Insulin regulates Glut1 activity in primary murine alveolar epithelial cells under hypoxic conditions

Background: Glucose transporters (GLUTs) are a family of membrane bound proteins responsible for the facilitative diffusion of glucose across cellular membranes. Under hypoxic conditions, upregulation of GLUTs can play important roles in maintaining homeostasis and cell survival. However, inappropriate increases in GLUT expression and glucose uptake (under normoxic and/or hypoxic conditions) can also lead to several human disorders. For example, GLUT1-4 have been widely investigated for their respective roles in seizures, developmental delays, diabetes, and solid tumors. It is therefore important to better understand the (patho)-physiological effects of GLUT isoforms. Since the current literature is unclear regarding the expression and physiological role of GLUT1 in lung epithelia, the objective of our research is to evaluate the functional expression and putative regulation of Glut1 by hypoxia and insulin using primary mouse alveolar epithelial cells (mAECs). Methods: Primary mAECs were isolated from WT C57Bl6 mice and cultured in epithelial growth media, then incubated with media containing 10mM (high) or 2mM (low) glucose or 10mM galactose. In high glucose media, mAECs were supplemented with insulin [0-5μM] and incubated for 18 hours. Glut1 and Glut4 expression were determined using standard qPCR protocols (n=3). Cell viability was measured by MTT normalized to total protein (measured by BCA) (n=6). ATP was measured by ATPlite kit (Perkin Elmer) and glucose uptake was determined using a colorimetric kit (Abcam). Results were normalized to % of Control. Statistical analysis was conducted using PRISM (GraphPad software); p<0.05 was considered statistically significant. Results: Under hypoxic conditions, we observed a significant increase in glucose uptake in mAECs (from 0.90x10 5 ± 0.07x10 5 RLUs under normoxia to 1.2x10 5 ± 0.02x10 5 RLUs under hypoxia); n=6, p<0.001. Glut1 transcript expression (but not Glut4 ) also significantly increased in mAECs cultured under hypoxic conditions; p<0.001. Moreover, 5μM insulin supplement significantly increased Glut1 transcript expression (p=0.001) but failed to induce Glut4 expression in hypoxic mAECs. Significance: The important implication from our research is that Glut1 is functionally expressed in the alveolar epithelium and plays an important role in maintaining cell viability and ATP production (as subsequently measured in our studies using mAECs). Based on our preliminary data, we conclude that hypoxia-induced alterations in the functional expression of Glut1 in the lung epithelium may be an important target for treatment of pulmonary diseases characterized by metabolic stress. This work is supported by the Huntsman Endowed Chair awarded to RP and R01HL137033 awarded to MH. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.