Glucose transporter 1 (GLUT1)

How is GLUT1 localization & gene expression affected by glucose throughout C2C12 skeletal muscle myogenesis?

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Background

Glucose Transporter 1 (GLUT1) is a concentration dependent, plasma membrane transport protein responsible for basal glucose uptake in cells (Wang et al. 2020). Due to this, the presence of GLUT1 is considered to be a marker for glycolysis (Liu et al., 2012; Wang et al. 2020) and therefore visualizing GLUT1 localization via immunocytochemistry and relative gene expression via qPCR may aid in further characterizing the preferential metabolic pathways utilized by C2C12 cells during the stages of myogenesis. High glucose levels have been shown to induce insulin resistance in C2C12 myotubes (Luo et al., 2019) and therefore elucidating how the frequently utilized 25 mM glucose concentration affects C2C12 myogenesis is important to address the reproducibility crisis currently seen in the scientific community.

hypothesis

Increased glucose availability is hypothesized to correspond with elevated GLUT1 glucose uptake in C2C12 cells, most specifically in the early stage of differentiation in where more energy is required for the cells to rapidly proliferate. Therefore, the overall relative expression of GLUT1 is expected to be higher in the cells differentiating in 25 mM glucose in comparison to 5 mM glucose, in which GLUT1 will be localized within the cell membrane and thus actively transporting glucose into the cell.

GLUT1 LABELLING PATTERN

DAPI stain for Nuclei (BLUE)

GLUT1 tagged with AlexaFluor 488 Goat-Anti-Rabbit (GREEN)

MitoTracker Red Stain for Mitochondria (RED)

Overlay of All Channels

Note: Images were obtained on day 7 of myogenic differentiation during trial 1 (Marin Flanagan, 2022).

Phase contrast images

Myoblast

Day 0

Day 4

Day 6

Day 11

Representative images of C2C12 cells grown in 5.5 mM glucose during myogenesis (Marin Flanagan, 2022).

Myoblast

Day 0

Day 4

Day 6

Day 11

Representative images of C2C12 cells grown in 25 mM glucose during myogenesis (Jenna Buragina, 2022).

results

Immunocytochemistry

Representative micrographs of the immunocytochemistry performed throughout myogenesis. Mitochondria are shown in red, nuclei in blue, and GLUT1 in green. (Images taken by Marin Flanagan for 5.5 mM glucose and Jenna Buragina for 25 mM glucose, 2022)

Mitochondria are present at all stages of myogenesis.
GLUT1 appears to be localized to the plasma membrane.
GLUT1 is present in all stages of myogenesis in both glucose conditions.

Relative gene expression

GLUT1 gene expression appears to remain constant across the stages of myogenesis in both 5.5 mM and 25 mM glucose growth conditions.
In general, GLUT1 expression is higher in the 5.5 mM glucose condition in comparison to 25 mM glucose.
GLUT1 expression appears to increase in the later stages of differentiation (D6 and D11) for the 5.5 mM glucose condition, however, the error bars either overlap or are very close and therefore no definite conclusions on this can be made.

Average relative gene expression of GLUT1 throughout myogenesis (n=2-4).

discussion

Since active mitochondria were present throughout all stages of myogenesis in both glucose conditions and therefore could be performing oxidative phosphorylation, GLUT1 one cannot be considered a reliable marker for detecting the preferential use of glycolysis as it is only representing glucose uptake.
This finding refutes the current hypothesis in the literature, potentially demonstrating that oxidative phosphorylation is utilized throughout the entirety of C2C12 myogenesis independent of glucose concentration.
There appears to be more GLUT1 in C2C12 cells grown in 5.5 mM which may be due to the cells downregulating GLUT1 gene expression in response to excess glucose concentrations (25 mM) to avoid unnecessary glucose uptake, and upregulating GLUT1 when glucose is more limited.
Given this and the consistency of C2C12 cell morphology throughout differentiation in both glucose conditions, 25 mM glucose is likely an acceptable glucose concentration to use when raising C2C12 cells for observing myogenesis, however, further studies are necessary to confirm whether 25 mM glucose media can confound results in drug development research given this study was limited in reliably establishing metabolic pathway preferences.

references

Liu, Y., Cao, Y., Zhang, W., Bergmeirer, S., Qian, Y., Akbar, H., Colvin, R., Dingm J., Tong, L., Qu, S., Hines, J., & Chen, X. (2012). A small-molecule inhibitor of glucose transporter 1 downregulations glycolysis, induces cell-cycle arrest, and inhibits cancer cell growth in vitro and in vivo. Molecular Cancer Therapeutics, 11(8), 1672-1682. https://doi.org/10.1158/1535-7163.MCT-12-0131

Luo, W., Ai, L., Wang, B. F., & Zhou, Y. (2019, December). High glucose inhibits myogenesis and induces insulin resistance by down-regulating AKT signaling. Biomedicine &Amp; Pharmacotherapy, 120, 109498. https://doi.org/10.1016/j.biopha.2019.109498

Wang, J., Xu, W., Wang, B., Lin, G., Wei, Y., Abudurexiti., Zhu, W., Liu, C., Qin, X., Dai, B., Wan, F., Zhang, H., Zhu, Y., & Ye, D. (2020). GLUT1 is an AR target contributing to tumor growth and glycolysis in castration-resistant and enzlutamide-resistant prostate cancers. Cancer Letters, 485, 45-55. https://doi.org/10.1016/j.canlet.2020.05.007

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