The neuronal nitric oxide synthase expression increases during satellite cell-derived primary myoblasts differentiation.

The neuronal nitric oxide synthase (nNOS; encoded by NOS1)-derived nitric oxide (NO) plays an important role in maintaining skeletal muscle mass. In adult skeletal muscle, nNOS localizes to the cell membrane, cytosol, and nucleus, and regulates muscle hypertrophy and atrophy in various subcellular fractions. However, its role in muscle stem cells (also known as muscle satellite cells), which provide myonuclei for postnatal muscle growth, maintenance, and regeneration, remains unclear. The present study aimed to determine nNOS expression in muscle satellite cell-derived primary myoblasts during differentiation and its DNA methylation levels, an epigenetic modification that controls gene expression. Undifferentiated and differentiated satellite cell-derived primary myoblasts were found to express nNOS. Immunohistochemical analysis revealed that nNOS colocalized with Pax7 (satellite cell marker) only in the undifferentiated myoblasts. Furthermore, nNOS immunoreactivity spread to the cytosol of Pax7-negative differentiated myotube-like cells. The level of Nos1µ mRNA, the main isoform of skeletal muscle nNOS, was increased in differentiated satellite cell-derived primary myoblasts compared to that in the undifferentiated cells. However, Nos1 methylation levels remained unchanged during differentiation. These findings suggest that nNOS induction and the appropriate transition of its subcellular localization may contribute to muscle differentiation.

Short-term running exercise alters DNA methylation patterns in neuronal nitric oxide synthase and brain-derived neurotrophic factor genes in the mouse hippocampus and reduces anxiety-like behaviors.

Running exercise has beneficial effects on brain health. However, the effects of relatively short-term running exercise (STEx) on behavior, and its underlying signaling pathways, are poorly understood. In this study, we evaluated the possibility that the regulation by STEx of brain-derived neurotrophic factor (BDNF) and neuronal nitric oxide synthase (nNOS, encoded by NOS1), which are important molecules for anxiety regulation, might involve mechanisms of epigenetic modification, such as DNA methylation. C57BL/6J male mice were divided into sedentary (SED, n = 12) and STEx (EX, n = 15) groups; STEx was conducted with the mice for a duration of 11 days. STEx reduced anxiety-like behaviors, and STEx reduced Nos1α and increased Bdnf exon I and IV mRNA levels in the hippocampus. Interestingly, behavioral parameters were associated with Bdnf exon I and IV and Nos1α mRNA levels in the ventral, but not dorsal, hippocampal region. However, STEx had no effect on peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc-1α) or fibronectin type III domain-containing 5 (Fndc5) mRNA levels, which are relatively long-term exercise-induced upstream regulators of BDNF. In parallel with gene expression changes, we found, for the first time, that STEx downregulated Bdnf promoter IV and upregulated Nos1 DNA methylation levels in the hippocampus, and these patterns were partially different between the dorsal and ventral regions. These findings suggest that the beneficial effects of running exercise on mood regulation may be controlled by alterations in epigenetic mechanisms, especially in the ventral hippocampus. These effects occur even after a relatively short-term period of exercise.

Association between the inability to identify particular odors and physical performance, cognitive function, and/or brain atrophy in community-dwelling older adults from the Fukuoka Island City study.

BACKGROUND: Olfactory dysfunction is associated with severe brain atrophy and cognitive impairment in Parkinson's disease. However, it remains unknown whether an inability to identify particular odors is associated with physical performance, cognitive function, and/or brain atrophy in community-dwelling older adults. METHODS: In this cross-sectional study, 44 community-dwelling older adults were included (14 males, 30 females; mean age: 72.4 ± 5.7 years, range: 63-85 years). The Odor Stick Identification Test for Japanese, consisting of 12 odors, was used to examine olfaction. Subjects also completed physical performance (lower limb function, balance, and gait speed) and cognitive function (global cognition, logical memory, and the Trail Making Tests). Additionally, magnetic resonance imaging was used to investigate brain atrophy in the bilateral medial temporal area (MTA) and whole gray matter using the voxel-based specific regional analysis system for Alzheimer's disease. RESULTS: Total olfaction was not significantly associated with physical performance, cognitive function, or brain atrophy. However, MTA atrophy was associated with an inability to identify Japanese orange (B: - 0.293; ß: - 0.347; p < .05) after adjusting for age and sex (R2: 0.328; adjusted R2: 0.277). Subjects who were unable to identify Japanese orange (n = 30) had worse MTA atrophy than those who were able to identify Japanese orange (n = 14), even after adjusting for covariates (p < .05). CONCLUSIONS: Total olfaction was not associated with physical performance, cognitive function, or brain atrophy. However, an inability to identify Japanese orange odor was independently associated with mild MTA atrophy among community-dwelling older adults.

One week, but not 12 hours, of cast immobilization alters promotor DNA methylation patterns in the nNOS gene in mouse skeletal muscle.

KEY POINTS: DNA methylation may play an important role in regulating gene expression in skeletal muscle to adapt to physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) in skeletal muscle is a key regulator of skeletal muscle mass; however, it is unclear whether nNOS expression is regulated by DNA methylation. We found that 1 week of cast immobilization increased nNOS DNA methylation levels and downregulated nNOS gene expression in atrophic slow-twitch soleus muscle from the mouse leg. These changes were not detected in non-atrophic fast-twitch extensor digitorum longus muscle. Twelve hours of cast immobilization decreased nNOS gene expression, whereas nNOS DNA methylation levels were unchanged, suggesting that downregulation of nNOS gene expression by short-term muscle inactivity is independent of the DNA methylation pattern. These findings contribute to a better understanding of the maintenance of skeletal muscle mass and prevention of muscle atrophy by epigenetic mechanisms via the nNOS/NO pathway. ABSTRACT: DNA methylation is a mechanism that controls gene expression in skeletal muscle under various environmental stimuli, such as physical activity and inactivity. Neuronal nitric oxide synthase (nNOS) regulates muscle atrophy in skeletal muscle. However, the mechanisms regulating nNOS expression in atrophic muscle remain unclear. We hypothesized that nNOS expression in atrophic muscle is regulated by DNA methylation of the nNOS promotor in soleus (Sol; slow-twitch fibre dominant) and extensor digitorum longus (EDL; fast-twitch fibre dominant) muscles. One week of cast immobilization induced significant muscle atrophy in Sol but not in EDL. We showed that 1 week of cast immobilization increased nNOS DNA methylation levels in Sol, although only a minor change was detected in EDL. Consistent with the increased DNA methylation levels in atrophic Sol, the gene expression levels of total nNOS and nNOSµ (i.e. the major splicing variant of nNOS in skeletal muscle) decreased. The abundance of the nNOS protein and cell membrane (especially type IIa fibre) immunoreactivity also decreased in atrophic Sol. These changes were not observed in EDL after 1 week of cast immobilization. Furthermore, despite the lack of significant atrophy, 12 h of cast immobilization decreased gene expression levels of total nNOS and nNOSµ in Sol. However, no association was detected between nNOS DNA methylation and gene expression. The expression of the nNOSß gene, another splicing variant of nNOS, in EDL was unchanged by cast immobilization, whereas its expression was not detected in Sol. We concluded that chronic adaptation of nNOS gene expression in cast immobilized muscle may involve nNOS DNA methylation.

Short- and long-term effects of high-fat diet feeding and voluntary exercise on hepatic lipid metabolism in mice.

Exercise is an effective tool for improving high-fat diet induced fat accumulation in the liver. However, the process of fat accumulation in the liver and the efficacy of early intervention with exercise remain unclear. The aim of this study was to investigate the short- and long-term effects of high-fat diet feeding and voluntary exercise on hepatic lipid metabolism in mice. Male C57BL/6J mice aged 6 weeks were randomly divided into two groups, the control group and high-fat diet feeding group, and fed a normal or high-fat diet for 12 weeks. After 6 weeks, mice in the high-fat diet feeding group were further divided into no exercise group and voluntary exercise training group, with mice in the exercise group provided a running wheel for 6 weeks. Body weight, food intake, and wheel rotation counts were measured every second day for 12 weeks. We found that voluntary exercise for 1 week (short-term exercise) significantly reduced fat accumulation in the liver by downregulating the expression of hepatic lipogenesis-associated proteins and upregulating the expression of hepatic lipolysis-associated proteins, as determined through western blotting and histology. Further, voluntary exercise for 6 weeks (long-term exercise) downregulated the expression of hepatic lipogenesis-associated proteins. These results suggest that hepatic lipogenesis and/or hepatic lipolysis mediate the beneficial effects of voluntary exercise on hepatic fat accumulation.

Exercise training rescues high fat diet-induced neuronal nitric oxide synthase expression in the hippocampus and cerebral cortex of mice.

Consumption of a high fat diet (HFD) and being overweight both induce functional deterioration and atrophy of the hippocampus. These alterations are associated with mental disorders such as depression and anxiety. Exercise combats obesity and enhances brain health. There is substantial evidence that neuronal nitric oxide synthase (nNOS) is a key regulator of affective behavior, and that increased brain nNOS leads to anxiety while environmental enrichment (EE), which reduces brain nNOS, has anxiolytic effects. In this study we investigated the effects of HFD with and without exercise on nNOS protein and gene expression levels in the brains of mice. Twelve weeks of HFD consumption increased body and mesenteric fat weight, as well as nNOS protein levels in the hippocampus and cerebral cortex. Six weeks of exercise training reduced body fat and rescued hippocampal and cortical nNOS expression levels in HFD-fed mice. Cerebellar nNOS expression was unaffected by HFD and exercise. Our results suggest that HFD-induced brain dysfunction may be regulated by hippocampal and/or cortical nNOS, and that exercise may have therapeutic potential for the treatment of HFD-induced depression and anxiety via the nNOS/NO pathway. In conclusion, exercise reverses HFD-induced changes in hippocampal and cortical nNOS protein levels in mice.

Effects of environmental enrichment in aged mice on anxiety-like behaviors and neuronal nitric oxide synthase expression in the brain.

Previous studies have shown that an enriched environment (EE) has an important effect on brain function via the neuronal nitric oxide synthase/nitric oxide (nNOS/NO) pathway in young and aged animals. However, whether EE induces its effect by altering nNOS expression levels and whether it lowers anxiety-like behaviors in aged mice remains unclear. Here, we show that nNOS expression levels increased with age in the hippocampus and cerebellum in aged mice, but not in the cortex. Moreover, EE reduced anxiety-like behaviors in aged mice and reduced nNOS expression levels in the cerebellum, but not in the cortex. The present study suggests that EE improves anxiety-like behaviors in aged mice by altering nNOS expression levels in the hippocampus or cerebellum.

Factors associated with water consumption measured using the stable isotope techniques among Japanese adults: a cross-sectional study.

BACKGROUND/OBJECTIVES: While some dietary guidelines have established daily water requirements for adults, those for older adults are not well defined. Factors associated with water turnover (WT), indicating water requirements and preformed water (PW), referring to fluids consumed from food and beverages, remain poorly understood. Therefore, we aimed to investigate these factors in Japanese adults. SUBJECTS/METHODS: This cross-sectional study included 57 participants aged 54-85 years, living in Fukuoka City, Japan. The WT and PW were measured using the doubly labelled water (DLW) methods from August 2015 to September 2015. Dietary intake and physical activity were evaluated using a 3-day dietary record (DR) and a validated triaxial accelerometer, respectively. Fasting blood and urine samples, as well as environmental conditions, were assessed using a validated technique. We evaluated the variables for predicting water consumption by using a multivariate generalised linear model, with forward stepwise selection using these covariates. RESULTS: The median WT and PW were 2842 ml/day and 2227 ml/day, respectively. In the multivariate model, WT (R2 = 0.629) and PW (R2 = 0.621) were moderately predicted by variables such as sex, body weight, moderate-to-vigorous physical activity, water consumption estimated by DR, urinary aldosterone and osmolality, creatinine clearance and wet-bulb globe temperature. The PW estimates from DR were ~40% lower than those from DLW method, despite a significant correlation between the values. CONCLUSIONS: WT and PW are associated not only with lifestyle and environmental factors but also with urinary markers and kidney function. These findings provide useful insights into the differences in each individual's water requirement.

Behavioral defects and downregulation of hippocampal BDNF and nNOS expression in db/db mice did not improved by chronic TGF-ß2 treatment.

Epidemiological evidence suggests that there is a link between diabetes and mood disorders, such as depression and anxiety. Although peripheral or central inflammation may explain this link, the molecular mechanisms are not fully understood and few effective treatments for diabetes or mood disorders are available. In the present study, we aimed to determine whether transforming growth factor (TGF)-ß2, an anti-inflammatory substance, might represent a potential therapeutic agent for diabetes-related mood behaviors. TGF-ß2 expression in the hippocampus is affected by anxiolytic drugs and stress exposure, it is able to cross the blood-brain barrier, and it is as an exercise-induced physiological adipokine that regulates glucose homeostasis. Therefore, we hypothesized that a chronic TGF-ß2 infusion would ameliorate diabetes-related glucose intolerance and mood dysregulation. To determine the effects of the chronic administration of TGF-ß2 on diabetes, we implanted osmotic pumps containing TGF-ß2 into type 2 diabetic mice (db/db mice), and age-matched non-diabetic control wild type mice and db/db mice were infused with vehicle (PBS), for 12 consecutive days. To assess anxiety-like behaviors and glucose homeostasis, the mice underwent elevated plus maze testing and intraperitoneal glucose tolerance testing. Hippocampal and perigonadal visceral white adipose tissue perigonadal white adipose tissue samples were obtained 12 days later. Contrary to our hypothesis, TGF-ß2 infusion had no effect on diabetes-related glucose intolerance or diabetes-related behavioral defects, such as inactivity. In db/db mice, the expression of inflammatory markers was high in pgWAT, but not in the hippocampus, and the former was ameliorated by TGF-ß2 infusion. The expression of brain-derived neurotrophic factor and neuronal nitric oxide synthase, important regulators of anxiety-like behaviors, was low in db/db mice, but TGF-ß2 infusion did not affect their expression. We conclude that although TGF-ß2 reduces the expression of pro-inflammatory markers in the adipose tissue of diabetic mice, it does not ameliorate their obesity or mood dysregulation.

Effects of Dietary Vitamin D Deficiency on Markers of Skeletal Muscle Mitochondrial Biogenesis and Dynamics.

We examined the effects of dietary vitamin D deficiency on markers of mitochondrial biogenesis and dynamics in rat soleus muscle. Male Wistar rats were fed a chow with no vitamin D (No-D; 0 IU/kg) or a moderate dose (Mod-D; 2,000 IU/kg) of vitamin D chow for 8 wk. Compared to the Mod-D group, at 8 wk the No-D group showed significantly lower serum 25(OH)D levels. Although vitamin D deficiency had no effect on body composition, the No-D rats showed significantly decreased levels of PGC-1α, a marker of skeletal muscle mitochondrial biogenesis, and DRP1, a marker of skeletal muscle mitochondrial fission. The change in the PGC-1α protein expression and the serum 25(OH)D concentrations were significantly correlated. The change in DRP1 protein expression and the serum 25(OH)D concentrations tended to be correlated. There was no significant between-group difference in markers of mitochondrial fusion (MFN2 and OPA1) and mitophagy (PARKIN) in soleus muscle, and no relationship with serum 25(OH)D concentrations. Collectively our findings suggest that dietary vitamin D deficiency decreased PGC-1α and DRP1 protein expression in rat soleus muscle.

Administration of Jerusalem artichoke reduces the postprandial plasma glucose and glucose-dependent insulinotropic polypeptide (GIP) concentrations in humans.

Background: The consumption of Jerusalem artichoke has multiple beneficial effects against diabetes and obesity. Objective: The aim of this study was to determine the effect of a single administration of Jerusalem artichoke tubers on postprandial glycemia and the concentrations of incretin hormones in humans. Method: Grated Jerusalem artichoke was administered prior to a meal (Trial 1; white rice for prediabetic participants, n = 10). Dose-dependent effect of Jerusalem artichoke (Trial 2; white rice for prediabetic participants, n = 4) and effect prior to the fat-rich meal were also investigated (Trial 3; healthy participants, n = 5) in this pilot study. Circulating glucose, insulin, triglyceride, glucagon, active glucagon-like peptide-1 (GLP-1), and active glucose-dependent insulinotropic polypeptide (GIP) concentrations were subsequently measured in all the trials. Results: Jerusalem artichoke significantly reduced the glucose and GIP concentrations after the consumption of either meal in Trial 1 and Trial 3, whereas there were no differences in the insulin, glucagon, and active GLP-1 concentrations. Also, there was no significant difference in the triglyceride concentration after the ingestion of the fat-rich meal in Trial 3. The glucose and GIP-lowering effects were dose-dependent, and the consumption of at least 100 g of Jerusalem artichoke was required to have these effects in Trial 2. Conclusion: This study demonstrates that a single administration of Jerusalem artichoke tubers reduces postprandial glucose and active GIP concentrations in prediabetic and healthy individuals.

HSPA8 Single-Nucleotide Polymorphism Is Associated with Serum HSC70 Concentration and Carotid Artery Atherosclerosis in Nonalcoholic Fatty Liver Disease.

There is an association between nonalcoholic fatty liver disease (NAFLD) and atherosclerosis, but the genetic risk of atherosclerosis in NAFLD remains unclear. Here, a single-nucleotide polymorphism (SNP) of the heat shock 70 kDa protein 8 (HSPA8) gene was analyzed in 123 NAFLD patients who had been diagnosed using a liver biopsy, and the NAFLD phenotype including the maximum intima-media thickness (Max-IMT) of the carotid artery was investigated. Patients with the minor allele (A/G or G/G) of rs2236659 showed a lower serum heat shock cognate 71 kDa protein concentration than those with the major A/A allele. Compared with the patients with the major allele, those with the minor allele showed a higher prevalence of hypertension and higher Max-IMT in men. No significant associations between the HSPA8 genotype and hepatic pathological findings were identified. In decision-tree analysis, age, sex, liver fibrosis, and HSPA8 genotype were individually associated with severe carotid artery atherosclerosis (Max-IMT ≥ 1.5 mm). Noncirrhotic men aged ≥ 65 years were most significantly affected by the minor allele of HSPA8. To predict the risk of atherosclerosis and cardiovascular disease, HSPA8 SNP genotyping might be useful, particularly for older male NAFLD patients.

Porphyromonas gingivalis Administration Induces Gestational Obesity, Alters Gene Expression in the Liver and Brown Adipose Tissue in Pregnant Mice, and Causes Underweight in Fetuses.

Preventing adverse pregnancy outcomes is crucial for maternal and child health. Periodontal disease is a risk factor for many systemic diseases including adverse pregnancy outcomes, such as preterm birth and low birth weight. In addition, the administration of the periodontopathic bacterium Porphyromonas gingivalis exacerbates obesity, glucose tolerance, and hepatic steatosis and alters endocrine function in the brown adipose tissue (BAT). However, the effects of having periodontal disease during pregnancy remain unclear. Thus, this study investigates the effect of P. gingivalis administration on obesity, liver, and BAT during pregnancy. Sonicated P. gingivalis (Pg) or saline (Co) was injected intravenously and administered orally to pregnant C57BL/6J mice three times per week. Maternal body weight and fetal body weight on embryonic day (ED) 18 were evaluated. Microarray analysis and qPCR in the liver and BAT and hepatic and plasma triglyceride quantification were performed on dams at ED 18. The body weight of Pg dams was heavier than that of Co dams; however, the fetal body weight was decreased in the offspring of Pg dams. Microarray analysis revealed 254 and 53 differentially expressed genes in the liver and BAT, respectively. Gene set enrichment analysis exhibited the downregulation of fatty acid metabolism gene set in the liver and estrogen response early/late gene sets in the BAT, whereas inflammatory response and IL6/JAK/STAT3 signaling gene sets were upregulated both in the liver and BAT. The downregulation of expression levels of Lpin1, Lpin2, and Lxra in the liver, which are associated with triglyceride synthesis, and a decreasing trend in hepatic triglyceride of Pg dams were observed. P. gingivalis administration may alter lipid metabolism in the liver. Overall, the intravenous and oral administration of sonicated P. gingivalis-induced obesity and modified gene expression in the liver and BAT in pregnant mice and caused fetuses to be underweight.

Effects of inosine monophosphate and exercise training on neuronal nitric oxide synthase in the mouse brain.

Recently, the purine nucleoside inosine has been demonstrated to have several neuroprotective effects. Similarly, exercise training has well-known beneficial effects on mental health and cognitive function. Neuronal nitric oxide synthase (nNOS) is a key neuronal messenger in several brain regions, and the downregulation of nNOS has been shown to improve brain function. However, whether inosine and exercise training have combined effects on nNOS pathway-related proteins in the brain remains unknown. We found, for the first time, that inosine monophosphate (IMP), which is a precursor of inosine, decreases nNOS levels in the ventral hippocampus (vHp) and the cerebellum (Ce), but not in the dorsal hippocampus (dHp). In the vHp, the phosphorylation of cAMP response element-binding protein (CREB) was also upregulated, which negatively correlated with nNOS protein levels. In the cerebral cortex (Cx), no significant activation of the nNOS pathway was observed. In the dHp, vHp, Cx, and Ce, no interactions between the effects of IMP and exercise on nNOS protein and CREB phosphorylation levels were observed. The phosphorylation of nNOS was regulated by the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway. Although IMP induced minor changes in Akt phosphorylation, nNOS phosphorylation was unchanged by either IMP or exercise. In conclusion, in the vHp, which is associated with emotional behavior, IMP decreased nNOS levels and activated CREB, suggesting that IMP can elicit anxiolytic effects.

Acute bout of exercise downregulates thioredoxin-interacting protein expression in rat contracting skeletal muscles.

We previously reported that in rat skeletal muscle, disuse (i.e., decreased muscle contractile activity) rapidly increases thioredoxin-interacting protein (TXNIP), which is implicated in the reduced glucose uptake. Accordingly, we sought herein to (a) determine the effect of exercise (i.e., increased muscle contractile activity) on muscle TXNIP protein expression, and (b) elucidate the mechanisms underlying the changes of TXNIP protein expression in response to exercise. Rat epitrochlearis and soleus muscles were dissected out after an acute bout of 3-hr swimming (without weight loading) or 3-hr treadmill running (15% grade at 9m/min). In a separate protocol, the isolated epitrochlearis and soleus muscles were incubated for 3 hr with AMP-dependent protein kinase activator AICAR. Immediately after the cessation of the 3-hr swimming, the TXNIP protein was decreased in epitrochlearis but not in soleus muscle. Conversely, 3-hr treadmill running decreased the TXNIP protein in soleus but not in epitrochlearis muscle. TXNIP protein was decreased concomitantly with reduced postexercise muscle glycogen, showing that a decrease in TXNIP protein expression occurs in muscles that are recruited during exercise. In addition, 3-hr incubation with AICAR decreased TXNIP protein in both isolated epitrochlearis and soleus muscles. Our results suggest that (a) an acute bout of exercise downregulates TXNIP protein expression in rat contracting skeletal muscles, and (b) the reduction in TXNIP protein expression in contracting muscles is probably mediated by AMPK activation, at least in part.

Anxiety-like behaviors and hippocampal nNOS in response to diet-induced obesity combined with exercise.

A high-fat diet (HFD) and overweight status can induce hippocampal dysfunction, leading to depression and anxiety. Exercise has beneficial effects on emotional behaviors. We previously reported that exercise training rescues HFD-induced excess hippocampal neuronal nitric oxide synthase (nNOS) expression, which is a key regulator of anxiety. Here, we investigated anxiety-like behaviors and hippocampal nNOS expression in response to HFD combined with exercise. Mice were assigned to standard diet, HFD, or HFD with exercise groups for 12 weeks. We found that exercise during the final 6 weeks of the HFD regime improved 12 weeks of HFD-induced defecation, accompanied by rescue of excess nNOS expression. However, anxiety indicators in the elevated plus maze were unchanged. These effects were not apparent after only 1 week of exercise. In conclusion, 6 weeks of exercise training reduced HFD-related anxiety according to one of our measures (defecation), and reversed changes in the hippocampal nNOS/NO pathway.