High D-glucose levels induce ACE2 expression via GLUT1 in human airway epithelial cell line Calu-3

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2) receptors. ACE2 is expressed on human airway epithelial cells. Increased ACE2 expression may be associated with potentially high risk of COVID-19. However, the factors responsible for the regulation of ACE2 expression in human airway epithelial cells are unknown. Furthermore, hyperglycemia is a risk factor for poor disease prognosis.

Results: In this study, we investigated the effects of D-glucose on ACE2 mRNA and protein expressions in Calu-3 bronchial submucosal cells. The cells were cultured in minimal essential medium containing different D-glucose concentrations. After 48 and 72 h of high D-glucose (1000 mg/dL) treatment, ACE2 mRNA expressions were significantly increased. ACE2 protein expressions were significantly increased after 24 h of high D-glucose treatment. ACE2 mRNA expression was enhanced by a D-glucose concentration of 550 mg/dL or more after 72 h of treatment. In addition, we investigated the role of glucose transporters (GLUTs) in Calu-3 cells. ACE2 mRNA and protein expressions were suppressed by the GLUT1 inhibitor BAY-876 in high D-glucose-treated Calu-3 cells. GLUT-1 siRNA was also used and ACE2 mRNA expressions were suppressed in high D-glucose-treated Calu-3 cells with GLUT-1 knockdown.

Conclusions: This is the first report indicating that high D-glucose levels induced ACE2 expression via GLUT1 in bronchial submucosal cells in vitro. As hyperglycemia can be treated appropriately, these findings could help reduce the risk of worsening of coronavirus disease 2019.

Keywords: ACE2; Angiotensin converting enzyme 2; COVID-19; Glucose transporters; SARS-CoV-2.

Fig. 1

Real-time quantitative polymerase chain reaction analysis of angiotensin-converting enzyme 2 (ACE2) mRNA expression. a Calu-3 cells were treated with normal D-glucose (NG, 100 mg/dL) or high D-glucose (HG, 1000 mg/dL) for 24, 48, and 72 h. b Calu-3 cells were treated with 100–5000 mg/dL D-glucose concentrations. ACE2 gene expression was normalized relative to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data indicated in the graph are mean fold increase ± SE over mean control value. Symbol (*) indicates statistical differences (p < 0.05). Data are presented as mean ± SD (n = 3)

Fig. 2

Western blot analysis of angiotensin-converting enzyme 2 (ACE2) expression in Calu-3 cells. a The cells were treated with normal D-glucose (NG) or high D-glucose (HG) for 24, 48, and 72 h. Whole cell lysates were collected and subjected to western blotting for ACE2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). b The bar graph was generated by quantifying western blots from three independent experiments. Data are presented as mean ± SD (n = 6). Symbol (*) indicates statistical differences (p < 0.05)

Fig. 3

Inhibition of Glucose transporter (GLUT) gene in Calu-3 cells. The effects of GLUT1 inhibitor (BAY-876) on ACE2 expression were analyzed by real-time PCR (a) and western blotting (b, c) after 72 h of treatment. Data are presented as mean ± SD (n = 4). Symbol (*) indicates statistical differences (p < 0.05). d GLUT-1 mRNA was suppressed after GLUT-1 siRNA treatment for 24 h. e SiRNA-mediated knockdown of GLUT1 suppressed ACE2 mRNA in Calu-3 cells. Data are presented as mean ± SD (n = 3). Symbol (*) indicates statistical differences (p < 0.05)

Fig. 4

Immunofluorescence analysis of ACE2 expression in Calu-3 cells. The cells were treated with normal D-glucose (NG), high D-glucose (HG), or HG and 100 nM of glucose transporter 1 inhibitor (BAY-876) for 72 h. Green color indicates ACE2 expression, which was enhanced by HG and suppressed by BAY-876 in Calu-3 cells. Nuclei are stained in blue with DAPI. Scale bar, 100 μm

Fig. 5

Analysis of Calu-3 cell viability and proliferation by lactate dehydrogenase assay and MTT assay. a, c Cell viability was measured by LDH assay after the cells were treated with different D-glucose concentrations and HG with BAY-876 for 72 h. b, d Cell proliferation was measured by MTT assay after the cells were treated with different D-glucose concentrations and HG with BAY-876 for 72 h. The figure shows that D-glucose levels and BAY-876 did not affect the viability of cultured cells. Data are represented as mean ± SD (n = 3 per group). NG, normal D-glucose; HG, high D-glucose