Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Methods - Supplemental Methods
• Supplemental Table 1 - Genes whose expression was increased in soleus muscle by exercise.
• Supplemental Table 2 - Genes whose expression was decreased in soleus muscle by exercise.
• Supplemental Figure 1 - Effects of exercise on the expression of HB-EGF in liver (A), epididymal white adipose tissue (B), or spleen (C). Total protein was extracted from each tissue of 15-week-old male C57BL/6 mice 3 h after they had completed the exercise protocol. The samples were then subjected to immunoblot analysis for determination of the amount of HB-EGF protein. The amount of α-tubulin was determined as a loading control. Data are from two animals for each condition and are representative of three independent experiments.
• Supplemental Figure 2 - Effects of muscle-specific overexpression of HB-EGF on tissue weight and myofiber size. (A) Weight of various tissues from 21-week-old HB-EGFmTg (#45) mice (n = 3) or WT mice (n = 5) fed a standard diet. (B) Size (cross-sectional area) of type I (left) or type II (right) myofibers in gastrocnemius muscle of 18-week-old HB-EGFmTg (#45) or WT mice (n = 4 for each genotype) maintained on a standard diet. Data are means ± SEM. NS, not significant.
• Supplemental Figure 3 - Locomotor activity counts (per hour) of 12-week-old HB-EGFmTg (#45) or WT mice (n = 10 for each genotype) fed a standard diet. Gray and open boxes indicate dark and light periods, respectively. Data are means ± SEM.
• Supplemental Figure 4 - Akt phosphorylation in the liver and adipose tissue of HB-EGFmTg mice. Lysates of the liver or epididymal white adipose tissue of mice treated as in Figure 5A were subjected to immunoblot analysis with antibodies to phosphorylated or total Akt.
• Supplemental Figure 5 - Suppression of fasting-induced expression of the MAFbx gene in skeletal muscle by HB-EGF. Total RNA was isolated from gastrocnemius muscle of 15-week-old HB-EGFmTg (#45) or WT mice (n = 3 for each group) that had been deprived (or not) of food for 8 h and was then subjected to reverse transcription and real-time quantitative PCR analysis for determination of the amount of MAFbx mRNA. Data were normalized by the amount of 36B4 mRNA, were expressed relative to the value for WT mice maintained with free access to food, and are means ± SEM. *P < 0.05 versus the value for fasted WT mice.
• Supplemental Figure 6 - Glucose homeostasis and Akt signaling in 21-week-old HB-EGFmTg mice (#45) fed a high-fat diet. (A) Blood glucose levels in HB-EGFmTg (n = 8) or age-matched WT (n = 9) mice fed ad libitum with a high-fat diet or deprived of the diet (fasted) for 12 h. (B) Plasma insulin levels in HB-EGFmTg (n = 8) or age-matched WT (n = 9) mice fed ad libitum with a high-fat diet. (C) Glucose tolerance test. HB-EGFmTg or age-matched WT mice (n = 6 for each genotype) were deprived of food overnight and then injected intraperitoneally with a bolus of glucose. Blood glucose and plasma insulin levels were determined at the indicated times thereafter. (D) Insulin tolerance test. HB-EGFmTg or age-matched WT mice (n = 6 for each genotype) were injected intraperitoneally with a bolus of insulin and blood glucose levels were monitored at the indicated times thereafter. (E) Mice of the two genotypes were injected intravenously with saline or insulin (0.2 U/kg) and killed 15 min later. Lysates prepared from gastrocnemius muscle were then analyzed for Akt phosphorylation by immunoblot analysis. Data in (A) through (D) are means ± SEM; those in (E) are representative of three independent experiments.*P < 0.05, **P < 0.01 versus the corresponding value for WT mice.