EPA inhibits the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 pathway in C2C12 myotubes in a PPARγ-dependent manner


Journal Title (Medline/Pubmed accepted abbreviation): Br. J. Nutr.
Year: 2011
Volume: 105
Page numbers: 348-356
doi (if applicable): 10.1017/S0007114510003703

Summary of Background and Research Design

Background:The NF-κB cell signaling pathway is activated under stress and affects transcription of genes involved in the immune system. One gene that is activated by the NF-κB pathway is MuRF1 (muscle RING finger 1). MuRF1 targets muscle protein for degradation.
     n-3 Polyunsaturated fatty acids (PUFAs), also known as omega-3 fatty acids, have been touted for having anti-inflammatory properties. n-3 PUFAs include α-linolenic acid (ALA, from plants such as flax seed), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), the latter two existing in fish.

Mechanism under investigation: n-3 PUFAs → ↑ expression of PPARγ, a nuclear receptor → ↓ degradation of IκBα (an inhibitor of NF-κB) → ↓ activity of NF-κB → ↓ expression of MuRF1 → ↓ degradation of muscle protein

RNA interference (RNAi)- a molecular biology technique in which a strand of RNA complementary to the gene of interest is synthesized and introduced to a cell culture. Expression of the gene is then downregulated.


Hypothesis: ALA and EPA will downregulate the NF-κB pathway. Consequently, an increase in total IΚBΑ, a decrease in NF-κB binding to DNA, and a decrease in expression of the MuRF1 gene will be observed. If PPARγ is downregulated artificially, these effects will not be seen.

Experimental protocol:Mouse myoblasts (precursors to muscle cells) were used in a cell culture.
1) ALA and EPA were introduced to myoblasts at 150, 300, and 600 µM. The amount of phosphorylated IκBα (p-IκBα, targets IκBα for degradation, indicates activation of the NF-κB pathway) and total IκBα were assessed along with the DNA-binding activity of NF-κB.

2) ALA and EPA were introduced at the same concentrations to assess expression of the MuRF1 and PPARγ genes

3) RNAi was used to knockdown PPARγ gene expression. Then 600 µM n-3 PUFAs were introduced and components of the NF-κB pathway were quantified.


Summary of research findings:
1) 600 µM of EPA decreased the amount of p-IκBα and caused an 86% increase in total IκBα levels. ALA nor lower concentrations of EPA showed an effect. 600 µM of EPA also lead to a decrease in NF-κB DNA-binding in the cell culture.

2) 600 µM of EPA caused MuRF1 mRNA quantities to decrease 3.38 fold and PPARγ mRNA levels to increase 2.3 fold. ALA showed no effect.

The results from EPA in experiments (1) and (2) corroborate their proposed mechanism that n-3 PUFAs downregulate the NF-κB pathway.

3) RNAi succeeded in knocking down PPARγ about 50-60%. Contrary to the cells with functional PPARγ, 600 µM EPA did not prevent the degradation of IκBα, decrease the binding of NF-κB to DNA, or reduce MuRF1 gene expression. ALA showed no significant effect here either. This result indicates that the mechanism of action of EPA is through PPARγ.


Interpretation of findings/Key practice applications:

600 µM of EPA showed to reduce activity of the NF-κB pathway, but not ALA. The results of this study provide evidence that EPA may work through PPARγ to manifest its anti-inflammatory effects. It is unknown why EPA was effective and ALA was not.

Limitations:

One must be cautious in translating this result into the clinical setting. It is unlikely that cells will ever be exposed to EPA levels of this magnitude. Even supplementation of 2.2 g EPA and 2.2 g docosahexaenoic acid (DHA) per day for 6 weeks in healthy men results in plasma EPA and DHA concentration of about 150?M each (Bloomer et al., Lipids Health Dis 2009;8:36 DOI 10.1186/1476-511X-8-36), which is substantially (4-fold) lower than the level of EPA used in this experiment.
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