LDL uptake-dependent phosphatidylethanolamine translocation to the cell surface promotes fusion of osteoclast-like cells.

Osteoporosis is associated with vessel diseases attributed to hyperlipidemia, and bone resorption by multinucleated osteoclasts is related to lipid metabolism. In this study, we generated low-density lipoprotein receptor (LDLR)/lectin-like oxidized LDL receptor-1 (LOX-1, also known as Olr1) double knockout (dKO) mice. We found that, like LDLR single KO (sKO), LDLR/LOX-1 dKO impaired cell-cell fusion of osteoclast-like cells (OCLs). LDLR/LOX-1 dKO and LDLR sKO preosteoclasts exhibited decreased uptake of LDL. The cell surface cholesterol levels of both LDLR/LOX-1 dKO and LDLR sKO osteoclasts were lower than the levels of wild-type OCLs. Additionally, the amount of phosphatidylethanolamine (PE) on the cell surface was attenuated in LDLR/LOX-1 dKO and LDLR sKO preosteoclasts, whereas the PE distribution in wild-type OCLs was concentrated on the filopodia in contact with neighboring cells. Abrogation of the ATP binding cassette G1 (ABCG1) transporter, which transfers PE to the cell surface, caused decreased PE translocation to the cell surface and subsequent cell-cell fusion. The findings of this study indicate the involvement of a novel cascade (LDLR∼ABCG1∼PE translocation to cell surface∼cell-cell fusion) in multinucleation of OCLs.

Molecular regulation of skeletal muscle mass and the contribution of nitric oxide: A review.

A variety of internal and external factors such as exercise, nutrition, inflammation, and cancer-associated cachexia affect the regulation of skeletal muscle mass. Because skeletal muscle functions as a crucial regulator of whole body metabolism, rather than just as a motor for locomotion, the enhancement and maintenance of muscle mass and function are required to maintain health and reduce the morbidity and mortality associated with diseases involving muscle wasting. Recent studies in this field have made tremendous progress; therefore, identification of the mechanisms that regulate skeletal muscle mass is necessary for the physical and nutritional management of both athletes and patients with muscle wasting disease. In this review, we present an overall picture of the interactions regulating skeletal muscle mass, particularly focusing on the insulin-like growth factor-I (IGF-I)/insulin-Akt-mammalian target of rapamycin (mTOR) pathway, skeletal muscle inactivity, and endurance and resistance exercise. We also discuss the contribution of nitric oxide (NO) to the regulation of skeletal muscle mass based on the current knowledge of the novel role of NO in these processes.

Dietary Nitrite Attenuates Elastase-Induced Pulmonary Emphysema in a Mouse Model.

Pulmonary emphysema (PE) is a major pathological feature of chronic obstructive pulmonary disease (COPD) and is characterized by proteolytic destruction of the alveolar structure and subsequent inflammation of the respiratory tract. We hypothesized that nitrite attenuates the development of PE via anti-inflammatory actions. PE was induced by intratracheal instillation of porcine pancreas elastase (PPE) in mice. Dietary nitrite dose-dependently (50 and 150 mg/L in drinking water) attenuated emphysematous development and macrophage accumulation in the alveolar parenchyma 21 d after PPE treatment. The present study shows that dietary nitrite might be a possible nutritional strategy in preventing the development of PE in mice.

The polymethoxy flavonoid sudachitin suppresses inflammatory bone destruction by directly inhibiting osteoclastogenesis due to reduced ROS production and MAPK activation in osteoclast precursors.

Inflammatory bone diseases, including rheumatoid arthritis, periodontitis and peri-implantitis, are associated not only with the production of inflammatory cytokines but also with local oxidative status, which is defined by intracellular reactive oxygen species (ROS). Osteoclast differentiation has been reported to be related to increased intracellular ROS levels in osteoclast lineage cells. Sudachitin, which is a polymethoxyflavone derived from Citrus sudachi, possesses antioxidant properties and regulates various functions in mammalian cells. However, the effects of sudachitin on inflammatory bone destruction and osteoclastogenesis remain unknown. In calvaria inflamed by a local lipopolysaccharide (LPS) injection, inflammation-induced bone destruction and the accompanying elevated expression of osteoclastogenesis-related genes were reduced by the co-administration of sudachitin and LPS. Moreover, sudachitin inhibited osteoclast formation in cultures of isolated osteoblasts and osteoclast precursors. However, sudachitin rather increased the expression of receptor activator of NF-κB ligand (RANKL), which is an important molecule triggering osteoclast differentiation, and the mRNA ratio of RANKL/osteoprotegerin that is a decoy receptor for RANKL, in the isolated osteoblasts, suggesting the presence of additional target cells. When osteoclast formation was induced from osteoclast precursors derived from bone marrow cells in the presence of soluble RANKL and macrophage colony-stimulating factor, sudachitin inhibited osteoclastogenesis without influencing cell viability. Consistently, the expression of osteoclast differentiation-related molecules including c-fos, NFATc1, cathepsin K and osteoclast fusion proteins such as DC-STAMP and Atp6v0d2 was reduced by sudachitin. In addition, sudachitin decreased activation of MAPKs such as Erk and JNK and the ROS production evoked by RANKL in osteoclast lineage cells. Our findings suggest that sudachitin is a useful agent for the treatment of anti-inflammatory bone destruction.

Protective effects of oral glutathione on fasting-induced intestinal atrophy through oxidative stress.

AIM: To determine whether oral glutathione (GSH) administration can alleviate the effects of fasting-induced intestinal atrophy in the small intestinal mucosa. METHODS: Rats were divided into eight groups. One group was fed ad libitum, another was fed ad libitum and received oral GSH, and six groups were administrated saline (SA) or GSH orally during fasting. Mucosal height, apoptosis, and cell proliferation in the jejunum were histologically evaluated. iNOS protein expression (by immunohistochemistry), nitrite levels (by high performance liquid chromatography, as a measure of NO production), 8-hydroxydeoxyguanosine formation (by ELISA, indicating ROS levels), glutathione/oxidized glutathione (GSH/GSSG) ratio (by enzymatic colorimetric detection), and γ-glutamyl transpeptidase (Ggt1) mRNA levels in the jejunum (by semi-quantitative RT-PCR) were also estimated. RESULTS: Oral GSH administration was demonstrated to drastically reduce fasting-induced intestinal atrophy in the jejunum. In particular, jejunal mucosal height was enhanced in GSH-treated animals compared to SA-treated animals [527.2 ± 6.9 for 50 mg/kg GSH, 567.6 ± 5.4 for 500 mg/kg GSH vs 483.1 ± 4.9 (µm), P < 0.01 at 72 h]. This effect was consistent with decreasing changes in GSH-treated animals compared to SA-treated animals for iNOS protein staining [0.337 ± 0.016 for 50 mg/kg GSH, 0.317 ± 0.017 for 500 mg/kg GSH vs 0.430 ± 0.023 (area of staining part/area of tissue), P < 0.01 at 72 h] and NO [2.99 ± 0.29 for 50 mg/kg GSH, 2.88 ± 0.19 for 500 mg/kg GSH vs 5.34 ± 0.35 (nmol/g tissue), P < 0.01 at 72 h] and ROS [3.92 ± 0.46 for 50 mg/kg GSH, 4.58 ± 0.29 for 500 mg/kg GSH vs 6.42 ± 0.52 (8-OHdG pg/µg DNA), P < 0.01, P < 0.05 at 72 h, respectively] levels as apoptosis mediators in the jejunum. Furthermore, oral GSH administration attenuated cell proliferation decreases in the fasting jejunum [182.5 ± 1.9 for 500 mg/kg GSH vs 155.8 ± 3.4 (5-BrdU positive cells/10 crypts), P < 0.01 at 72 h]. Notably, both GSH concentration and Ggt1 mRNA expression in the jejunum were also attenuated in rats following oral administration of GSH during fasting as compared with fasting alone [0.45 ± 0.12 vs 0.97 ± 0.06 (nmol/mg tissue), P < 0.01; 1.01 ± 0.11 vs 2.79 ± 0.39 (Ggt1 mRNA/Gapdh mRNA), P < 0.01 for 500 mg/kg GSH at 48 h, respectively]. CONCLUSION: Oral GSH administration during fasting enhances jejunal regenerative potential to minimize intestinal mucosal atrophy by diminishing fasting-mediated ROS generation and enterocyte apoptosis and enhancing cell proliferation.

Dietary nitrite supplementation attenuates cardiac remodeling in l-NAME-induced hypertensive rats.

Loss of nitric oxide (NO) bioavailability underlies the development of hypertensive heart disease. We investigated the effects of dietary nitrite on NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Sprague-Dawley rats were divided into five groups: an untreated control group, an l-NAME-treated group, and three other l-NAME-treated groups supplemented with 10 mg/L or 100 mg/L of nitrite or 100 mg/L of captopril in drinking water. After the 8-week experimental period, mean arterial blood pressure was measured, followed by sampling of blood and heart tissue for assessment of nitrite/nitrate levels in the plasma and heart, the plasma level of angiotensin II (AT II), and the heart transcriptional levels of AT II type 1 receptor (AT1R), transforming growth factor-ß1 (TGF-ß1), and connective tissue proteins such as type 1 collagen and fibronectin. Heart tissue was analyzed by histopathological morphometry, including assessments of ventricular and coronary vascular hypertrophy and fibrosis, as well as immunohistochemistry analyses of myocardial expression of AT1R. l-NAME treatment reduced the plasma nitrate level and led to the development of hypertension, with increased plasma levels of AT II and increased heart transcriptional levels of AT1R and TGF-ß1-mediated connective tissue proteins, showing myocardial and coronary arteriolar hypertrophy and fibrosis. However, dietary nitrite supplementation inhibited TGF-ß1-mediated cardiac remodeling by suppressing AT II and AT1R. These results suggest that dietary nitrite levels achievable via a daily high-vegetable diet could improve hypertensive heart disease by inhibiting AT II-AT1R-mediated cardiac remodeling.

Dietary nitrite reverses features of postmenopausal metabolic syndrome induced by high-fat diet and ovariectomy in mice.

Menopausal women are at greater risk of developing metabolic syndrome with reduced endothelial nitric oxide synthase (eNOS) activity. Hormone replacement therapy increases eNOS activity and normalizes some characteristics of metabolic syndrome. We hypothesized that nitric oxide (NO) supplementation should have a therapeutic effect on this syndrome. We examined the effect of dietary nitrite in a mouse model with postmenopausal metabolic syndrome induced by ovariectomy (OVX) and a high fat diet (HF). C57BL/6 female mice were divided into five groups, sham+normal fat diet (NF), sham+ HF, OVX+HF with or without sodium nitrite (50 mg and 150 mg/l) in the drinking water. Daily food intake and weekly body weight were monitored for 18 wk. OVX and HF significantly reduced plasma levels of nitrate/nitrite (NOx), and mice developed obesity with visceral hypertrophic adipocytes and increased transcriptional levels of monocyte chemoattractant protein-1, TNF-α, and IL-6 in visceral fat tissues. The proinflammatory state in the adipocytes provoked severe hepatosteatosis and insulin resistance in OVX+HF group compared with sham+NF group. However, dietary nitrite significantly suppressed adipocyte hypertrophy and transcriptions of proinflammatory cytokines in visceral fat in a dose-dependent manner. The improvement of visceral inflammatory state consequently reversed the hepatosteatosis and insulin resistance observed in OVX+HF mice. These results suggest that an endogenous NO defect might underlie postmenopausal metabolic syndrome and that dietary nitrite provides an alternative source of NO, subsequently compensating for metabolic impairments of this syndrome.

Inducible and neuronal nitric oxide synthases exert contrasting effects during rat intestinal recovery following fasting.

We investigated the effects of endogenous inducible (iNOS) and neuronal nitric oxide synthase on recovery from intestinal mucosal atrophy caused by fasting-induced apoptosis and decreased cell proliferation during refeeding in rats. Rats were divided into five groups, one of which was fed ad libitum, and four of which underwent 72 h of fasting, followed by refeeding for 0, 6, 24, and 48 h, respectively. iNOS and neuronal nitric oxide synthase mRNA and protein levels in jejunal tissues were measured, and mucosal height was histologically evaluated. Apoptotic indices, interferon-γ (IFN-γ) transcription levels, nitrite levels (as a measure of nitric oxide [NO] production),8-hydroxydeoxyguanosine formation (indicating reactive oxygen species [ROS] levels), crypt cell proliferation, and the motility indices (MI) were also estimated. Associations between mucosal height and NOS protein levels were determined using Spearman's rank correlation test. Notably, we observed significant increases in mucosal height and in neuronal nitric oxide synthase mRNA and protein expression as refeeding time increased. Indeed, there was a significant positive correlation between neuronal nitric oxide synthase protein level and mucosal height during the 48-h refeeding period ( r = 0.725, P < 0.01). Conversely, iNOS mRNA and protein expression decreased according to refeeding time, with a significant negative correlation between iNOS protein level and mucosal height being recorded during the 48-h refeeding period ( r = -0.898, P < 0.01). We also noted a significant negative correlation between jejunal neuronal nitric oxide synthase and iNOS protein concentrations over this same period ( r = -0.734, P < 0.01). Refeeding also restored the decreased jejunal MI caused by fasting. Our finding suggests that refeeding likely repairs fasting-induced jejunal atrophy by suppressing iNOS expression and subsequently inhibiting NO, ROS, and IFN-γ as apoptosis mediators, and by promoting neuronal nitric oxide synthase production and inducing crypt cell proliferation via mechanical stimulation. Impact statement Besides providing new data confirming the involvement of iNOS and nNOS in intestinal mucosal atrophy caused by fasting, this study details their expression and function during recovery from this condition following refeeding. We demonstrate a significant negative correlation between iNOS and nNOS levels during refeeding, and associate this with cell proliferation and apoptosis in crypts and villi. These novel findings elucidate the relationship between these NOS isoforms and its impact on recovery from intestinal injury. A mechanism is proposed comprising the up-regulation of nNOS activity by mechanical stimulation due to the presence of food in the intestine, restricting iNOS-associated apoptosis and promoting cell proliferation and gut motility. Our investigation sheds light on the molecular basis behind the repercussions of total parenteral nutrition on intestinal mucosal integrity, and more importantly, the beneficial effects of early enteral feeding.

Lectin-like oxidized low-density lipoprotein receptor-1 abrogation causes resistance to inflammatory bone destruction in mice, despite promoting osteoclastogenesis in the steady state.

Inflammatory bone diseases have been attributed to increased bone resorption by augmented and activated bone-resorbing osteoclasts in response to inflammation. Although the production of diverse proinflammatory cytokines is induced at the inflamed sites, the inflammation also generates reactive oxygen species that modify many biological compounds, including lipids. Among the oxidized low-density lipoprotein (LDL) receptors, lectin-like oxidized LDL receptor-1 (LOX-1), which is a key molecule in the pathogenesis of multifactorial inflammatory atherosclerosis, was downregulated with osteoclast differentiation. Here, we demonstrate that LOX-1 negatively regulates osteoclast differentiation by basically suppressing the cell-cell fusion of preosteoclasts. The LOX-1-deleted (LOX-1(-/-)) mice consistently decreased the trabecular bone mass because of elevated bone resorption during the growing phase. In contrast, when the calvaria was inflamed by a local lipopolysaccharide-injection, the inflammation-induced bone destruction accompanied by the elevated expression of osteoclastogenesis-related genes was reduced by LOX-1 deficiency. Moreover, the expression of receptor activator of NF-κB ligand (RANKL), a trigger molecule for osteoclast differentiation, evoked by the inflammation was also abrogated in the LOX-1(-/-) mice. Osteoblasts, the major producers of RANKL, also expressed LOX-1 in response to proinflammatory agents, interleukin-1ß and prostaglandin E2. In the co-culture of LOX-1(-/-) osteoblasts and wild-type osteoclast precursors, the osteoclastogenesis induced by interleukin-1ß and prostaglandin E2 decreased; this process occurred in parallel with the downregulation of osteoblastic RANKL expression. Collectively, LOX-1 abrogation results in resistance to inflammatory bone destruction, despite promoting osteoclastogenesis in the steady state. Our findings indicate the novel involvement of LOX-1 in physiological bone homeostasis and inflammatory bone diseases.

Dietary nitrite supplementation improves insulin resistance in type 2 diabetic KKA(y) mice.

BACKGROUND: Because insulin signaling is essential for endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production, the loss of bioavailable NO might be a common molecular mechanism underlying the development of insulin resistance and endothelial dysfunction. Although dietary nitrite acts as a substrate for systemic NO generation, thereby serving as a physiological alternative source of NO for signaling, it is not precisely known how dietary nitrite affects type 2 diabetes mellitus. Here we report the therapeutic effects of dietary nitrite on the metabolic and histological features of KKA(y) diabetic mice. METHODS: KKA(y) mice were divided into three groups (without nitrite, and with 50 mg/L and 150 mg/L nitrite in drinking water), and two groups of C57BL/6J mice served as controls (without nitrite and with 150 mg/L nitrite in drinking water). After 10 weeks, blood samples, visceral adipose tissues, and gastrocnemius muscles were collected after a 16-hour fast to assess the homeostasis model assessment of insulin resistance (HOMA-IR) levels, the histology of the adipose tissue, insulin-stimulated sequential signaling to glucose transporter 4 (GLUT4), and nitrite and nitrate contents in the muscle using an HPLC system. RESULTS: KKA(y) mice developed obesity with enhanced fasting plasma levels of glucose and insulin and exhibited increased HOMA-IR scores compared with the C57BL/6J control mice. Dietary nitrite dose-dependently reduced the size of the hypertrophic adipocytes and TNF-α transcription in the adipose tissue of KKA(y) diabetic mice, which also restored the insulin-mediated signal transduction, including p85 and Akt phosphorylation, and subsequently restored the GLUT4 expression in the skeletal muscles. CONCLUSIONS: These results suggest that dietary nitrite provides an alternative source of NO, and subsequently improves the insulin-mediated signaling and the metabolic and histological features in KKA(y) diabetic mice.

Osteocytes produce interferon-ß as a negative regulator of osteoclastogenesis.

Osteoclastogenesis is controlled by osteocytes; osteocytic osteoclastogenesis regulatory molecules are largely unknown. We searched for such factors using newly developed culture methods. Our culture system mimics the three-dimensional cellular structure of bone, consisting of collagen gel-embedded osteocytic MLO-Y4 cells, stromal ST2 cells on the gel as bone lining cells, and bone marrow cells. The gel-embedded MLO-Y4 cells inhibited the osteoclastogenesis induced by 1,25(OH)2D3 without modulating receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) production by ST2 cells, despite MLO-Y4 cells supported osteoclastogenesis in the absence of ST2 cells. In the bone marrow cell culture, the conditioned medium from MLO-Y4 cells decreased the capability of osteoclastic differentiation from the cells induced by macrophage colony-stimulating factor. This decreased capability was concomitant with an increase in protein kinase R mRNA expression and an inhibition of c-Fos translation. These changes were partially normalized by the simultaneous addition of an anti-interferon (IFN)-ß neutralizing antibody to MLO-Y4 cell conditioned medium. To study primary osteocytes, we prepared non-osteocytic cell-free osteocyte-enriched bone fragments (OEBFs). When osteoclast precursors were induced by macrophage colony-stimulating factor in the presence of OEBFs, the generated cells exhibited a diminished capacity for osteoclastogenesis. OEBFs prepared from OPG-knock-out mice exhibited a similar effect, indicating OPG-independent inhibition. The addition of anti-IFN-ß neutralizing antibody during the co-culture with OEBFs partially recovered the osteoclastogenic potential of the generated cells. The MLO-Y4 cells and OEBFs expressed IFN-ß mRNA. Although osteocytic RANKL is known to be important for osteoclastogenesis, our data suggest that osteocytes also produce IFN-ß as an inhibitor of osteoclastogenesis.

Low-density lipoprotein receptor deficiency causes impaired osteoclastogenesis and increased bone mass in mice because of defect in osteoclastic cell-cell fusion.

Osteoporosis is associated with both atherosclerosis and vascular calcification attributed to hyperlipidemia. However, the cellular and molecular mechanisms explaining the parallel progression of these diseases remain unclear. Here, we used low-density lipoprotein receptor knockout (LDLR(-/-)) mice to elucidate the role of LDLR in regulating the differentiation of osteoclasts, which are responsible for bone resorption. Culturing wild-type osteoclast precursors in medium containing LDL-depleted serum decreased receptor activator of NF-κB ligand (RANKL)-induced osteoclast formation, and this defect was additively rescued by simultaneous treatment with native and oxidized LDLs. Osteoclast precursors constitutively expressed LDLR in a RANKL-independent manner. Osteoclast formation from LDLR(-/-) osteoclast precursors was delayed, and the multinucleated cells formed in culture were smaller and contained fewer nuclei than wild-type cells, implying impaired cell-cell fusion. Despite these findings, RANK signaling, including the activation of Erk and Akt, was normal in LDLR(-/-) preosteoclasts, and RANKL-induced expression of NFATc1 (a master regulator of osteoclastogenesis), cathepsin K, and tartrate-resistant acid phosphatase was equivalent in LDLR-null and wild-type cells. In contrast, the amounts of the osteoclast fusion-related proteins v-ATPase V(0) subunit d2 and dendritic cell-specific transmembrane protein in LDLR(-/-) plasma membranes were reduced when compared with the wild type, suggesting a correlation with impaired cell-cell fusion, which occurs on the plasma membrane. LDLR(-/-) mice consistently exhibited increased bone mass in vivo. This change was accompanied by decreases in bone resorption parameters, with no changes in bone formation parameters. These findings provide a novel mechanism for osteoclast differentiation and improve the understanding of the correlation between osteoclast formation and lipids.

Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein.

Although extensive studies have done much to clarify the molecular mechanisms of osteoclastogenesis during the last ten years, there may still be unknown molecules associated with osteoclast differentiation. Thus, we used fluorescent differential display to screen for genes whose expression is induced by receptor activator of NF-κB ligand (RANKL), a crucial molecule for osteoclast formation. We identified caveolin-1 (Cav-1) as a RANKL-induced gene. Cav-1 is a major structural protein of caveolae and lipid rafts, cholesterol-enriched microdomains in the plasma membrane (PM). The RANKL-induced Cav-1 was immediately conveyed to lipid rafts. Conversely, expression of flotillin-1 (Flot-1), another scaffolding protein of lipid rafts, was reduced during osteoclastogenesis, indicating conversion of Flot-1-predominant rafts into Cav-1-enriched rafts. However, in vitro osteoclastogenesis of precursor cells from Cav-1-null mice was comparable to that of wild-type mice, while Cav-2 expression in the knockout osteoclasts was maintained. Conversely, Cav-2 gene silencing in Cav-1-null osteoclast precursors using siRNA for Cav-2 increased osteoclast formation, suggesting that the Cav-1/Cav-2 complex may act as a negative regulator for osteoclastogenesis. On the other hand, destruction of lipid rafts by removal of cholesterol from the PM by methyl-ß-cyclodextrin (MCD) treatment caused disordered signal transductions for osteoclastogenesis, such as hyperactivation of Erk1/2 and insensitivity of Akt to RANKL stimulus. The abnormal signaling was reproduced by deleting exogenous lipoproteins from the culture medium, which also resulted in reduced osteoclast formation. In addition, the deletion caused delayed expression of nuclear factor of activated T cells c1 (NFATc1), and depressed its activation in the cytosol and inhibited its translocation into nuclei. Simultaneously, the deletion reduced the level of FcRγ, a trigger protein for initiating the calcium signaling needed to activate NFATc1, and decreased Cav-1 in lipid rafts. These findings indicate that the molecular mechanisms of osteoclastogenesis are highly dependent on extracellular lipoprotein and the integrity of lipid rafts, and suggest possible involvement of cholesterol.

[Age-, period-, and birth-cohort-specific effects on the male proportion in Japanese newborns and projections for male proportion for 20 periods (2008-2027)].

OBJECTIVES: To determine the age-, period-, and cohort-specific effects on the male proportion in Japanese newborns, we performed an age-period-cohort (APC) analysis in this study. In addition, projections for the male proportion were analyzed. METHODS: We obtained data on live births of newborns for Japanese women in 1947-2007 from the National Vital Statistics. Cohort tables containing data on the male proportion were analyzed using a Bayesian APC model. Projections of the male proportion (2008-2027) were calculated. RESULTS: The age effect decreased when the mothers were 40-44 years old; however, the effect was relatively limited as compared with the period and cohort effects. The period effect increased from 1947 to 1969 and decreased thereafter. Analysis of the cohort effect on male proportion trends revealed a decreasing slope for birth cohorts born between 1905 and 1945 and a subsequent increase after 1958. The projections for male proportion indicated that the male proportion in 2027 would be similar to that in the 1970s. CONCLUSIONS: The age of the mother hardly affected the male proportion. The period effect started decreasing from the latter half of the 1960s. This may be attributable to the high economic growth since 1965 that promoted industrial development that led to environmental pollution, which in turn may have lead to the deterioration of the intrauterine environment. Cohort effects changed from 1958 and exhibited trends toward increase in male proportion; this may be due to improvements in obstetric care. Our results suggest that the male proportion in Japanese newborns will increase in the future.

Fasting-induced intestinal apoptosis is mediated by inducible nitric oxide synthase and interferon-{gamma} in rat.

Nitric oxide (NO) is associated with intestinal apoptosis in health and disease. This study aimed to investigate the role of intestinal NO in the regulation of apoptosis during fasting in rats. Male Wistar rats were divided into two groups and subcutaneously injected with saline (SA) or aminoguanidine (AG), followed by fasting for 24, 48, 60, and 72 h. At each time point, the jejunum was subjected to histological evaluation for enterocyte apoptosis by histomorphometric assessment and TUNEL analysis. We performed immunohistochemistry for inducible NO synthase (iNOS) expression in the jejunum and measured tissue nitrite levels using HPLC and 8-hydroxydeoxyguanosine adduct using ELISA, indicative of endogenous NO production and reactive oxygen species (ROS) production, respectively. Jejunal transcriptional levels of iNOS, neuronal NO synthase (nNOS), and interferon-gamma (IFN-gamma) were also determined by RT-PCR. Fasting caused significant jejunal mucosal atrophy due to attenuated cell proliferation and enhanced apoptosis with increase in iNOS transcription, its protein expression in intestinal epithelial cells (IEC), and jejunal nitrite levels. However, AG treatment histologically reduced apoptosis with inhibition of fasting-induced iNOS transcription, protein expression, and nitrite production. We also observed fasting-induced ROS production and subsequent IFN-gamma transcription, which were all inhibited by AG treatment. Furthermore, we observed reduced transcriptional levels of nNOS, known to suppress iNOS activation physiologically. These results suggest that fasting-induced iNOS activation in IEC may induce apoptosis mediators such as IFN-gamma via a ROS-mediated mechanism and also a possible role of nNOS in the regulation of iNOS activity in fasting-induced apoptosis.

Oral nitrite ameliorates dextran sulfate sodium-induced acute experimental colitis in mice.

Inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis are chronic inflammatory disorders of the intestinal tract with excessive production of cytokines, adhesion molecules, and reactive oxygen species. Although nitric oxide (NO) is reported to be involved in the onset and progression of IBDs, it remains controversial as to whether NO is toxic or protective in experimental colitis. We investigated the effects of oral nitrite as a NO donor on dextran sulfate sodium (DSS)-induced acute colitis in mice. Mice were fed DSS in their drinking water with or without nitrite for up to 7days. The severity of colitis was assessed by disease activity index (DAI) observed over the experimental period, as well as by the other parameters, including colon lengths, hematocrit levels, and histological scores at day 7. DSS treatment induced severe colitis by day 7 with exacerbation in DAI and histological scores. We first observed a significant decrease in colonic nitrite levels and increase in colonic TNF-alpha expression at day 3 after DSS treatment, followed by increased colonic myeloperoxidase (MPO) activity and increased colonic expressions of both inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) at day 7. Oral nitrite supplementation to colitis mice reversed colonic nitrite levels and TNF-alpha expression to that of normal control mice at day 3, resulting in the reduction of MPO activity as well as iNOS and HO-1 expressions in colonic tissues with clinical and histological improvements at day 7. These results suggest that oral nitrite inhibits inflammatory process of DSS-induced experimental colitis by supplying nitrite-derived NO instead of impaired colonic NOS activity.