Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells

Abstract

Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of estradiol (E2) and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1-10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/ml) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts (BFIBs) were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased levels of phospho-AKT, phospho-GSKβ, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

Endometrial glucose metabolism during early pregnancy

“…In addition, estradiol treatment increased glucose utilization and metabolism via glycolysis in uterine homogenates in immature rats ( Baquer & McLean 1972 ). Another study found that estradiol stimulated IGF1 in the uterine stroma of cows, which agrees with studies from mice ( Cooke et al 1997 , Gonzalez et al 2022 ). IGF1 increased the levels of 3-phosphoglycerate and lactic acid in immortalized bovine uterine epithelial (BUTE) cells ( Gonzalez et al 2022 ).…”

“…Another study found that estradiol stimulated IGF1 in the uterine stroma of cows, which agrees with studies from mice ( Cooke et al 1997 , Gonzalez et al 2022 ). IGF1 increased the levels of 3-phosphoglycerate and lactic acid in immortalized bovine uterine epithelial (BUTE) cells ( Gonzalez et al 2022 ). Collectively, these results suggest that estradiol increases glucose uptake and glycolytic metabolism by the uterine epithelium, perhaps indirectly.…”

“…In cows, the glycogen content of the uterine epithelium was higher near estrus than during the luteal phase ( Sandoval et al 2021 ). In agreement, estradiol stimulates the production of IGF1 in the uterine stroma, and a study showed that IGF1 increases glycogen levels in BUTE cells ( Cooke et al 1997 , Gonzalez et al 2022 ). Insulin has also been shown to increase glycogen levels in uterine epithelial cells ( Dean and Rose 2018 , Berg et al 2022 ).…”

“…These results showed that O -GlcNAcylation played an important role in promoting embryo implantation. A recent study found that IGF1 treatment of BUTE cells resulted in increased levels of N -acetyl-glucosamine (substrate for O -GlcNAcylation) and increased protein glycosylation as determined by Periodic acid–Schiff (PAS) staining ( Gonzalez et al 2022 ). In mice, inhibition of OGA by thiamet G promoted trophoblast differentiation and increased breaching of the endometrial epithelium in an in vitro model ( Ruane et al 2020 ).…”

The glycogenolytic enzyme acid α‐glucosidase is expressed in the bovine uterine endometrium

“…Western blots were performed as previously described (Gonzalez et al, 2022;Sandoval et al, 2021). Protein was separated on a 10% SDS-PAGE gel and was transferred onto a PVDF membrane, and membranes were blocked with 5% BSA in TBS-T. Anti-GAA (1:1000; ab137068, Abcam) or α-tubulin (1:500; 2144, Cell Signaling) antibodies were diluted in 5% BSA, added to the membranes and incubated overnight at 4°C.…”