RNA-sequencing and behavioral testing reveals inherited physical inactivity co-selects for anxiogenic behavior without altering depressive-like behavior in Wistar rats.

Physical inactivity is positively associated with anxiety and depression. Considering physical inactivity, anxiety, and depression each have a genetic basis for inheritance, our lab used artificial selectively bred low-voluntary running (LVR) and wild type (WT) female Wistar rats to test if physical inactivity genes selected over multiple generations would lead to an anxiety or depressive-like phenotype. We performed next generation RNA sequencing and immunoblotting on the dentate gyrus to reveal key biological functions from heritable physical inactivity. LVR rats did not display depressive-like behavior. However, LVR rats did display anxiogenic behavior with gene networks associated with reduced neuronal development, proliferation, and function compared to WT counterparts. Additionally, immunoblotting revealed LVR deficits in neuronal development and function. To our knowledge, this is the first study to show that by selectively breeding for physical inactivity genes, anxiety-like genes were co-selected. The study also reveals molecular insights to the genetic influences that physical inactivity has on anxiety-like behavior.

Sex dependent effects of physical activity on diet preference in rats selectively bred for high or low levels of voluntary wheel running.

Considering the current obesity epidemic is due in large part to an energy imbalance, it is crucial to explore biological mechanisms that mediate palatable high energy food intake and physical activity behavior levels. Previous research demonstrates a unique sex dependent influence of physical activity on diet preference, specifically changes in palatable high-fat diet intake. Therefore, factors of motivation may be underlying the differential effect of physical activity in male and female rats on their diet preference. The present study extends this hypothesis by assessing diet preference in male and female Wistar rats selectively bred for high (HVR) and low (LVR) levels of voluntary wheel running distances. HVR and LVR rats were housed under either sedentary (SED) or voluntary wheel running access (RUN) conditions for the duration of the study. 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); all 3 were provided in the home cage. Body weight, running distance, and intake of each diet was measured daily. At the conclusion of the 4 week diet preference test, animals were sacrificed and ventral striatum tissue was collected for later analysis. Results demonstrated intake patterns of diets were uniquely influenced by physical activity dependent on both the sex and the selectively bred line of rat. In addition, reward related ventral striatal mRNA expression was also dependent on both the sex and the selectively bred line of rat. Overall, the pattern of both behavioral and mRNA results suggest that voluntary wheel running behavior differentially mediates palatable diet consumption in males and females. Considering the pervasive abundance of both physical inactivity, combined with over-consumption of energy dense palatable diets, it is vital to understand the nature of these behavioral interactions.

Sex determines effect of physical activity on diet preference: Association of striatal opioids and gut microbiota composition.

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.

AMPK agonist AICAR delays the initial decline in lifetime-apex V̇o2 peak, while voluntary wheel running fails to delay its initial decline in female rats.

There has never been an outcome measure for human health more important than peak oxygen consumption (V̇o2 peak), yet little is known regarding the molecular triggers for its lifetime decline with aging. We examined the ability of physical activity or 5 wk of 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR) administration to delay the initial aging-induced decline in lifetime-apex V̇o2 peak and potential underlying molecular mechanisms. Experiment 1 consisted of female rats with (RUN) and without (NO RUN) running wheels, while experiment 2 consisted of female nonrunning rats getting the AMPK agonist AICAR (0.5 mg/g/day) subcutaneously for 5 wk beginning at 17 wk of age. All rats underwent frequent, weekly or biweekly V̇o2 peak tests beginning at 10 wk of age. In experiment 1, lifetime-apex V̇o2 peak occurred at 19 wk of age in both RUN and NO RUN and decreased thereafter. V̇o2 peak measured across experiment 1 was ∼25% higher in RUN than in NO RUN. In experiment 2, AICAR delayed the chronological age observed in experiment 1 by 1 wk, from 19 wk to 20 wk of age. RUN and NO RUN showed different skeletal muscle transcriptomic profiles both pre- and postapex. Additionally, growth and development pathways are differentially regulated between RUN and NO RUN. Angiomotin mRNA was downregulated postapex in RUN and NO RUN. Furthermore, strong significant correlations to V̇o2 peak and trends for decreased protein concentration supports angiomotin's potential importance in our model. Contrary to our primary hypothesis, wheel running was not sufficient to delay the chronological age of lifetime-apex V̇o2 peak decline, whereas AICAR delayed it 1 wk.

Fibroblast growth factor 2-stimulated proliferation is lower in muscle precursor cells from old rats.

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.

Age-dependent FOXO regulation of p27Kip1 expression via a conserved binding motif in rat muscle precursor cells.

Previously, we have demonstrated that forkhead box O3a (FOXO3a) overexpression increased p27(Kip1) 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 p27(Kip1) promoter using deletion analysis of a promoter/reporter construct and 2) determine if age-related differences exist in FOXO-induced p27(Kip1) expression. The full-length (-4.0/+0.4 kb) rat p27(Kip1) 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 p27(Kip1) promoter activity compared with MPCs isolated from young animals. Deletion of a 253-bp portion of the 5'-untranslated region (UTR) resulted in a significant decrease in FOXO-induced p27(Kip1) promoter expression. Site-specific mutation of a daf-16 family protein-binding element (DBE) within this 253-bp portion of the 5'-UTR also demonstrated a decrease in FOXO-induced p27(Kip1) promoter expression. These data suggest that a putative FOXO regulatory element located in the 5'-UTR of the rat p27(Kip1) gene plays a role in the age-dependent differences in FOXO3a-dependent p27(Kip1) promoter expression. These findings have implications for developing treatment strategies aimed at increasing the proliferation of MPCs and regenerative capacity of aged skeletal muscle.

Skeletal muscle IGF-binding protein-3 and -5 expressions are age, muscle, and load dependent.

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 x 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.