a1 Key Laboratory of Animal Genetics, Breeding and Reproduction of Ministry of Education and Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture, Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China
The present study was conducted to evaluate the mechanism by which n-3 PUFA regulates the inhibitor of κBα (IκBα)/NF-κB/muscle RING finger 1 (MuRF1) pathway in C2C12 myotubes. After treatment with 150, 300 or 600 μm-α-linolenic acid (ALA) or -EPA for 24 h in C2C12 myotubes, the levels of phosphorylated IκBα (p-IκBα) and total IκBα were measured by Western blot. Compared with the bovine serum albumin (BSA) control, 150 and 300 μm-ALA and -EPA, respectively, did not affect the total IκBα protein level (P>0·05). However, 600 μm-EPA, but not 600 μm-ALA, prevented IκBα phosphorylation and increased the total IκBα levels (P < 0·01). Furthermore, total nuclear protein was isolated and analysed by the electrophoretic mobility shift assay for NF-κB DNA-binding activity after treatment with 600 μm-ALA or -EPA for 24 h. EPA (600 μm), but not ALA (600 μm), decreased the NF-κB DNA-binding activity when compared with BSA (P < 0·01). It was further observed that 600 μm-EPA caused a 3·38-fold reduction in the levels of MuRF1 mRNA expression compared with BSA (P < 0·01). Additionally, 600 μm-EPA resulted in a 2·3-fold induction of PPARγ mRNA expression (P < 0·01). In C2C12 myotubes, PPARγ knockdown by RNA interference significantly decreased PPARγ mRNA and protein expression to approximately 50 and 60 % (P < 0·01), respectively. Interestingly, in C2C12 myotubes with PPARγ knockdown, 600 μm-ALA and -EPA did not affect the levels of p-IκBα and total IκBα, NF-κB DNA-binding activity or MuRF1 mRNA expression when compared with BSA (P>0·05). These results revealed that EPA, but not ALA, inhibited the IκBα/NF-κB/MuRF1 pathway in C2C12 myotubes in a PPARγ-dependent manner.
(Received May 17 2010)
(Revised August 23 2010)
(Accepted August 24 2010)
(Online publication October 19 2010)
Abbreviations: ALA, α-linolenic acid (C18 : 3n-3); BSA, bovine serum albumin; IκBα, inhibitor of κBα; MuRF1, muscle RING finger 1; p-IκBα, phosphorylated IκBα; RNAi, RNA interference