Glucose transporter 1 is important for the glycolytic metabolism of human endometrial stromal cells in hypoxic environment

Abstract

Aim: The study aimed to elucidate the glycolytic metabolism of human endometrial stromal cells (hESCs) in hypoxic environment.

Main methods: The hESCs were cultured in hypoxic environment, and their metabolic pathways were analyzed using metabolomics. We assessed glucose uptake using 2-deoxyglucose (2-DG) assay. The expression of glucose transporters (GLUTs) required for glucose uptake was determined using real-time quantitative polymerase chain reaction (qPCR) and western blotting. Furthermore, we knocked down GLUT1 and examined the uptake of 2-DG.

Key findings: Under hypoxia, glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate were significantly elevated in hESCs (P < 0.05). This finding indicated enhancement in glycolysis. The volume of glucose uptake increased significantly under hypoxia (P < 0.05). Hypoxia simultaneously induced the expression of GLUT1 and GLUT3 mRNA (P < 0.05) and attenuated the expression of GLUT8 (P < 0.05). Glucose uptake was significantly inhibited upon knockdown of GLUT1 (P < 0.0001).

Significance: These results demonstrated a very important role of glucose transport under hypoxia. Also, hESCs utilize glycolysis to adapt to hypoxic conditions that could occur in menstrual and implantation period. These findings pave the way to study implantation failure and tumors originating from the endometrium.

Keywords: Biological sciences; Cell biology; Cell culture; Endometrium; Glucose transporter; Glycolysis; Human endometrial stromal cells; Hypoxia; Metabolomics; Molecular biology; Oxidative stress; Reproductive system; Women's health.

Figure 1

Metabolic pathway in human endometrial stromal cells (hESCs). (A) A typical schematic of metabolic pathway. Glucose enters hESC through glucose transporters (GLUTs) and is converted to glucose-6-phosphate, fructose-6-phosphate, and fructose-1,6-diphosphate by glycolytic enzymes. Citric acid cycle intermediates are acetyl CoA, citric acid, cis-aconitic acid, isocitric acid, 2-oxoglutaric acid, succinic acid, fumaric acid, and malic acid. The hESCs were cultured under hypoxia (2% O₂) or normoxia (20% O₂) for 24 h before analysis. (B) Shown is the measured volume of metabolites involved in the glycolysis. (C) Shown is the measured volume of metabolites involved in the citric acid cycle. Results are combined data of three experiments with different cell preparations and each value represents mean ± SD; ∗P < 0.05.

Figure 2

The effect of hypoxia treatment on glucose uptake in human endometrial stromal cells (hESCs). Cells were cultured under hypoxia (2% O₂) or normoxia (20% O₂) and then incubated with the 2-deoxyglucose (2-DG) for 20 min. Results are combined data of three experiments with different cell preparations and each value represents mean ± SEM; ∗P < 0.05.

Figure 3

Effects of hypoxia on glucose transporters (GLUTs) expression in human endometrial stromal cells (hESCs). Cells were cultured under hypoxia (2% O₂) or normoxia (20% O₂) for 24 h. The mRNA levels of GLUT1 (A), GLUT3 (B), GLUT8 (C), GLUT9 (D), GLUT10 (E), and GLUT12 (F) were analyzed by real-time qPCR and normalized to elongation factor1α (EF1α) mRNA expression. Results are combined data of four experiments with different cell preparations and each value represents mean ± SEM; ∗P < 0.05.

Figure 4

Western blotting analysis for glucose transporter (GLUT) 1 protein expression. The protein obtained from human endometrial stromal cells (hESCs) cultured with or without echinomycin under hypoxic (2% O₂) or normoxic condition (20% O₂) for 48 h (A). The protein level of GLUT1 was quantified with ImageJ (B). Results are combined data of three experiments with different cell preparation. Columns and vertical bars represent the mean ± SEM for combined data; ∗P < 0.05. Original non-adjusted Western blotting analysis is shown in Supplementary Material.

Figure 5

Effects of silencing glucose transporter (GLUT) 1. The human endometrial stromal cells (hESCs) were transfected with GLUT1 small interfering RNA (siRNA; GLUT1-A or GLUT1-B) or non-silencing RNA (Control) and were cultured with 2% O₂ for 24 h. The mRNA levels of GLUT1 (A), GLUT3 (D) were analyzed by real-time qPCR and normalized to elongation factor1α (EF1α) mRNA expression under hypoxia (2% O₂). The protein levels of GLUT1 (B), GLUT3 (E) and β-actin were analyzed by western blotting. Total cell lysates were obtained from hESCs cultured under hypoxia (2% O₂) or normoxia (20% O₂) for 24 h. The protein levels of GLUT1(C) and GLUT3 (F) was quantified with ImageJ. Results are combined data of three experiments with different cell preparation. Columns and vertical bars represent the mean ± SEM for combined data; ∗P < 0.05. Original non-adjusted Western blotting analysis is shown in Supplementary Material.

Figure 6

Effects of silencing glucose transporter (GLUT) 1 on the other GLUTs and vascular endothelial growth factor (VEGF). The human endometrial stromal cells (hESCs) were transfected with GLUT1 small interfering RNA (siRNA; GLUT1-A or GLUT1-B) or non-silencing RNA (Control) and were cultured with 2% O₂ for 24 h. The mRNA levels of GLUT8 (A), GLUT9 (B), GLUT10 (C), GLUT12 (D), and VEGF (E) were analyzed by real-time qPCR and normalized to elongation factor1α (EF1α) mRNA expression under hypoxia (2% O₂). Results are combined data of three experiments with different cell preparation. Columns and vertical bars represent the mean ± SEM for combined data; ∗P < 0.05.

Figure 7

The effect of downregulated glucose transporter (GLUT) 1 on glucose uptake in human endometrial stromal cells (hESCs). Cells were transfected with GLUT1-A or GLUT1-B siRNA or non-silencing control and were cultured under hypoxia (2% O₂) or normoxia (20% O₂) for 24 h with or without cytochalasin B. The volume of glucose uptake was measured with 2-deoxyglucose uptake assay. Results are combined data of three experiments with different cell preparations and each value represents mean ± SEM; ∗P < 0.001, ∗∗P < 0.0001.

Figure 8

Effects of silencing GLUT1 on cell proliferation and apoptosis. The human endometrial stromal cells (hESCs) were transfected with GLUT1-A or GLUT1-B siRNA or non-silencing control and were cultured under hypoxia (2% O₂) or normoxia (20% O₂) for 24 h. The hESCs were added WST-8 (10μL) solution and then read at 450 nm (A). Apoptosis were determined by death detection ELISA (B). Results are combined data of three experiments with different cell preparations and each value represents mean ± SEM; ∗P < 0.001.