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. 2024 Apr 30;16(4):e59331.
doi: 10.7759/cureus.59331. eCollection 2024 Apr.

Dexamethasone and Insulin Modulate Alanine Aminotransferase (ALT) Activity and Alanine Oxidation in C2C12 Cells in a Dose-Dependent Manner

Affiliations

Dexamethasone and Insulin Modulate Alanine Aminotransferase (ALT) Activity and Alanine Oxidation in C2C12 Cells in a Dose-Dependent Manner

Saed Woraikat et al. Cureus. .

Abstract

Background: The muscle cells myocytes are differentiated for the purpose of contraction function, which plays a major role in body metabolism and energy haemostasis, through different metabolic pathways, such as glucose and protein metabolic pathways. Alanine aminotransferase (ALT) plays a crucial role by reversibly catalysing transamination between alanine and a-ketoglutarate to form pyruvate and glutamate and by mediating the conversion of these four major intermediate metabolites. ALT plays important roles for energy homeostasis during fasting and prolonged exercise anaerobically, when muscle protein must first be broken down into its constituent amino acids.

Methods: Mouse skeletal myoblast cell line C2C12 was cultured in Dulbecco's modified eagle medium (DMEM) growth medium, supplied with 2% horse serum supplemented with 1 uM insulin, 2 mM glutamine and penicillin and streptomycin antibiotics for seven days. The differentiation medium is refreshed every 24 hours. Then, C2C12 cells were treated with insulin and dexamethasone to examine their effects on myocytes' ALT activity.

Results: In our study, we found an impact on ALT activity under different influences, including C2C12 differentiation, dexamethasone and insulin treatments, which shed light on the dynamic interplay between ALT activity, alanine metabolism, and cellular states, like differentiation and stress responses.

Conclusion: The study provides valuable insights into the dynamic regulation of ALT activity and alanine metabolism in C2C12 cells across differentiation and drug treatments. Further research is encouraged to explore the underlying mechanisms and their implications for muscle function, differentiation and potential therapeutic interventions in metabolic disorders.

Keywords: alt; c2c12; dexamethasone; expression; insulin; myocytes.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ALT activity during C2C12 differentiation
A) Western blot representing the expression levels of ALT1 and ALT2, where β-actin serves as a loading control. B) Relative ALT activity compared to the control group. C) Quantitative analysis depicting the activity of ALT over a six-day period suggests an increase in ALT activity from day 0 to day 6, peaking on day 6. Data are expressed as mean ± S.E. * p < 0.05; **: p < 0.01; ****: p < 0.0001. ALT: alanine aminotransferase, SE: standard error.
Figure 2
Figure 2. Insulin and dexamethasone effects on ALT activity in C2C12 cells
Demonstrating ALT activity changes under the influence of insulin 0.1 μM and dexamethasone 1 μM treatments. Data are expressed as mean ± S.E. ****: p < 0.0001. ALT: alanine aminotransferase, SE: standard error.
Figure 3
Figure 3. C2C12 treated with high-dose dexamethasone (1 µM) and low-dose dexamethasone (0.1 µM)
A) Indicating the activity of ALT in C2C12 cells treated with two concentrations of dexamethasone during a period of 72 hours compared to untreated control cells. B) Western blot analysis of ALT1 and ALT2 protein expression in C2C12 cells following treatment with dexamethasone (1 µM and 0.1 µM) in comparison to control. β-actin is used as a loading control. Data are expressed as mean ± S.E. * p < 0.05; **: p < 0.01. ALT: alanine aminotransferase, SE: standard error.
Figure 4
Figure 4. Alanine oxidation in C2C12 treated with dexamethasone, insulin and D-cycloserine
A) Quantitative analysis representing the activity of ALT in C2C12 cells under various concentrations of d-cycloserine. B) representing the alanine oxidation in C2C12 cells treated with dexamethasone (Dex) or insulin (Ins) compared to the untreated control. C) Illustrating the effect of different concentrations of d-cycloserine on alanine oxidation in C2C12 cells. Data are expressed as mean ± S.E. * p < 0.05; ****: p < 0.0001. ALT: alanine aminotransferase, SE: standard error.
Figure 5
Figure 5. Alanine oxidation during C2C12 differentiation
Bar graph showing the relative alanine oxidation in C2C12 myoblasts at three different time points: initial (day 0), mid-differentiation (day 3) and late differentiation (day 6). The relative oxidation is normalised to the initial value on day 0. Data are expressed as mean ± S.E. ****: p < 0.0001. SE: standard error.
Figure 6
Figure 6. Alanine oxidation of different doses of alanine treatment
The graph depicts the relative alanine oxidation in response to different concentrations of alanine treatment. The oxidation is shown relative to the untreated control. Data are expressed as mean ± S.E. *** p < 0.001. SE: standard error.
Figure 7
Figure 7. Alanine oxidation of ALT overexpression
A) Western blot displaying the protein expression levels of ALT1 and ALT2 following transfection with their respective overexpression constructs (ALT1-AD and ALT2-AD) compared to a control vector (green fluorescent protein-adenovirus (GFP-AD)). β-actin is used as a loading control. B) Quantitative analysis of relative alanine oxidation in cells overexpressing either ALT1-AD or ALT2-AD, normalised to the control GFP overexpression. Data are expressed as mean ± S.E. ****: p < 0.0001. ALT: alanine aminotransferase, SE: standard error.

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Grants and funding

This study was supported by grants from the Natural Science Foundation Project of Chongqing (cstc2021jcyj-msxmX0286).

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