Simvastatin represses protein synthesis in the muscle-derived C2C12 cell line with a concomitant reduction in eukaryotic initiation factor 2B expression

Journal Title (Medline/Pubmed accepted abbreviation): Am. J. Physiol. Endocrin. Metab.
Year: 2011
Volume: 300
Page numbers: E564-E570
doi (if applicable): 10.1152/ajpendo.00383.2010

Summary of Background and Research Design

Background: Statins are drugs commonly used for lowering cholesterol. A negative side effect that has been reported while taking statins is muscle weakness. There is evidence that statins may interfere with expression of eukaryotic initiation factor 2B (eIF2B), which is cellular protein that promotes muscle protein synthesis.

Research goal: To better understand the relationship between statin-related reductions in protein synthesis and eIF2B in a muscle cell model.

Cell model: C2C12 are cells derived from murine (mouse) muscle cells.

Treatments/Protocol: Cells were treated with simvastatin (concentration of 10 µM), a statin drug, for 24 hrs. As a control, an equal volume of simvastatin-free medium was added to an identical culture. Also as a control, mevalonate was added to cells in the presence and absence of simvastatin. [NOTE: Mevalonate is the product of HMG-CoA reductase, the enzyme that statins inhibit. By adding mevalonate, the authors are “undoing” the effects of the statin inhibitor. As such, if simvastatin’s actions are important for reducing muscle protein synthesis, addition of mevalonate to simvastatin-treated cells should normalize protein synthesis]. During the last 30 min of incubation, 35S-labeled sulfur-containing amino acids (methionine and cysteine) were added to the media in order to measure its incorporation into protein and, therefore, the rate of protein synthesis. eIF2B activity was also assayed by looking at eIF2B guanine nucleotide exchange, the activity for which the eIF2B enzyme is responsible. Abundance and phosphorylation status (determines if enzymes are activated) of specific proteins were analyzed using Western blot and total RNA levels were analyzed using quantitative real-time polymerase chain reaction (PCR).

Summary of research findings:
  • Protein synthesis was substantially repressed when simvastatin was added to cells. When mevalonate was added, the rate of protein synthesis was rescued.
  • The quantity of all 5 subunits of eIF2B was reduced with simvastatin treatment, but the mRNA quantity was the same, meaning that the rate of protein translation was repressed and/or the rate of degradation was increased.
  • The activity of the eIF2B protein was reduced about 20% in the presence of simvastatin.
  • The authors suspected that the ubiquitin proteasome pathway (the complex that breaks down old, used, or unnecessary proteins) might be breaking down eIF2B, and that simvastatin may be influencing the ubiquitin proteasome pathway. When the cells were treated with both simvastatin and a compound that inhibits the ubiquitin proteasome pathway (MG-132), eIF2B expression was normalized. This suggests that eIF2B is getting degraded by the proteasome at a faster rate in the presence of simvastatin.

Interpretation of findings/Key practice applications:

Patients on statin medications sometimes experience muscle weakness and this study sheds light on the possible mechanism. Statins may cause eIF2B to be degraded more rapidly by the proteasome, therefore decreasing the rate of protein synthesis in muscle cells. A key limitation to this study, however, was the dose of simvastatin necessary to produce these effects. The authors found no effects at a concentration of 0.1µM, intermediate effects at a concentration of 1µM, and the above-mentioned effects at 10 µM. However, the blood levels of this drug in therapeutic usage in humans are generally in the submicromolar range. Thus, it is not clear if the findings of this study, while interesting from a basic science standpoint, can be extrapolated to the clinical setting. If a patient is on statins and is experiencing muscle weakness he or she should discuss it with a physician.
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