Weight Loss via exercise with controlled dietary intake may affect phospholipid profile for cancer prevention in murine skin tissues

Abstract

Exercise has been linked to a reduced cancer risk in animal models. However, the underlying mechanisms are unclear. This study assessed the effect of exercise with dietary consideration on the phospholipid profile in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced mouse skin tissues. CD-1 mice were randomly assigned to one of the three groups: ad libitum-fed sedentary control; ad libitum-fed treadmill exercise at 13.4 m/min for 60 min/d, 5 d/wk (Ex+AL); and treadmill-exercised but pair-fed with the same amount as the control (Ex+PF). After 14 weeks, Ex+PF but not Ex+AL mice showed approximately 25% decrease in both body weight and body fat when compared with the controls. Of the total 338 phospholipids determined by electrospray ionization-tandem mass spectrometry, 57 were significantly changed, and 25 species could distinguish effects of exercise and diet treatments in a stepwise discriminant analysis. A 36% to 75% decrease of phosphatidylinositol (PI) levels in Ex+PF mice occurred along with a significant reduction of PI 3-kinase in TPA-induced skin epidermis, as measured by both Western blotting and immunohistochemistry. In addition, approximately 2-fold increase of the long-chain polyunsaturated fatty acids, docosahexaenoic and docosapentaenoic acids, in phosphatidylcholines, phosphatidylethanolamines, and lysophosphatidylethanolamines was observed in the Ex+PF group. Microarray analysis indicated that the expression of fatty acid elongase-1 increased. Taken together, these data indicate that exercise with controlled dietary intake, but not exercise alone, significantly reduced body weight and body fat as well as modified the phospholipid profile, which may contribute to cancer prevention by reducing TPA-induced PI 3-kinase and by enhancing omega-3 fatty acid elongation.

Figure 1. Effects of exercise with or without controlled dietary intake on body weight and body fat

CD-1 mice at 8 weeks old were fed ad libitum or pair-fed the same amount as the sedentary control. They performed treadmill exercise at 13.4 m/min, 60 min/day, 5 days/week, for 14 weeks. A: Body weight (A) and body fat (B) were significantly lower in exercised and pair-fed mice in comparison with either sedentary control mice or exercised mice with ad libitum-feeding. Results are means ± SE, n = 10–15, *p ≤ 0.05 vs. sedentary control or exercised but ad libitum-fed mice.

Figure 2. Quantification of 22:5 and 22:6-containing acyl species in PC/PE 40:5 and PC/PE 40:6 phospholipids via production spectra for acyl group identification

CD-1 mice were exercised with controlled dietary intake for 14 weeks. The acyl groups of PC/PE 40:5 and PC/PE 40:6 in a lipid extract were identified as acyl anions from the appropriate negative ion precursors by ESI-MS/MS. PC and PE were analyzed as [M + OAc]⁻ and [M−H]⁻ ions, respectively. The lines show pairs of acyl species consistent with the observed PC or PE m/z. These product ion analyses were performed on selected molecular ions, indicating three major pairs of acyl composition (18:0–22:5 > 18:1–22:4 and 20:1–20:4) for PC40:5 (A), two major pairs of acyl composition (18:0–22:6 > 18:1–22:5) for PC 40:6 (B), two major pairs of acyl composition (18:0–22:5 > 18:1–22:4) for PE 40:5 (C), and three major pairs of acyl composition (18:0–22:6 > 18:1–22:5 and 18:2–22:4) for PE 40:6 (D). The fractions of the 40:5 and 40:6 species (determined from the lipid profile) that corresponded to the indicated 22:5 and 22:6-containing species were determined from the production spectra analysis (E). The PC/PE 18:0–22:5 and PC/PE 18:0–22:6 were significantly higher in exercise and pair fed mice, but not in exercise and ad libitum-fed mice when compared with the sedentary control. Results are means ± SE, n = 4. Means with different letters differ significantly, p ≤ 0.001.

Figure 3. Stepwise discriminant analysis of 25 phospholipid variables

CD-1 mice were exercised with or without controlled dietary intake for 14 weeks, and then quantitative profiling of phospholipids was performed by ESI-MS/MS in mouse skin tissues. Total 57 phospholipids were significantly changed among three treatment groups and 25 of them are indicated in the inserted table with mol% of total polar lipid content. Automatic backward stepwise variable selection in discriminant analysis identified 25 phospholipids as indicated in the inserted table that could successfully predict a treatment group with 92% correct classification (Wilks' lambda = 0.001 at p ≤ 0.00001) based upon the first two principal discriminant functions.

Figure 4. Effects of exercise with or without controlled dietary intake on PI3K protein expression in mouse skin tissues

CD-1 mice were exercised with or without controlled diet intake for 14 weeks. The level of PI3K protein in skin tissues was determined by western blotting and quantified by the FluorChem™ 8800 Advanced Imaging System. Results are means ± SE, n = 10–15. Means with different letters differ significantly, p ≤ 0.01.

Figure 5. Effects of exercise with or without controlled dietary intake on PI3K protein expression in skin epidermis

CD-1 mice were exercised with or without controlled dietary intake for 14 weeks. Representative histological skin sections with immunohistochemical staining for p110-PI3K in epidermis in acetone-treated control (A) and TPA-treated control (B), exercise with ad libitum-feeding (C), and exercise with pair-feeding (D) are shown. The arrows indicate representative staining of the target protein in epidermis (n = 3–5).

Figure 6. Effects of exercise with or without controlled dietary intake on Elovl gene expression in skin tissues

CD-1 mice were exercised with or without controlled dietary intake for 14 weeks. The expression of six Elovl family genes was measured by microarray analysis as described in the Materials and Methods. The accession number for these six genes are as follows, including both NM_001039176 (elongation of very long chain fatty acids-like 1 isoform 1) and NM_001039175 ( elongation of very long chain fatty acids-like 1 isoform 2 for Elovl1, NM_019423 (elongation of very long chain fatty acids-like 2) for Elovl2, NM_007703 (elongation of very long chain fatty acids-like 3) for Elovl3, NM_001145974 (elongation of very long chain fatty acids-like 4) for Elovl4, NM_134255 (elongation of very long chain fatty acids-like 5) for Elovl5, and NM_130450 (elongation of very long chain fatty acids-like 6) for Elovl6. Results are means ± SE (n = 4). Means with different letters differ significantly, p ≤ 0.05.