Short-Term High-Fat Feeding Does Not Alter Mitochondrial Lipid Respiratory Capacity but Triggers Mitophagy Response in Skeletal Muscle of Mice

Abstract

Lipid overload of the mitochondria is linked to the development of insulin resistance in skeletal muscle which may be a contributing factor to the progression of type 2 diabetes during obesity. The targeted degradation of mitochondria through autophagy, termed mitophagy, contributes to the mitochondrial adaptive response to changes in dietary fat. Our previous work demonstrates long-term (2-4 months) consumption of a high-fat diet increases mitochondrial lipid oxidation capacity but does not alter markers of mitophagy in mice. The purpose of this study was to investigate initial stages of mitochondrial respiratory adaptations to high-fat diet and the activation of mitophagy. C57BL/6J mice consumed either a low-fat diet (LFD, 10% fat) or high-fat diet (HFD, 60% fat) for 3 or 7 days. We measured skeletal muscle mitochondrial respiration and protein markers of mitophagy in a mitochondrial-enriched fraction of skeletal muscle. After 3 days of HFD, mice had lower lipid-supported oxidative phosphorylation alongside greater electron leak compared with the LFD group. After 7 days, there were no differences in mitochondrial respiration between diet groups. HFD mice had greater autophagosome formation potential (Beclin-1) and greater activation of mitochondrial autophagy receptors (Bnip3, p62) in isolated mitochondria, but no difference in downstream autophagosome (LC3II) or lysosome (Lamp1) abundance after both 3 and 7 days compared with the LFD groups. In cultured myotubes, palmitate treatment decreased mitochondrial membrane potential and hydrogen peroxide treatment increased accumulation of upstream mitophagy markers. We conclude that several days of high-fat feeding stimulated upstream activation of skeletal muscle mitophagy, potentially through lipid-induced oxidative stress, without downstream changes in respiration.

Keywords: autophagy; high-fat feeding; mitochondria; reactive oxygen species; respiration.

Figure 1

3 days of high-fat feeding lowered lipid substrate contribution to respiration and stimulated reactive oxygen species production. Mitochondrial respiration of isolated mitochondria from quadriceps muscle during sequential additions of substrates and inhibitors. (A) Rates of oxygen consumption expressed relative to protein content (pmol O₂/μg protein/sec) from mice fed low-fat or high-fat diet for 3 days. (B) Rates of oxygen consumption expressed relative to protein content (pmol O₂/μg protein/sec) from mice fed low-fat or high-fat diet for 7 days. (C) Contribution of lipid-supported oxidation expressed as a percentage of maximal respiration measured after FCCP. (D) Electron leak to H₂O₂ calculated from H₂O₂ emission during lipid-supported oxidative phosphorylation and expressed as a percentage of oxygen consumption. Data are means ± SD; n=4-5. Repeated measures ANOVA with Tukey’s post-hoc tests were used to analyze data in (A, B). Two-way ANOVA was used to test for effects of diet and time in (C, D). P values are main effects.

Figure 2

Short-term high-fat feeding did not alter mitochondrial enzyme abundance. Immunoblotting of lysates from quadricep muscle collected after a 4-hour fast from mice fed low-fat diet or high-fat diet for 3 days, 7 days, or 12 weeks. (A) Protein abundance of subunits in mitochondrial respiratory complexes I-V expressed in arbitrary units. (B) Hadh protein abundance in tissue lysates. (C) Representative blot images for (A, B). Data are means ± SD; n=4-5. Two-way ANOVA was used to test for effects of diet and time. P values are main effects.

Figure 3

Short-term high-fat feeding induced accumulation of mitophagy receptors. Autophagy protein markers in skeletal muscle of mice fed low-fat diet or high-fat diet for 3 days, 7 days, or 12 weeks. Quadriceps muscles were prepped as whole tissue lysates for immunoblotting of Beclin-1 (A). Gastrocnemius muscles were fractionated to prepare a mitochondrial-enriched fraction for immunoblotting of Parkin (B), Bnip3 (C), p62 (D), LC3II (E) and Lamp1 (F). Images below graphs are representative blot images. Protein targets in the mitochondrial fraction are normalized to Vdac abundance as a mitochondrial protein control. Panels (C–E) contain the same Vdac image because the targets are from the same membrane. Data are means ± SD; n=4-5. Two-way ANOVA was used to test for effects of diet and time. P values are main effects.

Figure 4

Short-term high-fat feeding altered fusion marker Mfn2. Fusion and fission markers in skeletal muscle of mice fed low-fat diet or high-fat diet for 3 days, 7 days, or 12 weeks. Quadriceps muscles were prepped as whole tissue lysates for immunoblotting. Gastrocnemius muscles were fractionated to prepare a mitochondrial-enriched fraction for immunoblotting of Mitofusion 2 (Mfn2) (A) and mitochondrial fission factor (Mff) (B). Drp1 protein abundance in whole tissue lysate (C). Images below graphs are representative blot images. Protein targets in the mitochondrial fraction are normalized to Vdac abundance as a mitochondrial protein control. Panels (A, B) contain the same Vdac image because the targets are from the same membrane. Data are means ± SD; n=4-5. Two-way ANOVA was used to test for effects of diet and time. P values are main effects.

Figure 5

Palmitate treatment in myotubes activated mitophagy. Autophagy protein markers in C2C12 myotubes treated with palmitate for 4, 8, or 24 hours. Cell lysates were fractionated to prepare a mitochondrial-enriched fraction for immunoblotting of Bnip3 (A) p62 (B) Parkin (C) Ulk1 pSer555 (D) and Beclin-1 (E). Representative blot images for (A–E) (F). JC-1 fluorescence indicating mitochondrial membrane potential (G). Mitochondrial localized LC3II (H) and Lamp-1 (I). Cells were treated with bafilomycin to inhibit autophagosome formation then mitochondria isolated and probed for LC3II (J) and Lamp1 (K). Representative blot images for (H–K) (L). Protein targets in the mitochondrial fraction are normalized to Vdac abundance as a mitochondrial protein control. Data are means ± SD; n=5-6 for immunoblots and n=11 for JC-1. Two-way ANOVA was used to test for effects of diet and time. Unpaired t-tests were used to test for JC-1 differences. P values are main effects.

Figure 6

Palmitate treatment in myotubes activated fusion/fission. Fusion and fission markers in C2C12 myotubes treated with palmitate for 4, 8, or 24 hours. Cell lysates were fractionated to prepare a mitochondrial-enriched fraction for immunoblotting for Mitofusion 2 (Mfn2) (A), mitochondrial fission factor (MFF) (B) and DRP1 (C). Images below graphs are representative blot images. Protein targets in the mitochondrial fraction are normalized to Vdac abundance as a mitochondrial protein control. Panels (B, C) contain the same Vdac image because the targets are from the same membrane. Data are means ± SD; n=5-6. Two-way ANOVA was used to test for effects of diet and time. P values are main effects.

Figure 7

Hydrogen peroxide treatment in myotubes activated mitophagy. Autophagy protein markers in C2C12 myotubes treated with specified concentrations of hydrogen peroxide for 1 hour. Cell lysates were fractionated to prepare a mitochondrial-enriched fraction and cytosolic fraction for immunoblotting. (A) JC-1 fluorescence indicating mitochondrial membrane potential. – Control is DMED and + Control is FCCP. (B) Ulk1 pS555 protein abundance in mitochondrial fraction. (C) Bnip3 protein abundance in mitochondrial fraction. (D) LC3II protein abundance in mitochondrial fraction. (E) LC3II protein abundance in mitochondrial fraction in bafilomycin treated cells. Images below graphs are representative blot images. Protein targets in the mitochondrial fraction are normalized to Vdac abundance as a mitochondrial protein control. Panels (D, E) are from the same membrane. Data are means ± SD; n=6 for immunoblots and n=4 for JC-1. Unpaired t-tests were used to test for differences between groups.