. To simulate E₂-priming, cells were treated with E₂ for 24 h, followed by E₂+P₄ during the second 24 h (E₂→P₄; Group 4). Cells were treated with insulin (15µg/ml) for 48 h (Group 5), insulin + E₂ for 48 h (Group 6), insulin + P₄ for 48 h (Group 7) and to simulate E₂-priming, insulin + E₂ for 24 h, followed by insulin + E₂ + P₄ for the second 24 h (I+ E₂→P_4; Group 8). Cells in Group 1 received treatment media only (Control). Three flasks of cells were grown for each treatment, resulting in 9 glycogen determinations for each treatment.

Fig. 2

GMMe cells were treated with insulin for the first 24 h (Groups 2,3 and 4). During the second 24 h, cells were treated with insulin (Group 3) or I + P₄ (Group 4). Cells in Group 1 received treatment media only (Control). Three flasks of GMMe cells were grown to confluence for each treatment, resulting in 9 glycogen measurements for each.

Fig. 3

Immunohistochemical localization of insulin receptor (INSR) protein in mink uterine cross-sections. Uteri were collected during estrus (March 6), diapause (March 19) and early-pregnancy (April 4). GE= Glandular epithelum; LE= Luminal epithelium. Three mink were sacrificed on each date and 9 images captured at 400 X for each animal. Data presented below the images represent the means (± S.E.) percent INSR protein content of each type of epithelium. The number of glands ranged from 10–18 per image. (ANOVA, F_{5, 12} = 107.05, P<0.001). Within a row, means without a common letter differ.

Fig. 4

Relative (mean ± SE) insulin receptor (Insr) mRNA levels in immortalized mink uterine epithelial cells (GMMe). Treatments were estradiol-17β (E₂; 10nM), progesterone (P₄; 10µM), insulin (I; 15µg/ml), I+E₂, or I+P₄, for 48 h, with a wash being conducted at the end of the first 24 h. To stimulate E₂-priming, cells were treated with E₂ or I+E₂ for the first 24 h then supplemented with fresh media containing those same hormones plus P₄ for the second 24 h (E₂→P₄ and I+ E₂→P₄). Control (CON) cells received incubation media only. After 48 h in culture, gene expression levels were determined in triplicate, for each of three flasks, resulting in nine measurements for each treatment. (ANOVA, F_{5, 12} = 107.05, P<0.05). Groups without a common letter differ at P≤ 0.05.

Fig. 5

Mean (± S.E.) glycogen concentrations (µ g/10⁷ cells) in immortalized mink uterine epithelial cells (GMMe). Cells were treated with estradiol-17β (E₂; 10nM), progesterone (P₄; 10µM), insulin (I;15µg/ml), I+E2, or I+P4, for 48 h. To stimulate E₂-priming, cells were treated with E₂ or I+E₂ for 24 h, then with the same hormones plus P₄ for the second 24 h (E₂→P₄ and I+ E₂→P₄). Control (CON) cells received media only. After 48 h, glycogen concentrations were determined in triplicate, resulting in nine measurements for each treatment. Due to contamination, only two flasks were analyzed for I, resulting in six determinations (ANOVA, F_{7, 22} = 58). Groups without a common letter differ at P ≤ 0.001.

Fig. 6

Mean (± S.E.) glycogen concentrations (µ g/10⁷ cells) in immortalized mink uterine epithelial cells (GMMe). Treatments were insulin (15 µ g/ml) for 24 h (I-24), 48 h (I-48), and I-48, with P₄ added during the last 24 h (I →P₄ + I). Control (CON) cells received incubation media only. After 48 h in culture, glycogen concentrations were determined in triplicate for each group, resulting in nine measurements for each treatment (ANOVA, F_{3, 14} = 155.6, P<0.001).