The influence of dietary lipid composition on skeletal muscle mitochondria from mice following 1 month of calorie restriction

Abstract

To investigate the role mitochondrial membrane lipids play in the actions of calorie restriction (CR), C57BL/6 mice were assigned to four groups (control and three 40% CR groups) and fed diets containing soybean oil (also in the control diet), fish oil, or lard. The fatty acid composition of the major mitochondrial phospholipid classes, proton leak, and H(2)O(2) production were measured in muscle mitochondria following 1 month of CR. The results indicate that phospholipid fatty acids reflected the polyunsaturated fatty acid profile of the dietary lipid sources. Capacity for Complex I- and III-linked H(2)O(2) production was decreased with CR, although there was no difference between CR groups. The CR lard group had lower proton leak than all other groups. The results indicate that a decreased degree of unsaturation in muscle mitochondrial membranes is not required for reduced H(2)O(2) production with CR. However, dietary lipids do have some influence on proton leak with CR.

Figure 1.

Hydrogen peroxide production in skeletal muscle mitochondria from mice consuming a control diet (Control n = 10) or calorie restriction (CR) diets containing lard (CR lard n = 6), soybean oil (CR soy n = 8), or fish oil (CR fish n = 9). All measurements were completed on freshly isolated mitochondria. H₂O₂ production was monitored in mitochondria respiring on succinate (Panel A), Pyruvate/Malate (Panel B) , or Succinate plus Pyruvate/Malate (Panel C). All comparisons were within substrate only or within the substrate and inhibitor combination. Bars that do not share a common letter indicate a significant difference (p < .05) between treatments. AA, Antimycin A; ROT, Rotenone.

Figure 3.

Thiobarbituric acid reactive substances (nmol/mg protein) production in hind-limb muscle mitochondria at the end of 1 mo calorie restriction (CR) from mice consuming a control diet (Control n = 8) or CR diets containing lard (CR lard n = 8), soybean oil (CR soy n = 8), or fish oil (CR fish n = 8). Bars that do not share a common letter indicate a significant difference (p < .05) between treatments.

Figure 2.

Proton leak kinetics curves in hind-limb muscle mitochondria at the end of 1 mo calorie restriction (CR) from mice consuming a control diet (Control n = 7) or CR diets containing lard (CR lard n = 8), soybean oil (CR soy n = 7), or fish oil (CR fish n = 7). Leak-dependent respiration and membrane potential were measured simultaneously using a Clark-type electrode and a TPMP⁺-sensitive electrode, respectively. The furthest point on the right in each panel represents State 4 respiration. All measurements were completed using mitochondria (0.25 mg/mL) in incubation medium (145 mM KCl, 5 mM KH₂PO₄, 30 mM Hepes, 3 mM MgCl₂, and 0.1 mM EGTA, pH 7.4). A methyl-triphenyl-phosphonium (TPMP⁺; 0–2.5 μM) standard curve was generated in each sample prior to the initiation of respiration and membrane potential measurements. All assays were performed in the presence of 5 mM succinate, 5 μM rotenone, oligomycin (8 μg/mL) and nigericin (0.08 μg/mL), and incremental additions of malonate (0.1–2.4 mM).