Glucosamine enhances proliferation, barrier, and anti-oxidative functions in porcine trophectoderm cells

Abstract

Trophectoderm (TE) is the first epithelium that appears during mammalian embryogenesis, and is a polarized transporting single cell layer that comprises the wall of the blastocyst. Previous studies have revealed the functional roles of glucose (Gluc), fructose (Fruc), and glutamine (Gln), which play a positive role in porcine trophectoderm (pTr) cell proliferation and migration, suggesting the importance of nutrients for normal development of the conceptus and implantation. This work was conducted to test the hypothesis that glucosamine (GlcN), which is synthesized from Gln and Fruc-6-phosphate through the hexosamine biosynthesis pathway (HBP), can stimulate proliferation and sustain the barrier and anti-oxidative functions of pTr cells. Cells were treated with 0, 0.25, or 0.5 mmol L⁻¹ GlcN in the presence or absence of adiquat (DQ) for the indicated time points. The results showed that 0.25 or 0.5 mmol L⁻¹ GlcN stimulated pTr cell viability and DNA replication compared to the control group. The addition of 0.25 mmol L⁻¹ GlcN enhanced the phosphorylation of mTOR signaling proteins, which can be inhibited by the inhibitor of phosphatidylinositol 3-kinase (PI3K), LY294002. Transepithelial electrical resistance (TEER) was increased, and paracellular permeability was correspondingly reduced in GlcN treatment. GlcN attenuated DQ-induced cell death and reduced the level of reactive oxygen species (ROS). The decreased TEER values and increased paracellular permeability caused by DQ treatment were also inhibited by GlcN treatment. The addition of 0.5 mmol L⁻¹ GlcN increased the protein expression of zonula occludens-3 (ZO-3), claudin-3, and claudin-4 in pTr cells, while inhibited the downregulation protein of claudin-1 and claudin-3 brought about by oxidative stress. Collectively, GlcN plays an important role in promoting proliferation and stimulating the mTOR cell signaling pathway, as well as ameliorating oxidative stress and augmenting barrier functions in pTr cells.