Mount Royal University - Laminin supplementation in cell culture and metabolic pathway indicators

Characterization of GLUT1 and CPT1B

Expression Patterns in C2C12 myogenesis

Meet The Team

Meet the line up behind the laminin lab!
Left to right: Chris Shaw, MaKayla Driedger, Charline Luong, Kim Nolasco

Figure 1. Proposed metabolic switch. Glucose transporter 1 (GLUT1) is a concentration-dependent plasma membrane transporter(1). Carnitine palmitoyl transferase 1B (CPT1B), located in the outer mitochondrial membrane, is the rate-limiting enzyme in mitochondrial FAO. Adapted from Relaix et al., 2021.

Research Purpose: This study aims to characterize the proposed metabolic shift using GLUT1 and CPT1B gene and protein expression/localization. Since the rate of myogenesis can be enhanced by laminin (an extracellular matrix protein), we further determined whether changes in the expression of GLUT1 and CPT1B were mirrored by the shift in myogenesis.

Keywords: C2C12, Laminin, Extracellular Matrix, Glucose transporter 1 (GLUT1), Carnitine palmitoyl transferase 1-B (CPT1B), Mitochondria, Fatty-Acid Oxidation, Metabolism, Cell Differentiation, Myogenesis, Morphology, Cellular and Molecular Biology, Cell Biology.

Rational

Muscle cell metabolism is an area of study where much is still unknown. To explore metabolic preferences during the stages of myogenesis, GLUT1 and CPT1B are proteins whose presence is suggestive of the cells' ability to use glucose and fatty acids for energy respectively. Current literature surrounding GLUT1 and CPT1B exists in different cell types where the knockout of GLUT1 has been shown to alter cellular glucose uptake(3). CPT1B knockout studies show fatty-acid import into the mitochondria in these knockouts does not occur(4).

Though previous studies show these transporters are useful indicators of glucose and fatty acid metabolism, studies examining muscle cell metabolism during myogenesis using these transporters are limited. Determining optimal cell culture conditions is critical when studying a cell line in vitro in order to obtain results that can be translated in vivo. Better understanding muscle cell metabolism during myogenesis can provide insight useful in the development of embryonic development, cell therapies, lab-grown meat(5) and treatments for disorders such as myogenic myopathies.

Figure 2. Potential markers of metabolic activity. Left: Depiction of GLUT1 transporter(8). Image made using Canva. Right: Depiction of CPT-1B. Note that fatty acid itself doesn't move across the membrane. It must be converted into acylcarnitine. Likewise, CPT1B doesn't transfer fatty acid itself; it catalyzes the fatty acid into acylcarnitine, which passively diffuses through the membrane(7). Image made using Canva.

Background

The formation of skeletal muscle during embryonic development occurs when star-shaped single nucleated myoblast cells (precursor) proliferate and differentiate into multinucleated tube-like cells called myotubes in a process called myogenesis(9). Myotubes will undergo fusion and mature to form a myofiber-the most basic unit of functional skeletal muscle(9).

The components of the cell microenvironment include physical fields, soluble factors, neighboring cells and extracellular matrix components(8). The current recommended cell culture conditions for the C2C12 cell line consider many of these factors, but do not consider cell interactions with the extracellular matrix(10) . Cells in vivo have the support of extracellular matrix proteins, which are critical for providing a protein scaffold which supports cell structure and function via cellular signalling events(11).

The 2013 study by Tixier et al., strongly suggests that the glycolysis-based pathway is favoured in the myogenic programming of syncytial muscle cells(12). The impact of aging in vivo alters the metabolic profile of the satellite cells, in turn, favouring the glycolytic pathways over oxidative phosphorylation in myoblast(12). Glycolysis is regulated indirectly by insulin due to the promotion of glucose uptake, and when this pathway was inhibited - results demonstrate the decline of glycolytic genes, in turn having decreased muscle growth and myoblast fusion arrest(12).

Mitochondria are well known to create energy in the form of ATP, primarily utilizing glucose(2). However, cell proliferation—an important part of myogenesis—requires more than just glucose. With just glucose, cells do not proliferate, and even die off. Fatty acids are also needed and can be used for ATP synthesis, as well as precursors for other macromolecules. As such, fatty acid oxidation can be a good indicator of cell metabolism.

The Metabolic Switch proposed by Relaix et al., suggests that oxidative metabolism does not occur during the early stages of myogenesis(2). It is suggested that muscle cells refrain from oxidative metabolism during the early stages, when cells are actively proliferating. This is beneficial as non-oxidative metabolism limits reactive oxygen species (ROS) production, thus decreasing cellular oxidative stress. As such, Relaix et al. suggest there are no active mitochondria present during the early stages of myogenesis(2). Considering this proposed metabolic switch, the use of cationic mitochondrial labels during immunofluorescence microscopy permit the visualization of active mitochondria during the various stages of myogenesis. Further, in assessing CPT1B protein expression using immunocytochemistry synergistically with these mitochondrial labels, CPT1B colocalization with mitochondria can be assessed.

Figure 3. Left: Potential metabolic pathway(s) of glucose. Glucose enters the cell via the GLUT1 transporter, located on the plasma membrane(6). From there, it may be used in the pentose phosphate pathway, glycolysis, or fermentation(6). The pyruvate resulting from glycolysis can be moved to the mitochondria for processes, such as the Krebs cycle, there(6). Image created using Canva. Right: A simplified depiction of the beginning of fatty acid oxidation/usage. Carnitine is connected to a fatty acid to create acylcarnitine via CPT1B on the cytosolic side of the outer mitochondrial membrane(7). This molecule then passively travels into the mitochondrial intermembrane space. From there, the acylcarnitine is transferred into the matrix by carnitine-acylcarnitine translocase (CAT) where it is cleaved apart again. The fatty acid then goes on to be used and Carnitine is then re-shuffled back outside to repeat the process(7). Image created using Canva.

Laminin

Laminin is known as a regulator of cell proliferation, differentiation, migration and adhesion. The laminin from Engelbreth-Holm Swarm murine sarcoma basement membrane will be used in this study(6). The known structures of laminin isolated from Engelbreth-Holm Swarm have heterotrimeric chains: the alpha-, beta-, and gamma-, held together by disulfide bonds(13). A simplified shape of laminin can be seen in figure 4, depicting three chains coiled together in an alpha-helical manner(13). The LG region (typically divided into LG 1-3 and LG 4-5) is at the c-terminal end of the chain(13). The first globular region of each chain is the N-terminal region (LN), and is similar in all three chains(13). Applications for this laminin have demonstrated an increased rate of muscle cell growth and differentiation(11).

Figure 4. Diagram depicting cells and their attachment to the basal membrane via laminin, and a close up of laminin's trimeric structure.

Experimental design

Fig 5. Flowchart showing the order of operations of the experiment. The top row shows the expected timeline of cell cultures. The middle row describes the experimental design for assessing GLUT1 and CPT1B protein expression and localization, and the bottom row describes the experimental design for assessing GLUT1 and CPT1B gene expression.

Results

GLUT1 Protein Expression and Localization

Figure 6. Epifluorescence micrographs of the myogenic process. Nuclei are labelled with DAPI, mitochondria with MitoTracker Red and GLUT1 with Alexa Fluor 488. All cells viewed with Olympus IX51 inverted microscope at 600x magnification and captured using an Olympus XM10 camera in conjunction with Olympus CellSens Standard.

GLUT1 Gene Expression

Figure 7. Relative expression of GLUT1 in C2C12 cells during myogenesis. GLUT1 expression for all samples was normalized against the non-laminin MB samples, using GAPDH as the reference gene. n=2

GLUT1 Key Results

GLUT1 gene expression was consistent throughout myogenesis.
GLUT1 protein was localized in the plasma membrane and its expression was unchanged throughout myogenesis.
Mitochondria were present at all stages of myogenesis.
Cells grown on laminin coating had myotube formation at day 4 compared to day 7 in controls.
GLUT1 gene expression remained relatively consistent throughout myogenesis in laminin-supplemented culture conditions. GLUT1 protein was expressed throughout myogenesis and localized to the plasma membrane

CPT1B Protein Expression and Localization

Figure 8. Epifluorescence micrographs of the myogenic process. Nuclei are labelled with DAPI, mitochondria with MitoTracker Red and CTP1B with Alexa Fluor 488. All cells viewed with Olympus IX51 inverted microscope at 600x magnification and captured using an Olympus XM10 camera in conjunction with Olympus CellSens Standard.

CPT1B Gene Expression

Figure 9. Relative expression of CTP1B in C2C12 cells showing an increasing trend over the days of differentiation. CTP1B expression for all samples was normalized against the non-laminin MB samples, using GAPDH as the reference gene. n=2

CTP1B Key Results

CPT1B gene expression does not change over the course of myogenesis.
CPT1B protein is expressed at earlier time points but only colocalized with mitochondria in day 7 and day 11 myotubes.
Mitochondria were present at all stages of myogenesis.
Cells grown on laminin coating had myotube formation at day 4 compared to day 7 in controls.
CPT1B protein was expressed in laminin-supplemented myoblasts compared to day 4 in controls.
CPT1B protein colocalized with mitochondria in day 4, day 7 and day 11 myotubes grown on laminin.

Discussion and Conclusion

Earlier myotube formation was seen in laminin supplemented cells which is consistent with an enhanced myogenic rate.
CPT1B protein expression also occurred at an earlier time point compared to controls. Thus it appears to indicate a metabolic switch, however this is not reflected in the gene expression.
Surprisingly, GLUT1 gene expression was upregulated in mature myotubes. Further studies are needed to confirm these results.
In contrast to the metabolic switch proposed by Relaix et al.(2), mitochondria were highly evident in myoblasts. This data is in line with previous studies suggesting mitochondria as a potential regulator of myogenesis(14).
This data suggests that to better understand the proposed metabolic switch, studies which measure glucose and fatty acid flux are required.

References

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(2) Relaix, F., Bencze, M., Borok, M.J. et al. Perspectives on skeletal muscle stem cells. Nature Communications 12, 692 (2021). https://doi.org/10.1038/s41467-020-20760-66

(3) Bao, Y. Y.; Zhong, J. T.; Shen, L. F.; Dai, L. B.; Zhou, S. H.; Fan, J.; Yao, H. T.; Lu, Z. J. Effect of Glut‐1 and Hif‐1α double knockout by CRISPR/Cas9 on radiosensitivity in laryngeal carcinoma via the PI3K/AKT/Mtor pathway. Journal of Cellular and Molecular Medicine 2022, 26 (10), 2881–2894.

(4) Zhang, Y.; Fang, X.; Dai, M.; Cao, Q.; Tan, T.; He, W.; Huang, Y.; Chu, L.; Bao, M. Cardiac-specific down-regulation of carnitine palmitoyltransferase-1B (CPT-1B) prevents cardiac remodeling in obese mice. Obesity 2016, 24 (12), 2533–2543.

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(7) Tourneau, C.; Raymond, E.; Faivre, S. Aplidine: A Paradigm of How to Handle the Activity and Toxicity of a Novel Marine Anticancer Poison. Current Pharmaceutical Design 2007, 13 (33), 3427–3439

(8) Warrick, J. W.; Murphy, W. L.; Beebe, D. J. Screening the Cellular Microenvironment: A Role for Microfluidics. Reviews in Biomedical Engineering 2008, 1, 75–93.

(9) Burattini, S., Ferri, P., Battistelli, M., Curci, R., Luchetti, F., & Falcieri, E. (2009). C2C12 murine myoblasts as a model of skeletal muscle development: morpho-functional characterization. European Journal of Histochemistry, 48(3). https://doi.org/10.4081/891

(9) C2c12 - crl-1772 | ATCC. https://www.atcc.org/products/crl-1772 (accessed Oct 31, 2022).

(10) Penton, C. M.; Badarinarayana, V.; Prisco, J.; Powers, E.; Pincus, M.; Allen, R. E.; August, P. R. laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion. Skeletal Muscle 2016, 6 (1).

(12) Tixier, V., Bataillé, L., Etard, C., Jagla, T., Weger, M., DaPonte, J. P., Strähle, U., Dickmeis, T., & Jagla, K (2013). Glycolysis supports embryonic muscle growth by promoting myoblast fusion. Proceedings of the National Academy of Sciences, 110(47), 18982–18987. https://doi.org/10.1073/pnas.1301262110.

(13) Hohenester, E. Structural Biology of Laminins. Essays in Biochemistry 2019, 63 (3), 285–295.

(14) Wagatsuma, A., Sakuma, K. Mitochondria as a potential regulator of myogenesis. Scientific World Journal. 2013; 2013:593267. doi: 10.1155/2013/593267. Epub 2013 Feb 3. PMID: 23431256; PMCID: PMC3574753.

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