High-active Mice Research Articles

Protein fractional synthesis rates within tissues of high- and low-active mice.

With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Biological factors regulating physical activity are studied to establish a possible target for improving the physical activity level. However, little is known about the role metabolism plays in physical activity regulation. Therefore, we studied protein fractional synthesis rate (FSR) of multiple organ tissues of 12-week-old male mice that were previously established as inherently low-active (n = 15, C3H/HeJ strain) and high-active (n = 15, C57L/J strain). Total body water of each mouse was enriched to 5% deuterium oxide (D2O) via intraperitoneal injection and maintained with D2O enriched drinking water for about 24 h. Blood samples from the jugular vein and tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected for enrichment analysis of alanine by LC-MS/MS. Protein FSR was calculated as -ln(1-enrichment). Data are mean±SE as fraction/day (unpaired t-test). Kidney protein FSR in the low-active mice was 7.82% higher than in high-active mice (low-active: 0.1863±0.0018, high-active: 0.1754±0.0028, p = 0.0030). No differences were found in any of the other measured organ tissues. However, all tissues resulted in a generally higher protein FSR in the low-activity mice compared to the high-activity mice (e.g. lung LA: 0.0711±0.0015, HA: 0.0643±0.0020, heart LA: 0.0649± 0.0013 HA: 0.0712±0.0073). Our observations suggest that high-active mice in most organ tissues are no more inherently equipped for metabolic adaptation than low-active mice, but there may be a connection between protein metabolism of kidney tissue and physical activity level. In addition, low-active mice have higher organ-specific baseline protein FSR possibly contributing to the inability to achieve higher physical activity levels.

Activated whole-body arginine pathway in high-active mice.

Our previous studies suggest that physical activity (PA) levels are potentially regulated by endogenous metabolic mechanisms such as the vasodilatory roles of nitric oxide (NO) production via the precursor arginine (ARG) and ARG-related pathways. We assessed ARG metabolism and its precursors [citrulline (CIT), glutamine (GLN), glutamate (GLU), ornithine (ORN), and phenylalanine (PHE)] by measuring plasma concentration, whole-body production (WBP), de novo ARG and NO production, and clearance rates in previously classified low-active (LA) or high-active (HA) mice. We assessed LA (n = 23) and HA (n = 20) male mice by administering a stable isotope tracer pulse via jugular catheterization. We measured plasma enrichments via liquid chromatography tandem mass spectrometry (LC-MS/MS) and body compostion by echo-MRI. WBP, clearance rates, and de novo ARG and NO were calculated. Compared to LA mice, HA mice had lower plasma concentrations of GLU (71.1%; 36.8 ± 2.9 vs. 17.5 ± 1.7μM; p<0.0001), CIT (21%; 57.3 ± 2.3 vs. 46.4 ± 1.5μM; p = 0.0003), and ORN (40.1%; 55.4 ± 7.3 vs. 36.9 ± 2.6μM; p = 0.0241), but no differences for GLN, PHE, and ARG. However, HA mice had higher estimated NO production ratio (0.64 ± 0.08; p = 0.0197), higher WBP for CIT (21.8%, 8.6 ± 0.2 vs. 10.7 ± 0.3 nmol/g-lbm/min; p<0.0001), ARG (21.4%, 35.0 ± 0.6 vs. 43.4 ± 0.7 nmol/g-lbm/min; p<0.0001), PHE (7.6%, 23.8 ± 0.5 vs. 25.6 ± 0.5 nmol/g-lbm/min; p<0.0100), and lower GLU (78.5%; 9.4 ± 1.1 vs. 4.1 ± 1.6 nmol/g lbm/min; p = 0.0161). We observed no significant differences in WBP for GLN, ORN, PHE, or de novo ARG. We concluded that HA mice have an activated whole-body ARG pathway, which may be associated with regulating PA levels via increased NO production.

Comparable Organ Protein Fractional Synthesis Rate of High and Low-Active Mice

Abstract Objectives With the rise in physical inactivity and its related diseases, it is necessary to understand the mechanisms involved in physical activity regulation. Scientists have explored physical activity regulation by investigating various physiological mechanisms involving hormones, neurotransmitters, and genetics; however, little is known about the role of metabolism on physical activity level. We hypothesize that protein turnover in specific organs like the muscle is higher in mice previously exhibiting high physical activity levels, as a mechanism to adapt to the increased demand. Therefore, we studied protein fractional synthesis rate (FSR) in tissues of inherently high and low active mice. Methods In order to study protein FSR of various organs, we assessed 12-week-old male inherently low-active (LA) mice (n = 23, lean body mass: 21.0 ± 1.1 g, C3H/HeJ strain) and high active (HA) mice (n = 20, lean body mass: 22.5 ± 1.3, C57L/J strain). One day before tissue collection, a D2O bolus was administered via intraperitoneal injection, and mice were provided D2O enriched drinking water to enrich the total body water to about 5% D2O. Eleven tissues (kidney, heart, lung, muscle, fat, jejunum, ileum, liver, brain, skin, and bone) were collected and analyzed for enrichment of alanine in the intracellular and protein-bound pool (LC-MS/MS). FSR was calculated as -ln(1-enrichment) as fraction per day. Data are mean ± SE (unpaired t-test: GraphPad Prism 8.2). Results We did not find significant differences between protein FSR of HA and LA mice in any measured organ. Example: Protein FSR (fraction/day): muscle (LA: 0.0326±-0.0026, HA: 0.0331 ± 0.0018, P = 0.8673), liver (0.3568 ± 0.0219, 0.3499 ± 0.0217, P = 0.8263), brain (0.0981 ± 0.0056, 0.1041 ± 0.0063, P = 0.4758). Conclusions The observed lack of significant differences in high and low-active mice suggests that differences in specific organ tissue protein turnover may not be a mechanism regulating inherent physical activity level. Since protein turnover is representative of the ability to adapt through upregulation and downregulation of metabolic processes, these results show that high-active mice are inherently no more equipped for metabolic regulation than the low active mice. Funding Sources Sydney and J.L. Huffines Institute for Sports Medicine, Human Performance Student Research Grant and CTRAL Grant.

High-active Mice Have Elevated Clearance Rate Of Bcaas Compared To Low-active Mice

Physical activity (PA) is associated with a diminished prevalence of hypokinetic related diseases and its worldwide economic burden on health care cost. For several decades, branched-chain amino acids (BCAAs; leucine [LEU], isoleucine [ILE], valine [VAL]) have been studied for muscle wasting disorders. However, little is known about the metabolic interactions between BCAAs and regulation of PA levels. PURPOSE: To assess BCAA metabolism by measuring plasma clearance rates in mice previously classified as either low-active (LA; C3H/HeJ) or high-active (HA; C57L/J). METHODS: 12-week-old male LA (n=23) mice (body weight: 25.8 ± 1.2g; lean body mass: 21.0 ± 1.1g; fat mass: 2.5 ± 0.5g) and HA (n=20) mice (27.5 ± 1.2; 22.5 ± 1.3; 2.5 ± 0.7) were used. Under anesthesia, a pulse of stable tracers (L-LEU[13C6], L-ILE [1-13C], and L-VAL [13C5]) was administered via the right jugular vein catheter. Subsequently, blood samples were taken (Time: 1, 3, 5, 7, 10, 15, 20, 25, 30, and 40 mins). Plasma enrichments and concentrations of LEU, ILE, and VAL were determined by LC-MS/MS. Whole-body production (WBP) was calculated from fitted area under the curve (AUC) as pulse/AUC, and clearance of stable tracers was calculated as WBP/plasma concentrations. Fitting and statistical analysis (unpaired student t-tests) were performed using GraphPad Prism 8 software. Data are expressed as mean ± SE. RESULTS: HA mice had significantly lower plasma concentrations for LEU (125.2 ± 4.8 vs 144.8 ± 6.4 μmol/l, p= 0.02), ILE (48.5 ± 1.9 vs 57.7 ± 2.3 μmol/l, p< 0.01) and VAL (156.8 ± 4.9 vs 187.9 ± 7.1 μmol/l, p< 0.01) and significantly higher WBP values for ILE (11.6 ± 0.6 vs 8.3 ± 1.1 nmol/g lbm/m, p= 0.02) and VAL (111.4 ± 5.4 vs 92.8 ± 2.8 nmol/g lbm/m, p< 0.01) compared to LA mice. No significant WBP differences were observed for LEU (75.5 ± 3.3 vs 72.7 ± 3.2 nmol/g lbm/m, p= 0.56). HA mice demonstrated higher percent clearance for LEU (40%), ILE (76%), and VAL (29%) compared to LA mice. CONCLUSIONS: The observed changes in plasma concentration, WBP, and clearance of BCAAs suggest modified metabolic pathways of LEU, VAL, and ILE in HA mice, compared to LA mice. FUNDING SOURCES: Texas A&M Vice President of Research Office, TACSM Student Research Development Award, College of Education & Human Development Student Research Grant, and funds from the Omar Smith Endowment.

Striatal transcriptome of a mouse model of ADHD reveals a pattern of synaptic remodeling.

Despite the prevalence and high heritability of Attention-Deficit/Hyperactivity Disorder (ADHD), genetic etiology remains elusive. Clinical evidence points in part to reduced function of the striatum, but which specific genes are differentially expressed and how they sculpt striatal physiology to predispose ADHD are not well understood. As an exploratory tool, a polygenic mouse model of ADHD was recently developed through selective breeding for high home cage activity. Relative to the Control line, the High-Active line displays hyperactivity and motor impulsivity which are ameliorated with amphetamine. This study compared gene expression in the striatum between Control and High-Active mice to develop a coherent hypothesis for how genes might affect striatal physiology and predispose ADHD-like symptoms. To this end, striatal transcriptomes of High-Active and Control mice were analyzed after mice were treated with saline or amphetamines. The pseudogene Gm6180 for n-cofilin (Cfl1) displayed 20-fold higher expression in High-Active mice corresponding with reduced Cfl1 expression suggesting synaptic actin dysregulation. Latrophilin 3 (Lphn3), which is associated with ADHD in human populations and is involved in synapse structure, and its ligand fibronectin leucine rich transmembrane protein 3 (Flrt3), were downregulated in High-Active mice. Multiple genes were altered in High-Active mice in a manner predicted to downregulate the canonical Wnt pathway. A smaller and different set of genes including glyoxalase (Glo1) were differentially regulated in High-Active as compared to Control in response to amphetamine. Together, results suggest genes involved in excitatory synapse regulation and maintenance are downregulated in ADHD-like mice. Consistent with the molecular prediction, stereological analysis of the striatum from a separate set of mice processed for imunohistochemical detection of synaptophysin revealed approximately a 46% reduction in synaptophysin immunoreactivity in High-Active relative to Control. Results provide a new set of molecular targets related to synapse maintenance for the next generation of ADHD medicines.

Differential miRNA expression in inherently high- and low-active inbred mice.

Despite established health benefits of regular exercise, the majority of Americans do not meet the recommended levels of physical activity. While it is known that voluntary activity levels are largely heritable, the genetic mechanisms that regulate activity are not well understood. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit transcription by binding to a target gene, inhibiting protein production. The purpose of this study was to investigate differential miRNA expression between inherently high- (C57L/J) and low- (C3H/HeJ) active inbred mice in soleus, extensor digitorum longus (EDL), and nucleus accumbens tissues. Expression was initially determined by miRNA microarray analysis, and selected miRNAs were validated by qRT-PCR. Expression of 13 miRNAs varied between strains in the nucleus accumbens, 20 in soleus, and eight in EDL, by microarray analysis. Two miRNAs were validated by qRT-PCR in the nucleus accumbens; miR-466 was downregulated (∼4 fold; P < 0.0004), and miR-342-5p was upregulated (∼115 fold; P < 0.0001) in high-active mice. MiR-466 was downregulated (∼5 fold; P < 0.0001) in the soleus of high-active mice as well. Interestingly, miR-466 is one of several miRNA families with sequence located in intron 10 of Sfmbt2; miRNAs at this locus are thought to drive imprinting of this gene. “Pathways in cancer” and “TGFβ signaling” were the most significant pathways of putative target genes in both the soleus and nucleus accumbens. Our results are the first to consider differential miRNA expression between high- and low-active mice, and suggest that miRNAs may play a role in regulation of physical activity.

Differential protein expression in the nucleus accumbens of high and low active mice

Physical inactivity is associated with the development of a variety of chronic illnesses. Literature has shown that physical activity is genetically regulated; however there is limited information on the mechanisms that influence this process with existing studies primarily focused on genomic and/or transcription association studies. There have been no studies to determine differential protein expression in the nucleus accumbens, the brain site thought to be involved in activity regulation, between high and low active animals. We compared the global nucleus accumbens proteome signature from known high- and low-active mice and identified seven differentially expressed proteins. Low active mice generally over expressed proteins associated with neural stress (Stress 70 protein and V type proton ATPase catalytic subunit A), and the high-active mice over expressed proteins associated with metabolism (creatine kinase B, succinyl-CoA ligase). Previously suggested mechanisms associated with activity regulation in the nucleus accumbens have centered on dopamine receptor 1 and endocannabinoid receptor 1. However, these proteins and the associated pathways were not differentially expressed between high and low active mice. In conclusion, protein expression must be determined as part of the effort to identify involved mechanisms in regulating activity and there appears to be separate nucleus accumbens proteome signatures associated with high- and low-active mice.

Differential skeletal muscle proteome of high- and low-active mice

Physical inactivity contributes to cardiovascular disease, type II diabetes, obesity, and some types of cancer. While the literature is clear that there is genetic regulation of physical activity with existing gene knockout data suggesting that skeletal muscle mechanisms contribute to the regulation of activity, actual differences in end-protein expression between high- and low-active mice have not been investigated. This study used two-dimensional differential gel electrophoresis coupled with mass spectrometry to evaluate the proteomic differences between high-active (C57L/J) and low-active (C3H/HeJ) mice in the soleus and extensor digitorum longus (EDL). Furthermore, vivo-morpholinos were used to transiently knockdown candidate proteins to confirm their involvement in physical activity regulation. Proteins with higher expression patterns generally fell into the calcium-regulating and Krebs (TCA) cycle pathways in the high-active mice (e.g., annexin A6, P = 0.0031; calsequestrin 1; P = 0.000025), while the overexpressed proteins in the low-active mice generally fell into cytoskeletal structure- and electron transport chain-related pathways (e.g., ATPase, P = 0.031; NADH dehydrogenase, P = 0.027). Transient knockdown of annexin A6 and calsequestrin 1 protein of high-active mice with vivo-morpholinos resulted in decreased physical activity levels (P = 0.001). These data suggest that high- and low-active mice have unique protein expression patterns and that each pattern contributes to the peripheral capability to be either high- or low-active, suggesting that different specific mechanisms regulate activity leading to the high- or low-activity status of the animal.

Activity Level in Mice is not Associated with Dopamine 2 Receptor Expression in Heart or Muscle Tissue

It has been suggested that the dopaminergic pathway is involved in regulating physical activity. PURPOSE: This study was conducted to assess the differential expression of Dopamine 2 Receptor (Drd2) between high active (HA) mice and low active (LA) mice. METHODS: F2 mice (n=310) derived from high-active C57L/J mice and low active C3H/HeJ mice were monitored daily for duration, distance, and velocity on a running wheel for 21 days starting at 9 weeks of age. Tissue was harvested from all animals and RNA isolated from the heart and gastroc/soleus tissues from the top 5 HA mice and bottom 5 LA mice. Drd2 expression was assessed by RT-PCR and band densities were quantified following gel electrophoresis. RESULTS: While no significant difference in weight (HA=23.9 ± 0.8gm vs. LA=25.5 ± 5.8gm, p=0.57), the HA mice ran significantly more per day (HA=13.0 ± 0.5km, LA=0.2 ± 0.2km, p=0.0001) than LA mice. There was no significant difference in Drd2 expression in the heart (p=.22) and gastroc/soleus (p=.87) tissues (n=10). However, the heart expressed significantly higher amounts of Drd2 than the gastroc/soleus (p=.029) within each mouse. CONCLUSIONS: The data indicate that there is no difference in Drd2 expression in the heart and gastroc/soleus tissues between HA mice and LA mice suggesting that Drd2 expression may not play a role in mediating physical activity levels in mice. More studies with larger numbers of mice would be needed to confirm this result. Therefore, further data needs to be collected to assess the exact role Drd2 plays in physical activity levels.

Dopaminergic Parameters During Social Isolation in Low- and High-Active Mice

Alterations induced by social isolation (1 day to 18 weeks) in low- and high-active mice (LAM and HAM) were studied in respect to locomotor activity, [3H]-spiperone binding in the striatum, striatal, and cortical dopamine metabolism, and presynaptic dopaminergic sensitivity to apomorphine (0.75 mg/kg; IP). Isolated HAM and LAM showed increased locomotor activity compared to group-housed mice after long-term isolation (6–18 weeks). Considering the studied dopaminergic parameters, it has been found that social isolation did not affect striatal D2 receptors, striatal and cortical dopamine metabolism, and apomorphine-mediated reduction of dopaminergic metabolism. The change of housing conditions was generally associated with an increase of cortical dopamine metabolism after 1 week. Activity type specific differences in group-housed LAM and HAM were found in the basal striatal dopamine metabolism and in the sensitivity of the nigrostriatal system to autoreceptor activation. The reduced striatal dopamine metabolism and the higher presynaptic sensitivity of HAM may be related to their high active running wheel behavior.

Dynamic Alterations of Serotonergic Metabolism and Receptors During Social Isolation of Low- and High-Active Mice

Alterations induced by social isolation (1 day to 18 weeks) in low- and high-active mice (LAM and HAM) were studied in respect to serotonin metabolism, [ 3H]-8-OH-DPAT binding of presynaptic (midbrain), postsynaptic (hippocampus) 5-HT 1A receptors and [ 3H]-ketanserin binding of cortical 5-HT 2A receptors. Individual housing of mice was associated with reduction of serotonin metabolism, depending on isolation time and brain structure. Whereas a transient decrease in the striatum and cortex was detected between 1 week and 6 weeks, reduction of cerebellar and hippocampal serotonin metabolism was found later (12–18 weeks). Serotonergic systems of HAM were found to be more reactive to environmental disturbances, and their serotonin metabolism was more affected by social isolation. Isolation-induced upregulation of cortical 5-HT 2A receptors was measured only in HAM. Densities of postsynaptic 5-HT 1A receptors in the hippocampus did differ either in grouped or isolated mice. However, there were significant differences in hippocampal 5-HT 1A receptor affinity, especially between 1 day and 3 weeks. Transient downregulation of presynaptic 5-HT 1A receptors in the midbrain was found in isolated mice between 3 and 6 weeks. These results are discussed in terms of interactions between serotonergic alterations and isolation-induced aggression.

Individual motor activity — Relationships to dopaminergic responses

Owing to motor activity mice were divided into two groups in a running-wheel test: low-active mice (MAM) and high-active mice (HAM). Locomotor activities in the running wheel and in glass boxes are compared. The HAM showed a more intensive explorative behavior than LAM and were also more responsive in terms of exogenous factors than LAM. In contrast, LAM showed higher locomotor activity than HAM after habituation. Analyzing the response of LAM and HAM to dopaminergic agonists such as apomorphine, bromocriptine, and amphetamine, the role of specific dopaminergic mechanisms for the two types is discussed. Although apomorphine mainly stimulated the climbing activity in HAM, bromocriptine (climbing activity) and amphetamine (locomotion) had stronger effects in LAM. Differences may be assumed between LAM and HAM concerning the nigrostriatal and/or mesolimbic dopaminergic mechanisms. On the one hand, climbing activity following apomorphine application accompanied by stereotypes may suggest a stronger activation of striatal dopaminergic mechanisms in HAM. On the other hand, climbing activity following bromocriptine accompanied by jumping behavior, as well as the stimulation of locomotion after amphetamine, suggests a more effective activation of mesolimbic dopaminergic structures in LAM.

The influence of brain damage on locomotor behavior of mice selectively bred for high or low activity in the open field

Locomotor activity was measured in mice with lesions restricted to the neocortex, in animals with hippocampal damage, and in sham-operated control mice of the selectively bred high and low open-field activity lines developed by J. C. DeFries. Post-operatively, the high-activity animals with hippocampal lesions showed reduced activity relative to those with only neocortical damage, and their postoperative activity levels were uncorrelated with those obtained preoperatively. Testing the animals under reduced illumination enhanced locomotor activity, with the greatest increase demonstrated by high-activity mice with hippocampal damage. While increases in activity under low illumination did occur in the low-activity line, no significant group differences were observed. Amphetamine did not affect locomotor behavior of the high-activity control animals, whereas doses of 1.0 and 10.0 mg/kg reduced the activity of the low-activity mice. High-activity mice with hippocampal damage evidenced a significant increase in locomotor behavior after the 10.0 mg/kg dose. These results emphasize that the behavioral effects of brain lesions and pharmacological manipulations cannot be adequately assessed without regard to genotype of the animals under investigation.