Endoplasmic Reticulum Stress and Autophagy Markers in Soleus Muscle Disuse-Induced Atrophy of Rats Treated with Fish Oil

Abstract

Endoplasmic reticulum stress (ERS) and autophagy pathways are implicated in disuse muscle atrophy. The effects of high eicosapentaenoic (EPA) or high docosahexaenoic (DHA) fish oils on soleus muscle ERS and autophagy markers were investigated in a rat hindlimb suspension (HS) atrophy model. Adult Wistar male rats received daily by gavage supplementation (0.3 mL per 100 g b.w.) of mineral oil or high EPA or high DHA fish oils (FOs) for two weeks. Afterward, the rats were subjected to HS and the respective treatments concomitantly for an additional two-week period. After four weeks, we evaluated ERS and autophagy markers in the soleus muscle. Results were analyzed using two-way analysis of variance (ANOVA) and Bonferroni post hoc test. Gastrocnemius muscle ω-6/ω-3 fatty acids (FAs) ratio was decreased by both FOs indicating the tissue incorporation of omega-3 fatty acids. HS altered (p < 0.05) the protein content (decreasing total p38 and BiP and increasing p-JNK2/total JNK2 ratio, and caspase 3) and gene expressions (decreasing BiP and increasing IRE1 and PERK) of ERS and autophagy (decreasing Beclin and increasing LC3 and ATG14) markers in soleus. Both FOs attenuated (p < 0.05) the increase in PERK and ATG14 expressions induced by HS. Thus, both FOs could potentially attenuate ERS and autophagy in skeletal muscles undergoing atrophy.

Keywords: docosahexaenoic acid; eicosapentanoic acid; hindlimb suspension; skeletal muscle atrophy; unfolded protein response; ω-3 fatty acids.

Figure 1

(A) Soleus phosphorylated IRE 1 (p-IRE1) content. Membranes stained with Ponceau S to assess total protein loading (n = 7–8). Results (mean ± SEM) were compared using two-way ANOVA. # For treatments with fish oils (p < 0.05). The six groups are: MO-C; control receiving mineral oil-MO, MO-HS; HS receiving MO, EPA-C; control treated with high EPA fish oil, EPA-HS; HS treated with high EPA fish oil, DHA-C; control treated with high DHA fish oil, and DHA-HS; HS treated with high DHA fish oil. (B) Complementary cDNA bands of not spliced XBP1 (uXBP1) (top band) and spliced XBP1 (sXBP1) (bottom band). Results obtained from 3 independent experiments. C+: positive control (insulin-producing beta-cell line, INS-1E treated with 1 μM thapsigargin, 1 h). MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.

Figure 2

Soleus phosphorylated and total JNK 1 and 2 contents: (A) p-JNK 1, (B) total JNK 1, (C) p-JNK 1/total JNK 1 ratio, (D) p-JNK 2, (E) total JNK 2, (F) p-JNK 2/total JNK 2 ratio, (G) western blotting representative images of JNK 1 and 2. Results (mean ± SEM) were compared using two-way ANOVA (n = 7–8). * Hindlimb suspension effect (p < 0.05). The six groups are: MO-C; control receiving mineral oil-MO, MO-HS; HS receiving MO, EPA-C; control treated with high EPA fish oil, EPA-HS; HS treated with high EPA fish oil, DHA-C; control treated with high DHA fish oil, and DHA-HS; HS treated with high DHA fish oil. MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.

Figure 3

Soleus p38 protein content. (A) Phosphorylated p-38 (p-p38), (B) total p38, (C) p-p38/total p38 ratio, (D) western blotting representative images of p38. Results (mean ± SEM) were compared using two-way ANOVA (n = 6–8). * Hindlimb suspension effect (p < 0.05). The six groups are: MO-C; control receiving mineral oil-MO, MO-HS; HS receiving MO, EPA-C; control treated with high EPA fish oil, EPA-HS; HS treated with high EPA fish oil, DHA-C; control treated with high DHA fish oil, and DHA-HS; HS treated with high DHA fish oil. MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.

Figure 4

Soleus content of endoplasmic reticulum stress markers: (A) binding immunoglobulin protein, BIP, (B) protein disulfide isomerase, PDI, (C) CCAAT/enhancer-binding protein homologous protein, CHOP, (D) caspase 3 (intact protein form). Results (mean ± SEM) were compared using two-way ANOVA (n = 6–8). * Hindlimb suspension effect (p < 0.05). The six groups are: MO-C; control receiving mineral oil-MO, MO-HS; HS receiving MO, EPA-C; control treated with high EPA fish oil, EPA-HS; HS treated with high EPA fish oil, DHA-C; control treated with high DHA fish oil, and DHA-HS; HS treated with high DHA fish oil. MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.

Figure 5

Effects of hindlimb suspension (HS) and oral fish oil (FO) supplementations (High EPA or High DHA) on soleus expression of endoplasmic reticulum stress markers: (A) BiP, (B) CHOP, (C) IRE1, (D) PERK. Results are reported as mean ± SEM (n = 6–9). RPL37A and HPRT1 genes are internal controls. BiP, CHOP, and IRE1 results were analyzed using two-way ANOVA. * HS effect (p < 0.05). PERK results were analyzed using two-way ANOVA and Bonferroni post hoc test. # FO treatment effects (p < 0.05). α Different from MO-C (p < 0.001). β Different from MO-HS (p < 0.01). The six groups are: MO-C, control receiving mineral oil-MO; MO-HS, HS receiving MO; EPA-C, control treated with high EPA fish oil; EPA-HS, HS treated with high EPA fish oil; DHA-C, control treated with high DHA fish oil; and DHA-HS, HS treated with high DHA fish oil. MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.

Figure 6

Effects of hindlimb suspension (HS) and oral fish oil (FO) supplementations (High EPA or High DHA) on autophagy gene expressions: (A) Beclin, (B) LC3 II, (C) ATG14. Results (mean ± SEM) were compared using two-way ANOVA (n = 6–9). * HS effect (p < 0.05). # FO treatment effect (p < 0.05). The six groups are: MO-C; control receiving mineral oil-MO, MO-HS; HS receiving MO, EPA-C; control treated with high EPA fish oil, EPA-HS; HS treated with high EPA fish oil, DHA-C; control treated with high DHA fish oil, and DHA-HS; HS treated with high DHA fish oil. MO: Mineral oil supplementation; EPA: High eicosapentaenoic acid fish oil supplementation; DHA: High docosahexaenoic acid fish oil supplementation; HS: hindlimb suspension; SEM: Standard Error of the Mean.