Activation of the IGF1 receptor stimulates glycogen synthesis by mink uterine epithelial cells

Abstract

Glycogen synthesis by mink uterine epithelial cells is stimulated by estradiol (E ) during estrus, although the mechanism/s through which the steroid promotes glycogen accumulation are unknown. Our aim was to determine if insulin is required for E induced glycogen synthesis by an immortalized mink uterine epithelial cell line (GMMe). We show that the cells expressed the genes for glycogen metabolizing enzymes (hexokinase 1, glucose-6-phosphatase 3, glycogen synthase 1, and glycogen phosphorylase-muscle), receptors for insulin, insulin-like growth factor 1 and E (Esr1). Interestingly, treatment of cells with E alone failed to stimulate glycogen production, whereas supraphysiological concentrations of insulin (50 μg/ml) only, significantly increased glycogen content. Moreover, insulin + E increased glycogen content when compared to insulin alone (p < 0.05), an affect that was blocked when cells were treated with the pure E receptor antagonist ICI 182,780. Glycogen synthesis in response to insulin was significantly inhibited when cells were pre-treated with picropodophyllotoxin, an IGF1R antagonist. Treatment of cells with LY294002, a phosphatidylinositol 3-kinase (PI3K) antagonist, blocked insulin's effects on glycogen production whereas treatment with U0126, an inhibitor of mitogen activated kinase-kinase (MEK1/2) was without effect. These findings suggest to us that the affects of E on glycogen synthesis by GMMe cells is mediated through Esr1 and increased responsiveness of the cells to insulin. Because picropodophylotoxin blocked the effects of insulin on glycogen production, and both insulin and IGF1 act through PI3K, it is possible that IGF1 plays a role in glycogen production by these cells.

Estradiol and progesterone affect enzymes but not glucose consumption in a mink uterine cell line (GMMe)

“…Furthermore, it is the first time for such complete measurements in uterine cells derived from an animal with an obligate diapause phase of reproduction. The results are markedly different from other mammals in vivo but consistent with patterns observed previously for in vitro studies and GMMe [22,24]. While these hormones alone in vitro did not alter glycolytic flux, metabolite levels and the glucose consumption in the GMMe cells, treatment with E 2 induced a decrease in most enzyme activities, while P 4 alone induced an increase in several K m s.…”

“…For the last 24 h, the medium was replaced with DMEM/F-12 (5 mM glucose) every 12 h, with 10 nM E 2 , 10 μM P 4 or dimethyl sulfoxide (DMSO; control) vehicle. These hormone levels were shown to be the minimum necessary to affect insulin receptor and, with insulin, glycogen levels in GMMe cells [22,23]. These levels were several orders of magnitude smaller than what was measured in vivo in mink [25].…”

“…E2 (E2758, Sigma-Aldrich) and P 4 (P783; Sigma-Aldrich) were dissolved in 100% DMSO (Sigma-Aldrich, Hybri-Max™) to make stock solutions (50 μM E 2 and 100 mM P 4 ). Medium glucose at 5 mM was used [22], which is similar to blood glucose in mink during the breeding season (∼5.3 mM) [26,27].…”

“…Though P 4 and E 2 are essential for changing carbohydrate usage and enzymes in mink endometrial tissue, in vitro it has been shown that E 2 , at least, must act in coordination with other hormones (primarily insulin) to affect glycogen levels. In GMMe, E 2 enhances insulin's stimulatory effect on glycogen storage, while E 2 alone has no effect on glycogen levels [22]. Thus, any changes to glycolytic enzymes by P 4 and E 2 alone may not affect carbohydrate usage directly, but rather play a larger role in priming the system for hormonal regulation by insulin.…”

“…Mink immortalized endometrial cells (GMMe) [21] provide an ideal model system to study hormonal regulation of glycolysis in the uterus, not only because carbohydrate metabolism has already been studied in mink uterus and in GMMe, but also because they are one of the only immortalized endometrial cell lines that are commercially available. They respond to E 2 and P 4 [22,23] and express estrogen receptor Type 1 [22]. They also represent a source of mink enzymes, for which few K m s have been determined under any conditions [24].…”