For each PCR reaction, 18S (with a 324-bp product) was co-amplified with each target cDNA
(mRNA) to express each as a ratio of target mRNA/18S. Images were captured under UV, and mRNA expressions were analyzed via the Bio-Rad ChemiDoc™ XRS imaging system and the Bio-Rad QuantityOne® software (Bio-Rad Laboratories, Hercules, GSK2126458 solubility dmso CA, USA) as described previously [29]. mRNA expression of 4EBP1 was used as a negative marker of protein synthesis, while the E3 ligase atrogin-1 was used as a positive regulator of protein degradation. Mitogenic factors, IGF-IEa and its isoform IGF-IEb(mechano growth factor (MGF)), were used as positive regulators of mitogenesis and myogenesis. Myostatin and its receptor activin IIB were Selumetinib supplier measured as negative regulators of myogenesis. Muscle cell regeneration was analyzed by transcriptional levels of the myogenic regulatory factors (MRFs): myogenin and myogenic differentiation factor
(MyoD). Statistical analysis Lean body mass, FM, TBM, functionality (grip strength and incline plane, MR-determined myofiber dimensions and target genes associated with myofiber size were analyzed using one way ANOVA across six groups including 1 young baseline (44 wks), 2 middle aged (60 wks, control and HMB), 1 old (86 wks.), and 2 very old (102 wks. control and HMB) groups using Statistica (StatSoft®, Tulsa, OK, USA) (Figure 1). Significance was set at p ≤ 0.05, and a tukey post hoc analysis was used to determine which specific mean values differed from others for each variable. The overarching goal of this project was to use MR to examine the impacts of age and HMB on skeletal muscle cells during the aging process. Myofiber size was therefore one of the primary outcome measures in this project and provided the basis for the sample sizes as determined by the G*Power
analysis software [30, 31]. Our rationale for sample size was based on a study by Payne et al. [32]. These investigators found ID-8 that Fisher 344 rats 102 wks of age demonstrated significant atrophy in the soleus than young adult animals (Effect size (ES) of 3.7). Based on an alpha level of 0.05, a power of 80 and an ES of 3.7, a total of 30 rats (5 per experimental group) were needed to have sufficient power to detect age related changes in myofber dimensions. Results Food and HMB consumption All values for food consumed are presented in Table 1. Average total Kcals and Kcals for carbohydrates, protein, and fat were not different between groups. Table 1 Average Kcal consumption for among conditions Kcals Kcals (CHO) Kcals (PRO) Kcals (Fat) 44 wks Baseline 67.3 ± 4.1 38.9 ± 2.4 19.2 ± 1.2 9.0 ± 0.6 60 wks Control 66.8 ± 1.8 38.7 ± 1.1 19.0 ± 0.5 8.9 ± 0.3 60 wks HMB 65.9 ± 1.5 38.2 ± 0.9 18.7 ± 1.2 8.8 ± 0.6 86 wks Baseline 62.3 ± 6.5 35.5 ± 3.64 17.4 ± 2.0 8.2 ± 0.9 102 wks Control 62.5 ± 5.8 36.1 ± 2.4 17.8 ± 1.0 8.4 ± 0.5 102 wks HMB 63.2 ± 6.19 36.8 ± 3.6 18.1 ± 1.8 8.5 ± 0.