Carnitine Palmitoyltransferase 1B (CPT1B)

How is CPT1B protein localization and gene expression affected throughout C2C12 skeletal muscle myogenesis?

Image obtained from Beger et al. (2018)

Background

Carnitine-palmitoyl transferase (CPT) is a rate-limiting enzyme with type I located on the outer mitochondrial membrane and type II on the inner membrane (McGarry & Brown, 1997). CPT has three isoforms, the one of importance to this study is the muscle isoform, M-CPT or CPT1B. CPT1B converts acyl-CoA into acylcarnitine which can then be transported into the mitochondria for B-oxidation of fatty acids, which inevitably leads to oxidative phosphorylation (McGarry & Brown, 1997; Beger et al., 2018). Therefore, CPT1B is a good marker of capacity of cells for fatty acid uptake as it controls mitochondrial β-oxidation and has been used as a marker in multiple studies (Schreurs et al., 2009; Ashmore et al., 2015; Sasaki et al., 2021; Holst et al., 2003).

HYPOTHESIS

CPT1B will be used as a marker of the cells’ capability to take up fatty acids; hence tracking the expression of CPT1B in C2C12 cells correlates to levels of β-oxidation, and in turn oxidative phosphorylation, revealing insight into the metabolism of differentiating myoblasts. If grown in a low glucose environment, then C2C12 cells are hypothesized to have a high expression level of CPT1B because there is not enough glucose for the cells to exclusively run glycolysis; therefore, the cells might resort to fatty acid oxidation for ATP production, increasing the expression of CPT1B to increase the cells’ capacity of fatty acid uptake.

CPT1B LABELLING PATTERN

DAPI stain for nuclei (BLUE)

CPT1B antibody tagged with AlexaFluor-488 (GREEN)

Mitochondria stained with MitoTracker Red (RED)

Overlay of all channels

Images obtained at the myoblast stage.

Phase contrast images

Myoblast

Day 0

Day 4

Day 6

Day 11

Representative images of C2C12 cells grown in 25 mM glucose during myogenesis. Images were taken by Nicelle Chua, 2023.

Myoblast

Day 0

Day 4

Day 6

Day 11

Representative images of C2C12 cells grown in 5.5 mM glucose during myogenesis. Images were taken by Reem Amin, 2023.

results

Immunocytochemistry

Representative images of the immunocytochemistry data throughout myogenesis. Mitochondria are shown in red, nuclei in blue, and CPT1B in green (n=3-4). The 5.5 mM glucose images were taken by Reem Amin (2023), and the 25 mM glucose images were taken by Nicelle Chua (2023).

CPT1B and mitochondria are present at all stages of myogenesis.
CPT1B does not colocalize to the mitochondria until day 7 in 5.5 mM glucose and on day 4 for 25 mM glucose as indicated by yellow.

Relative gene expression

CPT1B gene expression remains relatively constant throughout myogenesis.
The 5.5 mM and 25 mM glucose conditions have relatively similar expression levels of CPT1B, except at day 6, where the gene expression drops under 25 mM glucose.
The expression pattern of CPT1B in 5.5 mM and 25 mM glucose is relatively similar.
The onset of CPT1B protein expression was shifted to an earlier time point in 5.5 mM glucose relative to 25 mM.

Average relative gene expression of CPT1B throughout myogenesis normalized to the 25 mM myoblast with error bars representing the SEM (n=2-4).

discussion

Data obtained contradicts the proposed hypothesis by Relaix et al. (2021) as mitochondria are seen in early stages of differentiation, such as the myoblast.
This data suggests a possible difference in the hypothesized metabolic preferences when cells are raised in physiological glucose levels compared to 25 mM glucose.
Due to a small number of trials and large error bars, a conclusion is hard to come to as more trials must be conducted.

REFERENCES

Ashmore, T., Roberts, L. D., Morash, A. J., Kotwica, A. O., Finnerty, J., West, J. A., ... & Murray, A. J. (2015). Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism. BMC biology, 13(1), 1-17.

Beger, R. D., Bhattacharyya, S., Gill, P. S., & James, L. P. (2018). Acylcarnitines as translational biomarkers of mitochondrial dysfunction. Mitochondrial Dysfunct. Caused Drugs Environ. Toxic, 1, 383-393.

Holst, D., Luquet, S., Nogueira, V., Kristiansen, K., Leverve, X., & Grimaldi, P. A. (2003). Nutritional regulation and role of peroxisome proliferator-activated receptor δ in fatty acid catabolism in skeletal muscle. Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1633(1), 43-50.

McGarry, J. D., & Brown, N. F. (1997). The mitochondrial carnitine palmitoyltransferase system—from concept to molecular analysis. European journal of biochemistry, 244(1), 1-14.

Relaix, F., Bencze, M., Borok, M. J., Der Vartanian, A., Gattazzo, F., Mademtzoglou, D., ... & Rotini, A. (2021). Perspectives on skeletal muscle stem cells. Nature Communications, 12(1), 1-11.

Sasaki, Y., Kojima-Yuasa, A., Tadano, H., Mizuno, A., Kon, A., & Norikura, T. (2022). Ursolic acid improves the indoxyl sulfate-induced impairment of mitochondrial biogenesis in C2C12 cells. Nutrition Research and Practice, 16(2), 147-160.

Schreurs, M., Kuipers, F., & Van Der Leij, F. R. (2010). Regulatory enzymes of mitochondrial β-oxidation as targets for treatment of the metabolic syndrome. obesity reviews, 11(5), 380-388.

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