Variable Glucose Microenvironments

Research Question

How do physiological glucose conditions (5.5 mM) impact the protein and gene expression of GLUT1 and CPT1B in comparison to the standard culture conditions (25 mM) used throughout C2C12 Myogenesis?

Rationale & purpose

The purpose of this study is to determine the localization of Glucose Transporter 1 (GLUT1) and Carnitine Palmitoyltransferase 1B (CPT1B) throughout C2C12 myogenesis under standard cell culture (25 mM) and physiological (5.5 mM) glucose media conditions to help elucidate the preferred metabolic pathways utilized during skeletal muscle myogenic differentiation. This is also necessary to address the current reproducibility crisis in which researchers are utilizing different glucose conditions to observe C2C12 myogenesis.

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Image created via BioRender.com.

MYOGENESIS

The C2C12 muscle cell growth and differentiation timeline. Micrographs demonstrating changes in cell morphology and confluency were captured via low-power Phase Contrast microscopy. Due to limitations in the first trial, the representative day zero image is not at the desired 100% confluency, however, the morphology of the cells is accurate.

The ATCC primary cell culture of C2C12 cells is a prime model for this study as it is well-researched and has been extensively utilized in studies on glucose metabolism and insulin resistance (Wong et al., 2020).

Metabolism

The metabolism of C2C12 muscle cells relies on the catabolism of carbohydrates, lipids, and proteins for the generation of ATP predominantly through the process of mitochondrial oxidative phosphorylation. Whether oxidative phosphorylation is used consistently throughout skeletal muscle myogenesis is currently under debate, however, most of the literature supports the theory that glycolysis is preferred during myoblast proliferation and oxidative phosphorylation takes precedence upon the onset of differentiation (Relaix et al., 2021). Leary et al. (1998) emphasizes this differential use of metabolic pathways throughout C2C12 myogenesis by showing that proliferating myoblasts obtain only 30% of their ATP from oxidative phosphorylation, while day 12 myotubes depended on this pathway for 61% of their ATP. Specifically, once proliferation ceased, cells became increasingly reliant on mitochondrial pathways as myogenesis progressed (Leary et al., 1998). This metabolic switch from glycolysis to oxidative phosphorylation during myogenesis is further supported by Barbieri et al. (2011) which demonstrated a 2-fold increase in mitochondrial biogenesis after differentiation.

The currently proposed metabolic switch hypothesis in which glycolysis is hypothesized to be preferentially used during the proliferation stage of muscle cells, where upon differentiation, active mitochondria are present and oxidative phosphorylation takes precedence. Image obtained from Relaix et al. (2021).

Hypothesis diagram adapted from Relaix et al. (2021) and modified to include GLUT1 and CPT1B used in this study.

Image obtained from Baker (2016).

REPRODUCIBILITY CRISIS

The scientific community is currently faced by a reproducibility crisis in which inconsistent study design, reporting, and a lack of reproduction has translated to little advancement in various disciplines (Baker, 2016; Begley & Ioannidis, 2015; Block, 2021; Laraway et al., 2019). Regarding the C2C12 model, the glucose concentration utilized in cell culture media across studies is inconsistent, and high glucose conditions exceeding the physiological level of 5 mM is commonly employed due to this concentration enhancing the rate of C2C12 differentiation (Seyer et al., 2011). The ATCC even specifically recommends culturing C2C12 muscle cells in 25 mM of glucose despite being evidently unrepresentative of in vivo skeletal muscle conditions. Additionally, applications within this field of research have been stated as unrepresentative of animal models due to the use of exaggerated conditions and neglect of systems like metabolism during experimentation (Drucker, 2016). Overall, drawing conclusions based on myogenesis observed at 25 mM glucose conditions is unreliable for clinical implications and therefore it is crucial to establish how high and low glucose culture conditions affect C2C12 cell metabolic processes and proliferation throughout myogenic differentiation.

Experimental Flow: ImmunoCytochemistry

C2C12 cells were grown in high (25 mM) and low (5.5 mM) glucose conditions and observed at the myoblast stage, days 0, 4, 7, and 11 of myogenesis. Immunocytochemistry was used to visualize both the localization and presence of the cellular markers, CPT1B and GLUT1.

Staining of mitochondria with MitoTracker Red will occur before fixation. Cells were permeabilized and blocked, then the nuclei were stained with DAPI. One set of low glucose and one set of high glucose coverslips were each subjected to GLUT1 and CPT-1B separately. Imaging using an epifluorescence microscope was performed on each of the days and compared to identify the differences in relative concentrations of these cellular markers and their localization throughout myogenesis. Control coverslips will also be prepared for each set and will include an autofluorescence control (blank), a non-specific binding control (secondary antibody only) and controls to test for staining bleed-through (DAPI only and MitoTracker Red only).

Experimental Flow: Gene expression

C2C12 cells were grown in high (25 mM) and low (5.5 mM) glucose conditions and isolated at the myoblast stage, days 0, 4, 7, and 11 of myogenesis. Primers were designed via DNAstar SeqBuilder Pro software. The efficiency of the primers for the target genes (CPT1B & GLUT1) and the housekeeping gene (GAPDH) was evaluated using quantitative polymerase chain reaction (qPCR). RNA was extracted from all samples, and then assessed for quality and concentration using gel electrophoresis, spectrophotometry, and fluorometry. Following RNA assessment, RNA was reverse transcribed into complementary DNA (cDNA), which was used to run qPCR for the target (CPT1B & GLUT1) and housekeeping (GAPDH) genes. Relative gene expression of CPT1B and GLUT1 was determined using the Livak method, where the myoblast grown in 25 mM glucose served as the calibrator sample (n=2-4).

Video methods for the preparation of coverslips for immunocytochemistry

Video Methods

Cell Culture Video Final.mp4

Information about the markers used & Results

Carnitine Palmitoyltransferase 1B

Glucose transporter 1

conclusions/Future directions

The currently proposed hypothesis by Relaix et al. (2021) is inconsistent with the findings of this study as active mitochondria were detected in all stages of differentiation, including myoblasts.
Since there are mitochondria present in the early stages, glucose can go through either glycolysis or oxidative phosphorylation making GLUT1 only a marker of glucose uptake, and not an accurate marker of glycolysis.
Culturing cells in different glucose conditions did not show drastic differences, so the glucose conditions cells are grown in is not important given they will respond to their environment by adjusting gene expression to allow for only the necessary amount of glucose to enter the cell.
Further studies must focus on using more reliable methods for tracking glycolysis and oxidative phosphorylation, such as quantifying metabolic byproducts of these metabolic pathways, in order to confirm the conclusions made in this study.

Research Poster Presented at the Faculty of Science and Technology Research Day

Group 1 Poster.pptx

group members

From L to R: Jenna Buragina, Reem Amin, and Nicelle Chua in B141 lab.

From L to R: Jenna Buragina, Marin Flanagan, Nicelle Chua, and Reem Amin at 2023 MRU Research Days.

REFERENCES

Baker, M. (2016). 1,500 scientists lift the lid on reproducibility. Nature, 533, 452-454.

Barbieri, E., Battistelli, M., Casadei, L., Vallorani, L., Piccoli, G., Guescini, M., . . . Falcieri, E. (2011). Morphofunctional and biochemical approaches for studying mitochondrial changes during myoblasts differentiation. Journal of Aging Research, 2011 doi:10.4061/2011/845379

Begley, C. G., & Ioannidis, J. P. A. (2015). Reproducibility in science: Improving the standard for basic and preclinical research. Circulation Research, 116(1), 116-126. https://doi.org/10.1161/CIRCRESAHA.114.303819

Block., J. A. (2021). The reproducibility crisis and statistical review of clinical and translational studies. Osteoarthritis and Cartilage, 29(7), 937-938. https://doi.org/10.1016/j.joca.2021.04.008

Drucker, D. (2016). Never waste a good crisis: confronting reproducibility in translational research. Cell metabolism, 24(3), 348-360.

Laraway, S., Snycerski, S., Pradhan, S., & Huitema, B. E. (2019). An overview of scientific responsibility: Consideration of relevant issues for behaviour science/analysis. Perspectives on Behavior Science, 42, 33-37. https://doi.org/10.1007/s40614-019-00193-3

Leary, S. C., Battersby, B. J., Hansford, R. G., & Moyes, C. D. (1998). Interactions between bioenergetics and mitochondrial biogenesis. Biochimica et biophysica acta (BBA)-bioenergetics, 1365(3), 522-530.

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.

Seyer, P., Grandemange, S., Rochard, P., Busson, M., Pessemesse, L., Casas, F., Cabello, G., & Wrutniak-Cabello, C. (2011). P43-dependent mitochondrial activity regulates myoblast differentiation and slow myosin isoform expression by control of calcineurin expression. Experimental Cell Research, 317(4), 2059-2071. https://doi.org/10.1016/j.yexcr.2011.05.020

Wong, C., Al-Salami, H., & Dass, C. (2020). C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceutical development at the preclinical stage. Journal of Pharmacy and Pharmacology, 72(12), 1667-1693.

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