Fig 1.
IWT, interval walking training.
Table 1.
Physical characteristics and fitness at baseline and changes after training.
Table 2.
Training achievements over 5 months.
Table 3.
Nutritional components of the milk products.
Table 4.
Dietary intake per day during the training period.
Fig 2.
Percent changes after training in muscle strength (A) and methylation of the NFKB1 and NFKB2 promoter regions assessed by pyrosequencing (B). The data were adjusted for pretraining values by ANCOVA. The mean and SE bars are presented for 12, 12, and 13 subjects in the IWT control (CNT), IWT + low-dose (LD) and IWT + high-dose milk product intake (HD) groups, respectively. **Significant differences from pretraining values, P<0.01. Significant differences from the CNT group, †P<0.05 and †††P<0.001. Significant differences from the LD group, ‡P<0.05 and ‡‡P<0.01. A: Average percent changes in isometric knee extension (ΔFEXT) and flexion (ΔFFLX) forces are presented. B: Average percent changes in CpG sites 1–7 for NFKB1 (upper) and average percent changes in CpG sites 1–6 for NFKB2 (lower) are presented.
Table 5.
Primers used for the pyrosequencing assay.
Fig 3.
Flow chart diagram showing the genome-wide DNA methylation analysis.
DMPs, differentially methylated CpG positions; Ratio1, the ratio of the β-value after training to that before training.
Fig 4.
The hyper- or hypo-methylated positions induced by milk product intake during IWT in whole blood cells.
A: DNA methylation levels of CpG sites identified by the Infinium 450K methylation assay. Typical examples of normalized β-values before vs after training in a CNT subject and an HD subject, which were used to determine the ratio (Ratio1) of the β-value after training to that before training in each CpG site. B: Ratio2 on a base-2 logarithmic scale (y) plotted against 19,371 differentially methylated CpG positions (DMPs) after training in the HD group (n = 12) compared with the CNT group (n = 12) (x). Ratio2 indicates the ratio of the median value of Ratio1 for the HD group to that for the CNT group at each DMP. The number of DMPs on the x-axis is an arbitrary unit, which was ranked by the Ratio2 values. The upper and lower areas of the gray lines in the figure indicate the Ratio1 values of the HD that group are >1.2-fold higher (indicated by red) and lower (indicated by green) than that of the CNT group, respectively. C: Pie chart displaying the genomic location of the 1,448 positions in which more hyper- or hypo-methylation occurred in the HD group than in the CNT group by >1.2-fold in relation to genes (i) and CpG (ii) context.
Fig 5.
Hierarchical clustering of the hyper- or hypo-methylated positions induced by milk product intake during IWT.
Among the 1,448 positions in which more hyper- or hypo-methylation occurred in the HD group than in the CNT group by >1.2-fold, those within gene-associated regions (1,243 positions) were subjected to an ingenuity pathway analysis (IPA). Hierarchical clustering analysis was then performed on the dataset obtained from the results of the IPA, which included 83 genes in the top network, canonical pathways, or physiological functions.
Fig 6.
IPA-derived top-scoring network: a cancer-, tumor morphology-, and tissue development-related network (score = 42).
Colored genes are hyper- (red) or hypo-methylated (green). A solid line indicates a direct interaction, and a dashed line indicates an indirect relationship between nodes. The shape of the node indicates its function. More information is available at the Ingenuity Inc. website (http://www.ingenuity.com).
Table 6.
Methylation of the NFKB1 and NFKB2 promoter regions at baseline and changes after training assessed by pyrosequencing.