The purpose of the current study was to examine IGFBP-3, -4, and -5 mRNA and protein expression levels as a function of muscle type, age, and regrowth from an immobilization-induced atrophy in Fischer 344 × Brown Norway rats. IGFBP-3 mRNA expression in the 4-mo-old animals was significantly higher in the red and white portions of the gastrocnemius muscle compared with the soleus muscle. However, there were no significant differences in IGFBP-3 mRNA expression among any of the muscle groups in the 30-mo-old animals. There were no significant differences in IGFBP-5 mRNA expression in any of the muscle groups, whereas in the 30-mo-old animals there was significantly less IGFBP-5 mRNA expression in the white gastrocnemius compared with the red gastrocnemius muscles. Although IGFBP-3 and -5 proteins were detected in the type I soleus muscle with Western blot analyses, no detection was observed in the type II red and white portions of the gastrocnemius muscle. Aging from adult (18 mo) to old animals (30 mo) was associated with decreases in IGFBP-3 mRNA and protein and IGFBP-5 protein only in the soleus muscle. After 10 days of recovery from 10 days of hindlimb immobilization, IGFBP-3 mRNA and protein increased in soleus muscles from young (4-mo) rats; however, only IGFBP-3 protein increased in the old (30-mo) rats. Whereas there were no changes in IGFBP-5 mRNA expression during recovery, IGFBP-5 protein in the 10-day-recovery soleus muscle did increase in the young, but not in the old, rats. Because one of the functions of IGFBPs is to modulate IGF-I action on muscle size and phenotype, it is hypothesized that IGFBP-3 and -5 proteins may have potential modulatory roles in type I fiber-dominated muscles, aging, and regrowth from atrophy.

These findings are considered important because they reveal p21(Cip1) as a critical regulatory protein that needs to be considered when interpreting proliferation data from MPCs studied in culture. In addition, O2-dependent regulation of MPC proliferation is relevant to conditions, including sarcopenia, heart failure, cancer and muscular dystrophy, where increased oxidative stress exists.

promoter activity and protein expression, whereas it decreased proliferation in muscle precursor cells (MPCs). The objectives of the present study were to 1) locate and identify FOXO regulatory elements in the rat p27Kip1 promoter using deletion analysis of a promoter/reporter construct and 2) determine if age-related differences exist in FOXO-induced p27Kip1 expression. The full-length (Ϫ4.0/ϩ0.4 kb) rat p27Kip1 promoter construct revealed that both FOXO1 and FOXO3a induced an increase in transcriptional activity. Interestingly, MPCs isolated from old animals exhibited an increased FOXO3a-induced p27Kip1 promoter activity compared with MPCs isolated from young animals. Deletion of a 253-bp portion of the 5Ј-untranslated region (UTR) resulted in a significant decrease in FOXO-induced p27Kip1 promoter expression. Site-specific mutation of a daf-16 family protein-binding element (DBE) within this 253-bp portion of the 5Ј-UTR also demonstrated a decrease in FOXO-induced p27Kip1 promoter expression. These data suggest that a putative FOXO regulatory element located in the 5Ј-UTR of the rat p27 Kip1 gene plays a role in the age-dependent differences in FOXO3a-dependent p27Kip1 promoter expression. These findings have implications for developing treatment strategies aimed at increasing the proliferation of MPCs and regenerative capacity of aged skeletal muscle. satellite cell; skeletal muscle; proliferation; 5Ј-untranslated region; daf-16 family protein-binding element; forkhead box O EMERGING EVIDENCE suggests that forkhead box O (FOXO) transcription factors are at the nexus of aging, metabolism, and cell fate/function (9). The primary regulation of FOXO family proteins is downstream of insulin and IGF signaling. Aktmediated phosphorylation of FOXO results in nuclear exclusion and inhibition of transcriptional activity (7,8,23,30,33). However, despite extensive study into the multitude of functional roles, the details of the regulatory regions for targets of FOXO are still mostly elusive.FOXO proteins play a key role in multiple conditions of skeletal muscle wasting. Several models of skeletal muscle atrophy cause upregulation of the transcripts of the musclespecific ubiquitin ligases muscle ring finger-1 (MuRF-1) and muscle atrophy F-box (MAFbx/atrogin-1) (5). Followup experiments revealed that overexpression of MAFbx induced myotube atrophy in culture, whereas mice lacking either the MuRF-1 or MAFbx genes were resistant to denervation-induced muscle atrophy (5). A mutant FOXO that was constitutively active induced MAFbx expression and caused atrophy in myotubes in culture (34). Moreover, RNA inhibitor knock down of FOXO3a in vivo decreased MAFbx expression (34). Interestingly, recent findings have revealed increased FOXO3a mRNA (32) in sarcopenia, which is defined as an age-associated loss of skeletal muscle mass and strength.

In aged skeletal muscle, impairments in regrowth and regeneration may be explained by a decreased responsiveness of muscle precursor cells (MPCs) to environmental cues such as growth factors. We hypothesized that impaired responsiveness to fibroblast growth factor 2 (FGF2) in MPCs from old animals would be explained by impaired FGF2 signalling. We determined that 5-bromo-2′-deoxyuridine (BrdU) incorporation and cell number increase less in MPCs from 32- compared with 3-month-old rats. In the presence of FGF2, we demonstrated that there were age-associated differential expression patterns for FGF receptor 1 and 2 mRNAs. Measurement of downstream signalling revealed that that mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2)–extracellular signal-regulated kinase 1/2, protein kinase C and p38 were FGF2-driven pathways in MPCs. Uniquely, protein kinase C signalling was shown to play the largest role in FGF2-stimulated proliferation in MPCs. c-Jun N-terminal kinase (JNK) signalling was ruled out as an FGF2-stimulated proliferation pathway in MPCs. Inhibition of JNK had no effect on FGF2 signalling to BrdU incorporation, and FGF2 treatment was associated with increased phosphorylation of p38, which inhibits, rather than stimulates, BrdU incorporation in MPCs. Surprisingly, the commonly used vehicle, dimethyl sulphoxide, rescued proliferation in MPCs from old animals. These findings provide insight for the development of effective treatment strategies that target the age-related impairments of MPC proliferation in old skeletal muscle.

Previous studies suggest an interaction between the level of physical activity and diet preference. However, this relationship has not been well characterized for sex differences that may exist. The present study examined the influence of sex on diet preference in male and female Wistar rats that were housed under either sedentary (no wheel access) (SED) or voluntary wheel running access (RUN) conditions. Following a 1 week acclimation period to these conditions, standard chow was replaced with concurrent ad libitum access to a choice of 3 pelleted diets (high-fat, high-sucrose, and high-corn starch) in the home cage. SED and RUN conditions remained throughout the next 4 week diet preference assessment period. Body weight, running distance, and intake of each diet were measured daily. At the conclusion of the 4 week diet preference test, animals were sacrificed and brains were collected for mRNA analysis. Fecal samples were also collected before and after the 4 week diet preference phase to characterize microbiota composition. Results indicate sex dependent interactions between physical activity and both behavioral and physiological measures. Females in both RUN and SED conditions preferred the high-fat diet, consuming significantly more high-fat diet than either of the other two diets. While male SED rats also preferred the high-fat diet, male RUN rats consumed significantly less high-fat diet than the other groups, instead preferring all three diets equally. There was also a sex dependent influence of physical activity on both reward related opioid mRNA expression in the ventral striatum and the characterization of gut microbiota. The significant sex differences in response to physical activity observed through both behavioral and physiological measures suggest potential motivational or metabolic difference between males and females. The findings highlight the necessity for further exploration between male and female response to physical activity and feeding behavior.