Dietary nitrate supplementation increases nitrate and nitrite concentrations in human skin interstitial fluid.

Acute dietary nitrate (NO3-) supplementation can increase [NO3-], but not nitrite ([NO2-]), in human skeletal muscle, though its effect on [NO3-] and [NO2-] in skin remains unknown. In an independent group design, 11 young adults ingested 140 mL of NO3--rich beetroot juice (BR; 9.6 mmol NO3-), and 6 young adults ingested 140 mL of a NO3--depleted placebo (PL). Skin dialysate, acquired through intradermal microdialysis, and venous blood samples were collected at baseline and every hour post-ingestion up to 4 h to assess dialysate and plasma [NO3-] and [NO2-]. The relative recovery rate of NO3- and NO2- through the microdialysis probe (73.1% and 62.8%), determined in a separate experiment, was used to estimate skin interstitial [NO3-] and [NO2-]. Baseline [NO3-] was lower, whereas baseline [NO2-] was higher in the skin interstitial fluid relative to plasma (both P < 0.001). Acute BR ingestion increased [NO3-] and [NO2-] in the skin interstitial fluid and plasma (all P < 0.001), with the magnitude being smaller in the skin interstitial fluid (e.g., 183 ± 54 vs. 491 ± 62 µM for Δ[NO3-] from baseline and 155 ± 190 vs. 217 ± 204 nM for Δ[NO2-] from baseline at 3 h post BR ingestion, both P ≤ 0.037). However, due to the aforementioned baseline differences, skin interstitial fluid [NO2-] post BR ingestion was higher, whereas [NO3-] was lower relative to plasma (all P < 0.001). These findings extend our understanding of NO3- and NO2- distribution at rest and indicate that acute BR supplementation increases [NO3-] and [NO2-] in human skin interstitial fluid.

Soy protein intake increased bone mineral density under nonenergy-deficiency conditions but decreased it under energy-deficiency conditions in young female rats.

Many young individuals attempt to lose too much weight because of a false body image, which induces low bone mineral density (BMD) resulting from energy restriction. In addition, a decrease in estrogen has been observed along with the decrease in BMD. Estrogen is responsible for maintaining bone mass, and soybeans contain high levels of isoflavones, which have estrogen-like effects. Thus, we hypothesized that soy protein prevents low BMD caused by energy deficiency in young female rats. The purpose of this study was to examine the effect of soy protein intake on bone loss by energy deficiency in young female rats. Female Sprague-Dawley rats (6 weeks old) were randomly divided into the following 4 experimental groups: ad libitum feeding and casein diet (AL-Cas); ad libitum feeding and soy diet (AL-Soy); 40% energy restriction and casein diet (ER-Cas); and 40% energy restriction and soy diet (ER-Soy). The experimental period was 10.5 weeks. The AL-soy group had significantly higher BMD of the femur than the AL-Cas group (AL-Cas = 156 ± 5 mg/cm2, AL-Soy = 165 ± 7 mg/cm2; P < .05). Meanwhile, the ER-Soy group had significantly lower BMD of the tibia, femur, and lumbar spine than the ER-Cas group (ER-Cas = 147 ± 7 mg/cm2, ER-Soy = 133 ± 10 mg/cm2; P < .01). These results show that compared with ad libitum control groups, soy protein resulted in higher BMD under nonenergy deficiency, but under energy-deficiency conditions, it resulted in lower BMD.

Renalase is localized to the small intestine crypt and expressed upon the activation of NF-κB p65 in mice model of fasting-induced oxidative stress.

AIMS: Renalase expression is regulated by Nuclear Factor (NF)-κB and hypoxia inducible factor (HIF)-1α, and antioxidative stress function in renal cells were reported. However, dynamics of renalase and localizes in intestine were remain unknown. We evaluated the effects of oxidative stress on renalase expression and localization using model of fasting induced oxidative stress and Caco-2 cell, and examined the its physiological effects. MAIN METHODS: 24 male mice were divided into three groups: Control (Con), 72 h fasting (Fast), and 24 h refeeding after fasting (Refeed). Jejunum and ileum were collected respectively. The structure of jejunum and ileum were observed by hematoxylin and eosin (HE) stain. The expression levels of carbonylated protein, renalase, NF-κB p65 and HIF-1α were measured by immunoblotting. Localization of renalase was observed by immunofluorescent. in vitro assay was performed using Caco-2 cell. Renalase was overexpressed using adenovirus. After that, Caco-2 cell was treated with 2 mM H2O2 for 30 min or 24 h. KEY FINDINGS: Renalase was increased in Fast and it was localized in crypt. HIF-1α did not increase, but NF-κB p65 increased in Fast. Renalase overexpression protects the Caco-2 cells against H2O2 induced oxidative stress. SIGNIFICANCE: Renalase was localized in crypt and increased in Fast. This increase suggested protect response to oxidative stress because undifferenced cells were localized in crypt and need to be protected. Actually, renalase protected Caco-2 cells against H2O2 induced oxidative stress. Small intestinal renalase expression was regulated by NF-κB p65 and was considered to be a defense mechanism against oxidative stress.

The effect of dehydroepiandrosterone administration on intestinal calcium absorption in ovariectomized female rats.

PURPOSE: Dehydroepiandrosterone (DHEA) administration reportedly recovers osteoporosis, a bone disorder associated with bone deficiency in postmenopausal women. However, the physiological mechanism of DHEA in osteoporosis remains elusive, especially in terms of intestinal calcium absorption. Therefore, we investigated the effect of DHEA administration on calcium absorption in ovariectomized (OVX) female rats using an estrogen receptor antagonist. METHODS: Female Sprague-Dawley rats (n=23, 6 weeks old) were randomized into three groups: OVX control group (OC, n=7), OVX with DHEA treatment group (OD, n=8), and OVX with DHEA inhibitor group (ODI, n=8) for 8 weeks. RESULTS: Intestinal calcium accumulation, as well as the rate of absorption, demonstrated no significant differences during the experimental period among investigated groups. The bone mineral density (BMD) of the tibia at the proximal metaphysis was higher in the OD group than that in the OC group (p<0.05); however, BMD of the ODI group showed no significant difference from investigated groups. Furthermore, the BMD of the tibia at the diaphysis did not significantly differ among these groups. CONCLUSION: We revealed that DHEA administration does not involve intestinal Ca absorption, although this treatment improves BMD levels in OVX rats. These observations indicate that the effect of DHEA on the bone in postmenopausal women is solely due to its influence on bone metabolism and not intestinal calcium absorption.

Sleep disorder risk factors among student athletes.

OBJECTIVE: To clarify sleep disorder risk factors among student athletes, this study examined the relationship between lifestyle habits, competition activities, psychological distress, and sleep disorders. METHODS: Student athletes (N = 906; male: 70.1%; average age: 19.1 ± 0.8 years) in five university sports departments from four Japanese regions were targeted for analysis. Survey items were attributes (age, gender, and body mass index), sleep disorders (recorded through the Pittsburgh Sleep Quality Index), lifestyle habits (bedtime, wake-up time, smoking, drinking alcohol, meals, part-time jobs, and use of electronics after lights out), competition activities (activity contents and competition stressors), and psychological distress (recorded through the K6 scale). The relation between lifestyle habits, competition activities, psychological distress, and sleep disorders was explored using logistic regression analysis. RESULTS: Results of multivariate logistic regression analysis with attributes as adjustment variables showed that "bedtime," "wake-up time," "psychological distress," "part-time jobs," "smartphone/cellphone use after lights out," "morning practices," and "motivation loss stressors," were risk factors that were independently related to sleep disorders. CONCLUSIONS: Sleep disorders among student athletes are related to lifestyle habits such as late bedtime, early wake-up time, late night part-time jobs, and use of smartphones/cellphones after lights out; psychological distress; and competition activities such as morning practices and motivation loss stressors related to competition. Therefore, this study suggests the importance of improving these lifestyle habits, mental health, and competition activities.

The Impact of Different Amounts of Calcium Intake on Bone Mass and Arterial Calcification in Ovariectomized Rats.

Reduced estrogen secretion and low calcium (Ca) intake are risk factors for bone loss and arterial calcification in female rodents. To evaluate the effects of Ca intake at different amounts on bone mass changes and arterial calcification, 8-wk-old female Wistar rats were randomly placed in ovariectomized (OVX) control and OVX with vitamin D3 plus nicotine (VDN) treatment groups. The OVX with VDN rats were then divided into six groups to receive different amounts of Ca in their diets: 0.01%, 0.1%, 0.3%, 0.6%, 1.2%, or 2.4% Ca. After 8 wk of administration, low Ca intake groups with 0.01% and 0.1% Ca diets had significantly reduced bone mineral density (BMD) and bone mechanical properties as compared with those of the other groups, whereas high Ca intake groups with 1.2% and 2.4% Ca diets showed no differences as compared with the 0.6% Ca intake group. For both the 0.01% and 2.4% Ca intake groups, Ca levels in their thoracic arteries were significantly higher as compared with those of the 0.6% Ca diet group, and that was highly correlated with serum PTH levels. An increase in relative BMP-2 mRNA expression in the arterial tissues of the 0.01% and 2.4% Ca diet groups was also observed. These results suggested that extremely low Ca intake during periods of estrogen deficiency may be a possible risk for the complications of reduced BMD and arterial calcification and that extremely high Ca intake may promote arterial calcification with no changes in BMD.

The relationship between salivary calcium concentration and differences in bone mineral density level in female rats.

It has not yet been examined whether salivary calcium levels reflect changes in bone mass. The purpose of this study was to investigate the relationship between salivary calcium concentration and differences in bone mineral density due to estrogen deficiency and/or different calcium intake levels in female rats. In Experiment 1, the animals (n=14) were divided into an ovariectomized group (OVX) (n=8, 0.6% calcium diet) and a sham-operated group (Sham) (n=6, 0.6% calcium diet). The bone mineral density (BMD) levels of the tibia and lumbar spine were significantly lower in the OVX group than in the Sham group (p<0.001 and p<0.01, respectively), whereas there was no significant difference in the salivary calcium concentration between the two groups. In Experiment 2, after an ovariectomy operation, the animals (n=42) were randomized into five groups that received 0.01%, 0.1%, 0.6%, 1.2%, and 2.4% calcium diets (n=10, 10, 6, 8, and 8, respectively). The BMD levels of the tibia and lumbar spine were significantly lower in the 0.01% or 0.1% calcium diet intake groups than in the 0.6%, 1.2%, 2.4% calcium diet intake groups (all p<0.001), whereas there were no differences in the salivary calcium concentration among the groups. In conclusion, the salivary calcium level did not change during periods of decreasing BMD and bone strength induced by estrogen deficiency and/or calcium intake restrictions in female rats.

The effect of different amounts of calcium intake on bone metabolism and arterial calcification in ovariectomized rats.

Low calcium (Ca) intake is the one of risk factors for both bone loss and medial elastocalcinosis in an estrogen deficiency state. To examine the effect of different amounts of Ca intake on the relationship between bone mass alteration and medial elastocalcinosis, 6-wk-old female SD rats were randomized into ovariectomized (OVX) control or OVX treated with vitamin D(3) plus nicotine injection (VDN) groups. The OVX treated with VDN group was then divided into 5 groups depending on the different Ca content in their diet, 0.01%, 0.1%, 0.6%, 1.2%, and 2.4% Ca intakes. After 8 wk of experimentation, the low Ca intake groups of 0.01% and 0.1% showed a low bone mineral density (BMD) and bone properties significantly different from those of the other groups, whereas the high Ca intake groups of 1.2% and 2.4% showed no difference compared with the OVX control. Only in the 0.01% Ca intake group, a significantly higher Ca content in the thoracic artery was found compared with that of the OVX control. Arterial tissues of the 0.01% Ca intake group showed an increase of bone-specific alkaline phosphatase (BAP) activity, a marker of bone mineralization, associated with arterial Ca content. However, the high Ca intake did not affect arterial Ca content nor arterial BAP activity. These results suggested that a low Ca intake during periods of rapid bone loss caused by estrogen deficiency might be one possible cause for the complication of both bone loss and medial elastocalcinosis.

Effect of different intake levels of dietary protein and physical exercise on bone mineral density and bone strength in growing male rats.

Having higher bone mineral density (BMD) during growth is complexly influenced by many factors. For example, nutrition and physical exercise are key factors. However, few studies have investigated the combined effects of these factors. In this study, we investigated the effect of physical exercise and different levels of protein intake on BMD and bone strength of growing male rats. Forty-seven male Wistar rats (5 wk old) were randomized into 10% (Low), 20% (Moderate) and 40% (High) protein diet groups, and each group was further divided into exercise groups (LEx, MEx, HEx) or non-exercise groups (L, M, H). Exercise group rats were trained 6 d per week on a treadmill (25-30 m/min, 60 min) for 60 d. After being sacrificed, their BMD and bone strength were evaluated. The BMD of tibia, femoral breaking force and energy were significantly lower in the low protein diet groups than the other diet groups. In particular, the femoral breaking energy was significantly lower in the HEx group than in the H group, while there were no differences between LEx and L or MEx and M. Taken together, our data suggests that a low protein intake could suppress acquisition of bone mass and increasing bone strength during growth. Moreover, a high protein intake could also suppress bone strength during growth in which physical activity was vigorously performed. Therefore, sustaining an adequate protein intake level, around 20% protein intake, may be of significance for increasing not only bone mass but bone strength during growth.

DHEA administration activates local bioactive androgen metabolism in cancellous site of tibia of ovariectomized rats.

It is not known whether local androgen metabolism is involved in the mechanisms underlying the dehydroepiandrosterone (DHEA) administration-induced improvement of bone mineral density (BMD) in an estrogen-deficiency state. The aim of the present study was to clarify whether DHEA administration would improve local androgen metabolism and BMD in cancellous site of tibia of ovariectomized (OVX) rats. Twenty-two female rats, 6 weeks old, were randomized into three groups: sham-operated rats, OVX control rats, and OVX rats that received DHEA treatment. DHEA was administered intraperitoneally at 20 mg/kg body weight for 8 weeks. The concentrations of free testosterone and dihydrotestosterone (DHT) in cancellous site of tibia did not change as a result of ovariectomy, while the DHT concentration increased following DHEA administration. We revealed that DHEA administration improved the reduction of 17ß- and 3ß-hydroxysteroid dehydrogenases and clearly reversed the reduction of 5α-reductase types 1 and 2 and androgen receptor in the cancellous site of tibia of OVX rats. DHEA administration suppressed estrogen deficiency relative to the decrease in the cancellous BMD, which was positively associated with local DHT concentration. These findings indicate that DHEA administration enhances local bioactive androgen metabolism in the cancellous tibia of young OVX rats, suggesting that local DHT may play a part in the DHEA administration-induced improvement of cancellous BMD.

Relationship between arterial calcification and bone loss in a new combined model rat by ovariectomy and vitamin D(3) plus nicotine.

Epidemiological studies have reported an association between arterial calcification and bone loss after menopause. However, the underlying mechanism of the association remains unclear. Therefore, to explore the possible mechanisms of the association, we tried to develop a new combined model rat of ovariectomy (OVX, an animal model of osteoporosis) and vitamin D(3) plus nicotine (VDN rat, an animal model of arterial calcification). We tested them by using sham-operated control rats (SC), OVX control rats (OC), and OVX plus VDN-treated rats (OVN). Dissections were performed twice at 4 (4SC, 4OC, and 4OVN) and 8 (8SC, 8OC, and 8OVN) weeks after treatment. 8OVN showed bone loss and arterial calcification, although 8OC showed only bone loss. Moreover, arterial calcium content was associated with indexes of bone loss at 8 weeks. Thus, the OVN rat is considered a good model to examine the relationship of the two disorders after menopause. Additionally, the arterial endothelin-1 (ET-1, a potent regulator of arterial calcification) levels increased in both 4OVN and 8OVN, and the level was associated with arterial calcium content at 8 weeks. Furthermore, the arterial endothelial nitric oxide synthase (eNOS) protein, which is an enzyme that produces nitric oxide (an antiatherosclerotic substance), was significantly reduced in only 8OVN. Estrogens affect the alterations of the eNOS and ET-1 proteins. Therefore, we suggest that impairment of the ET-1- and NO-producing system in arterial tissue during periods of rapid bone loss by estrogen deficiency might be a mechanism of the relationship between the two disorders seen in postmenopausal women.

Estrogen deficiency and low-calcium diet increased bone loss and urinary calcium excretion but did not alter arterial stiffness in young female rats.

Many epidemiological studies have reported that the severity of arterial diseases such as arterial calcification and stiffness is inversely related to bone loss, i.e., osteoporosis. However, the nature of this relationship is unclear. The purpose of the present study was to examine the influences of estrogen deficiency and/or low-calcium diet (0.1% Ca) on bone metabolism and calcium balance, as well as aortic wall composition and stiffness in young female rats. Twenty-eight 6-week-old female rats were randomized into four groups: OVX-Low calcium (OL) and OVX-Normal calcium groups (ON) were ovariectomized, and Sham-Low calcium (SL) and Sham-Normal calcium groups (SN) were sham-operated. After 12 weeks, the bone mineral density of the lumbar spine and tibial proximal metaphysis were significantly lower in ON than in SN, and also significantly lower in OL than in ON. Additionally, OL rats had significant higher (vs. SN and SL) urinary deoxypyridinoline, but not urinary calcium, excretion at 4 weeks after ovariectomy. However, at 12 weeks after ovariectomy, urinary calcium excretion was significantly higher in OL than in SL, with corresponding increases in two bone turnover markers, bone-type alkaline phosphatase and tartrate-resistant acid phosphatase. Neither estrogen deficiency nor low-calcium diet affected aortic stiffness or elastin degeneration and calcium deposition over the course of the present study, although changes of bone metabolism occurred rapidly. Taken together, these results show that bone loss and arterial stiffness did not progress simultaneously in the present experimental protocol.