Polyunsaturated fatty acid deficiency affects sulfatides and other sulfated glycans in lysosomes through autophagy-mediated degradation.

Metabolic changes in sulfatides and other sulfated glycans have been related to various diseases, including Alzheimer's disease (AD). However, the importance of polyunsaturated fatty acids (PUFA) in sulfated lysosomal substrate metabolism and its related disorders is currently unknown. We investigated the effects of deficiency or supplementation of PUFA on the metabolism of sulfatides and sulfated glycosaminoglycans (sGAGs) in sulfatide-rich organs (brain and kidney) of mice. A PUFA-deficient diet for over 5 weeks significantly reduced the sulfatide expression by increasing the sulfatide degradative enzymes arylsulfatase A and galactosylceramidase in brain and kidney. This sulfatide degradation was clearly associated with the activation of autophagy and lysosomal hyperfunction, the former of which was induced by suppression of the Erk/mTOR pathway. A PUFA-deficient diet also activated the degradation of sGAGs in the brain and kidney and that of amyloid precursor proteins in the brain, indicating an involvement in general lysosomal function and the early developmental process of AD. PUFA supplementation prevented all of the above abnormalities. Taken together, a PUFA deficiency might lead to sulfatide and sGAG degradation associated with autophagy activation and general lysosomal hyperfunction and play a role in many types of disease development, suggesting a possible benefit of prophylactic PUFA supplementation.

Daily Ingestion of Eggplant Powder Improves Blood Pressure and Psychological State in Stressed Individuals: A Randomized Placebo-Controlled Study.

Eggplant (Solanum melongena) is a globally popular vegetable and its significant health effect has not been reported in randomized controlled trials. Recently, we reported that eggplant was rich in choline esters, including acetylcholine (ACh), and had an antihypertensive effect in spontaneously hypertensive rats. Here, we evaluated the effects of a continuous intake of eggplant powder on blood pressure (BP), stress, and psychological state (PS) in 100 stressed participants with normal-high BP or grade 1 hypertension in a randomized, double-blind, placebo-controlled, parallel-group comparative study. The participants were randomly assigned to the eggplant or placebo group. Participants in the eggplant group ingested capsules containing eggplant powder (1.2 g/day; 2.3 mg of ACh/day) for 12 weeks, whereas participants in the placebo group ingested placebo capsules. The primary outcome assessed was hospital BP. Secondary outcomes were stress and PS. Eggplant powder intake significantly decreased the hospital diastolic blood pressure (DBP) at week 8 overall and in the normal-high BP group, and the systolic blood pressure (SBP) and DBP at week 12 overall and in the grade 1 hypertension group, compared to those of the placebo group. It also improved negative PSs at week 8 or 12 in the normal-high BP group. This is the first evidence of the BP- and PS-improving effects of eggplant intake in humans. The functional substance responsible for the effects was estimated to be eggplant-derived choline ester, namely ACh.

Preventive effect of Eucommia leaf extract on aortic media hypertrophy in Wistar-Kyoto rats fed a high-fat diet.

Eucommia ulmoides Oliver leaf extract (ELE) has been shown to have anti-hypertensive and anti-obesity effects in rats that are fed a high-fat diet (HFD). To explore the effects of chronic administration of ELE on body weight, blood pressure and aortic media thickness, 7-week-old male Wistar-Kyoto (WKY) rats were orally administered a normal diet, a 30% HFD, or a 5% ELE plus HFD ad libitum for 10 weeks. The HFD treatment caused mild obesity and hypertension in the normotensive rats, while rats receiving both ELE and the HFD had significantly lower body weights, less visceral and perirenal fat, lower blood pressure and thinner aortic media than the control rats receiving the HFD only. The plasma adiponectin/leptin ratio also improved in ELE-treated rats. Although plasma leptin levels were elevated in all HFD rats, adiponectin levels increased only in the ELE-treated rats. Anti-hypertensive and anti-obesity effects may be caused by the geniposidic acid (GEA) and/or asperuloside present in ELE. These findings suggest that chronic ELE administration prevents aortic media hypertrophy in early-stage obesity with hypertension. Long-term administration of ELE might inhibit the development of arteriosclerosis.

The Restorative Effects of Eucommia ulmoides Oliver Leaf Extract on Vascular Function in Spontaneously Hypertensive Rats.

Eucommia ulmoides Oliv. leaf is a traditional Chinese antihypertensive and antidiabetic medicine. We examined the effects of chronic Eucommia leaf extract (ELE) administration on artery function and morphology in spontaneously hypertensive rats (SHRs). ELE was orally administered via normal diet ad libitum to six-week-old male SHRs at a concentration of 5% for seven weeks. Acetylcholine (ACh)-induced endothelium-dependent relaxation, sodium nitroprusside (SNP)-induced endothelium-independent relaxation, plasma nitric oxide (NO) levels, and media thickness were assessed. ELE significantly improved ACh-induced aortic endothelium-dependent relaxation but did not affect SNP-induced endothelium-independent relaxation in the SHRs, as compared to the animals receiving normal diet. Plasma NO levels and media thickness were significantly increased and decreased, respectively, in the ELE-treated SHRs. Therefore, long-term ELE administration may effectively improve vascular function by increasing plasma NO levels and bioavailability, and by preventing vascular hypertrophy in the SHR aorta.

Blood pressure-lowering peptides from neo-fermented buckwheat sprouts: a new approach to estimating ACE-inhibitory activity.

Neo-fermented buckwheat sprouts (neo-FBS) contain angiotensin-converting enzyme (ACE) inhibitors and vasodilators with blood pressure-lowering (BPL) properties in spontaneously hypertensive rats (SHRs). In this study, we investigated antihypertensive mechanisms of six BPL peptides isolated from neo-FBS (FBPs) by a vasorelaxation assay and conventional in vitro, in vivo, and a new ex vivo ACE inhibitory assays. Some FBPs demonstrated moderate endothelium-dependent vasorelaxation in SHR thoracic aorta and all FBPs mildly inhibited ACE in vitro. Orally administered FBPs strongly inhibited ACE in SHR tissues. To investigate detailed ACE-inhibitory mechanism of FBPs in living body tissues, we performed the ex vivo assay by using endothelium-denuded thoracic aorta rings isolated from SHRs, which demonstrated that FBPs at low concentration effectively inhibited ACE in thoracic aorta tissue and suppressed angiotensin II-mediated vasoconstriction directly associated with BPL. These results indicate that the main BPL mechanism of FBP was ACE inhibition in living body tissues, suggesting that high FBP's bioavailability including absorption, tissue affinity, and tissue accumulation was responsible for the superior ACE inhibition in vivo. We propose that our ex vivo assay is an efficient and reliable method for evaluating ACE-inhibitory mechanism responsible for BPL activity in vivo.

Purification and identification of antihypertensive peptides from fermented buckwheat sprouts.

Buckwheat (Fagopyrum esculentum) is rich in antihypertensive compounds. This study investigated the effect of lactic-fermented buckwheat sprouts (neo-FBS) on level, identification, and potency of blood pressure-lowering (BPL) compounds. A single oral dose of 1.0 mg/kg body weight buckwheat sprouts (BS) in spontaneously hypertensive rats did not show significant BPL activity, whereas neo-FBS significantly decreased blood pressure. HPLC of neo-FBS identified two peaks absent in the profile of BS. The peak exhibiting potent BPL activity was fractionated, and six peptides (DVWY, FDART, FQ, VAE, VVG, and WTFR) and tyrosine were identified by LC-MS/MS and Edman degradation. Single oral dose administration of the peptides revealed significant BPL effect of all the peptides, with the most potent being DVWY, FQ, and VVG. DVWY, VAE, and WTFR are novel. This study demonstrates that lactic fermentation of BS produces new, highly potent antihypertensive peptides and increases active compounds GABA and tyrosine already present in BS.

Changes in serum levels of biochemical markers of bone turnover during 10 years among Japanese men and women: associated factors and birth-cohort effect. The Taiji Study.

We aimed to clarify changes in biochemical markers of bone turnover (BTMs) over 10 years, associations with changes in bone mineral density (BMD), and birth-cohort effects in a Japanese community. We randomly selected 400 individuals (age, 40-79 years; 50 of each gender and age stratum) from a list of registered residents in 1993. We measured BMD of the spine and hip, and serum concentrations of total osteocalcin (OC), beta-C-terminal cross-linking telopeptide of type I collagen (beta-CTX), and N-terminal cross-linking telopeptide of type I collagen (NTX), in 1993 and 2003. Of the 400 subjects, 322 (153 men, 169 women) completed the 10-year follow-up. Mean change rates (standard deviation) for serum total OC, beta-CTX, and NTX over 10 years were -1.00 (3.74)%/year, 5.10 (22.48)%/year, and 0.40 (3.41)%/year, respectively, in men, and 0.02 (5.32)%/year, 5.53 (14.54)%/year, and 0.62 (3.26)%/year, respectively, in women. Change rates of BTMs were higher for women in their forties than for women in their fifties to seventies (P < 0.05), and higher in the menstrual transition group than in pre- and postmenopausal groups (P < 0.001). Changes in levels of BTMs over 10 years in women were significantly associated with change rates of BMDs at L2-L4 and total hip after adjusting for potential confounders. A significant birth-cohort effect was observed among women in their fifties. We concluded that change rates of BTMs during the 10 years were influenced by menstrual transition, age, and sex and associated with bone loss at L2-L4 and total hip.

Biochemical markers of bone turnover as predictors of osteoporosis and osteoporotic fractures in men and women: 10-year follow-up of the Taiji cohort.

We aimed to assess the capacity of biochemical markers of bone turnover (BTMs) to predict bone loss, osteoporosis (OP), and osteoporotic fractures. We randomly selected 400 individuals (age 40-79 years in 1993; 50 of each gender and age stratum) from a list of registered residents. In the years 1993, 1996, 2000, and 2003, bone mineral density (BMD) of the spine and hip were measured by dual-energy X-ray absorptiometry. The BTMs assessed at baseline were serum intact osteocalcin (OC), total OC, bone-specific alkaline phosphatase, C-terminal propeptide of type I procollagen, N-terminal propeptide of type I procollagen (PINP), C-terminal cross-linking telopeptide of type I collagen generated by matrix metalloproteinase, C-terminal cross-linking telopeptide of type I collagen (beta-CTX), N-terminal cross-linking telopeptide of type I collagen (NTX), urinary pyridinoline, and deoxypyridinoline (DPD). For 307 completers, multivariate analysis after adjusting for confounders revealed that serum PINP levels in men [hazard ratio (HR) 2.80, P < 0.05] and serum PINP (HR 1.65, P < 0.05), beta-CTX (HR 1.80, P < 0.001), NTX (HR 1.96, P < 0.01), and urinary DPD levels (HR 1.40, P < 0.05) in women were significantly related to the occurrence of spinal OP. In addition to adjustment for the baseline status of BMD, i.e., osteopenia or normal range, PINP, beta-CTX, and NTX in women could significantly predict the future occurrence of spinal OP. BTMs were not significant predictors of bone loss, femoral OP, or osteoporotic fractures. In conclusion, various BTMs in women can predict the occurrence of spinal OP.

Capacity of endogenous sex steroids to predict bone loss in Japanese men: 10-year follow-up of the Taiji Cohort Study.

This prospective cohort study aimed to evaluate the capacity of endogenous sex steroids to predict male osteoporosis (OP) among community-dwelling inhabitants. Among 1,028 male residents aged 40-79 years, 50 men belonging to each age stratum (200 in total) were randomly selected from a resident registration list. In the years 1993, 1996, 2000, and 2003, bone mineral density (BMD) of the lumbar spine and proximal femur was measured by dual-energy X-ray absorptiometry. Serum total estradiol (E(2)) and free testosterone (FT) were measured using samples extracted in 1993. Among the 200 participants at baseline, 153 subjects completed 10-year follow-ups. Mean values of serum E(2) and FT were 22.4 and 9.4 pg/ml, respectively. Rates of change for BMD at the lumbar spine and femoral neck were 0.8% and 0.5% during the first 3 years, 0.0% and 0.5% during 7 years, and 0.8% and -0.3% over 10 years, respectively. According to multivariate regression analysis after adjusting for age and body mass index, mean values of FT were significantly related to the rate of change of BMD at the femoral neck at 3 years (beta = 0.21; r (2) = 0.05; P < 0.01), but not at 7 or 10 years. Serum FT level could offer a useful predictor of bone loss within 3 years.

Phenolic compounds responsible for the superoxide dismutase-like activity in high-Brix apple vinegar.

High-Brix apple vinegar (HBAV) with palatable drinking qualities has been developed using a greater amount of apple ingredients. In HBAV and in regular apple vinegar (RAV), constituents of 4 kinds of organic acids, 20 kinds of amino acids, 3 kinds of sugars, 4 kinds of minerals, and phenols were determined. These constituents, except for acetic acid, in HBAV are of higher abundance than in RAV. HBAV had a 7.1 times greater superoxide dismutase (SOD)-like activity compared with RAV. Those constituents, except for phenols, had very low SOD-like activity, and total phenol levels in HBAV were comparable to 181 mg of gallic acid equivalents/100 mL, which was 6.0 times more abundant than in RAV. Nine kinds of phenols including two kinds of hydroxycinnamates, two kinds of hydroxybenzoates, and five kinds of hydroxycinnamoyl quinates, originating from raw material were determined, but there were no ascorbic acid and flavonoids in HBAV. Chlorogenic acid, 4-p-coumaroylquinic acid, and caffeic acid were the three major phenols, and their content levels were 19.6, 13.5, and 0.76 mg in 100 mL of HBAV, respectively. Sum of contents of chlorogenic acid and the isomers was 24.0 mg/100 mL, and the percentage was 56.9% in the total identified phenols in HBAV. In RAV, only chlorogenic acid was determined as phenols, and the content was 3.1 mg/100 mL. SOD-like activities of the constituents of HBAV were obtained through high-accuracy assays using vinegar reconstitutions, and each contribution to the total SOD-like activity was found. As a result, 77.2% for all SOD-like activity of HBAV was reconstituted using the determined nine phenols and other constituents. Chlorogenic acids were the most effective, and the contribution to the total activity was 41.7%. The most abundant phenols, chlorogenic acids, were the most important contributors to the SOD-like activity. These SOD-active phenols originated from raw material and remained through the acetic acid fermentation processes. In the fermentation process of HBAV, the active constituents were well maintained, providing an advantage in the production of a phenol-rich product.

Identification of oxytetracycline as a chondrogenic compound using a cell-based screening system.

To effectively treat degenerative joint diseases including osteoarthritis (OA), small chemical compounds need to be developed that can potently induce chondrogenic differentiation without promoting terminal differentiation. For this purpose, we screened natural and synthetic compound libraries using a Col2GFP-ATDC5 system and identified oxytetracycline (Oxy) as a chondrogenic compound. Oxy induced cartilaginous matrix synthesis and mRNA expressions of chondrocyte markers in ATDC5 cells. In addition, Oxy suppressed mineralization and mRNA expressions of terminal chondrocyte differentiation markers in ATDC5 cells, primary chondrocytes, and cultured metatarsal bones. Oxy's induction of Col2 mRNA expression was decreased by the addition of Noggin and was increased by the addition of BMP2. Furthermore, Oxy increased mRNA expression of Id1, Bmp2, Bmp4, and Bmp6. These data suggest that Oxy induces chondrogenic differentiation in a BMP-dependent manner and suppresses terminal differentiation. Oxy may be useful for treatment of OA and also for regeneration of cartilage tissue.

Orphan receptor tyrosine kinase ROR2 as a potential therapeutic target for osteosarcoma.

Osteosarcoma is the most prevalent bone malignant tumor in children and adolescents, and displays heterogeneous histology and high propensity for distant metastasis. Although adjuvant chemotherapy remarkably improved treatment outcome over the past few decades, prognosis for osteosarcoma patients with pulmonary metastasis is still unsatisfactory. To identify novel therapeutic targets for osteosarcoma, we investigated the gene expression profile of osteosarcomas by cDNA microarray analysis and found transactivation of receptor tyrosine kinase-like orphan receptor 2 (ROR2) expression in the majority of osteosarcoma samples. Treatment of osteosarcoma cell lines with siRNA against ROR2 significantly inhibited cell proliferation and migration. We also identified wingless-type MMTV integration site family, member 5B (WNT5B) as a putative ROR2 ligand and that the physiological interaction of WNT5B and ROR2 could enhance cell migration, indicating the possible roles of ROR2 and WNT5B in the metastatic property of osteosarcoma cells. Taken together, our findings revealed that the WNT5B/ROR2 signaling pathway is a promising therapeutic target for osteosarcoma.

Diet and lifestyle associated with increased bone mineral density: cross-sectional study of Japanese elderly women at an osteoporosis outpatient clinic.

BACKGROUND: Several studies have already demonstrated that lifestyle characteristics, such as physical activity, smoking, and alcohol intake, are associated with bone mineral density (BMD). Coffee intake was shown to be negatively associated with BMD, whereas tea drinking was reported to be associated with increased BMD. A review of the literature, however, revealed that few studies have described the association between BMD and lifestyle, including characteristic Japanese foods such as fish, natto, and Japanese green tea. The aim of this study was to identify lifestyle factors associated with BMD. METHODS: A total of 632 women age > or =60 years were enrolled in this study. Subjects were interviewed about their lifestyle by means of a questionnaire regarding the consumption pattern of dietary items. BMD was measured at the lumbar spine by dual energy X-ray absorptiometry. RESULTS: The BMD was higher in subjects with the habits of alcohol drinking, green tea drinking, and physical activity and lower in those with the habits of smoking and cheese consumption. Multiple regression analysis showed that factors associated with BMD were smoking, alcohol consumption, green tea drinking, and physical activity after adjusting for age and body mass index (BMI). CONCLUSIONS: In this cross-sectional study at an osteoporosis outpatient clinic, patients with the habits of alcohol drinking, green tea drinking, and physical activity had significantly higher BMD, and those who smoked had significantly lower BMD than patients without each habit after adjusting for age, BMI, and other variables regarding lifestyle.

Polyphenolic content and physiological activities of Chinese hawthorn extracts.

Hawthorn polyphenol (HP) was prepared by ethyl acetate treatment of the ethanol extract (HE) of Chinese hawthorn fruit. The concentrations of 15 polyphenols in the HP, HE, extraction residue (HJ), and a hawthorn leaf extract (HF) were determined by HPLC. For HP, the total content of the 15 polyphenols was 21.4%, comprised of 19.7% of procyanidins, 1.21% of chlorogenic acid, and 0.48% of flavonoids, compared to 2.55% for the HE. The yields of procyanidin monomer, dimer, trimer, tetramer, and pentamer were 50.5%, 30.3%, 23.0%, 14.6%, and 12.5% respectively, and the mean degree of polymerization was reduced to 1.39 (HP) from 1.65 (HE). Seven different physiological actions of the four extracts were investigated. The HP showed strong O(2)(-) and (.-)OH scavenging capacities (IC(50) values of 6.3 microg/ml and 1.1 microg/ml respectively), as well as selective prolyl endopeptidase inhibition (IC(50) of 60 microg/ml). The active constituents appeared to be procyanidins.

Identification of an alternatively spliced variant of Ca2+-promoted Ras inactivator as a possible regulator of RANKL shedding.

The receptor activator of NF-kappaB ligand (RANKL), a critical regulator of osteoclastogenesis, is synthesized as a membrane-anchored protein and cleaved into a soluble form by ectodomain shedding. We developed an assay system to identify molecules regulating the RANKL shedding. Using this system, we found that a splice variant of Ca2+-promoted Ras inactivator (CAPRI), deltaCAPRI, which is expressed in primary osteoblasts, promoted the RANKL shedding. The wild type CAPRI is a member of the Ras GTPase-activating protein (GAP) family and suppresses Ca2+-dependent Ras activation, whereas deltaCAPRI, which lacks one exon in the GAP-related domain, activated the Ras pathway. Overexpression of deltaCAPRI or a constitutive active form of Ras up-regulated the expression level of matrix-metalloproteinase 14 (MMP14), which directly cleaves the ectodomain of RANKL, whereas Erk activation by expressing the constitutive active Mek1 did not affect the MMP14 expression or RANKL shedding. These results suggest that deltaCAPRI is a possible regulator of RANKL shedding by modulating MMP14 expression through Ras signaling cascades other than the Erk pathway.

Cystatin 10, a novel chondrocyte-specific protein, may promote the last steps of the chondrocyte differentiation pathway.

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.