Cholesterol-Lowering and Antioxidant Effects of Pork-Liver Protein Hydrolysate in Otsuka Long-Evans Tokushima Fatty Rats.

In this study, we investigated the effects of a porcine liver protein hydrolysate (PLH) diet on lipid metabolism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type II diabetes. OLETF rats (20-wk-old males) were pair-fed with either a PLH diet containing 20% PLH or a casein diet for 14 wk. Dietary PLH significantly lowered serum cholesterol and phospholipid concentrations, mainly by decreasing low-density lipoprotein and high-density lipoprotein fractions. Fecal cholesterol was significantly increased in the PLH diet group; however, the total bile acid concentration in the feces was not significantly different between the groups. In addition, the PLH diet significantly decreased serum thiobarbituric acid reactive substance concentrations. These results suggest that dietary PLH exerts hyperlipidemic and antioxidant effects, indicating that it is a novel functional food ingredient.

Effects of Pawpaw (Asimina triloba) Seed Extract on the Differentiation and Fat Accumulation of 3T3-L1 Cells under Different Glucose Conditions.

Asimina triloba (pawpaw) contains various bioactive alkaloids and acetogenins. In the present study, the effects of pawpaw seed extract (PSE) on adipocyte differentiation and fat accumulation were investigated in 3T3-L1 cells under different glucose conditions. Treatment of undifferentiated cells with 10 ng/mL PSE increased lactic acid production, suggesting enhanced anaerobic glycolysis. PSE treatment also suppressed cell proliferation and decreased the nicotinamide adenine dinucleotide (NAD)+/NADH ratio in low-glucose medium; however, this effect was not observed in high-glucose medium. Additionally, PSE treatment under low-glucose conditions resulted in reduced accumulation of triglycerides and decreased expression of peroxisome proliferator-activated receptor (PPAR)-γ, CAAT/enhancer-binding protein (C/EBP)-α, and sterol regulatory element binding protein (SREBP)-1c in adipocyte-differentiated cells. PSE exerted greater effects on adipocyte differentiation and triglyceride content in 3T3-L1 cells under low-glucose conditions than under high-glucose conditions. These findings indicate that PSE enhances anaerobic glycolysis and inhibits adipocyte differentiation and fat accumulation in 3T3-L1 cells under glucose-restricted conditions.

Effects of visual and aromatic stimulations on the perception of five fundamental tastes.

Taste perception is affected by various environmental factors. In the present study, we investigated the effects of visual and aromatic stimulations on stress responses and taste perception. Fourteen young healthy participants were assessed for stress levels and taste intensities under 5 different conditions: normal (no stimuli), watching an action scene, watching a forest scene, sniffing a rosemary aroma, and sniffing a lavender aroma. Compared to participants under the action scene condition, participants under the forest scene or under the rosemary aroma condition showed significantly lower stress levels. Furthermore, the forest scene condition significantly increased the saltiness intensity, whereas the rosemary aroma condition significantly increased the bitterness intensity. A positive or negative correlation was observed between the stress level and taste intensity of sourness and saltiness, respectively. These findings indicate that visual image and aroma have the potential to change taste perception as well as modulate stress conditions.

Genistein inhibits chondrogenic differentiation and mineralization of ATDC5 cells.

Isoflavones are phytoestrogens abundant in leguminous crops and are used to prevent a variety of hormonal disorders. In the present study, the effects of genistein and daidzein on the chondrogenic differentiation of ATDC5 cells were investigated. Genistein (10 µM) treatment markedly reduced production of sulfated proteoglycans and collagen fibers in the ATDC5 cells. Genistein suppressed the expression of genes involved in chondrocyte differentiation such as Sox9, Col2a1, Col10a1, Acan, and Tgfb1. Additionally, genistein significantly decreased calcium deposition in ATDC5 cells during chondrogenic differentiation; however, it increased calcification under non-chondrogenic mineralizing conditions. Daidzein exhibited a similar effect of suppressing chondrogenesis in ATDC5 cells, although its efficacy was 10-times lower than that of genistein. These findings suggest that a high concentration of genistein inhibits chondrogenesis and chondrogenic mineralization, whereas it enhances non-chondrogenic mineralization.

Effects of Collagen Peptide Administration on Visceral Fat Content in High-Fat Diet-Induced Obese Mice.

Collagen peptides (CPs) are bioactive molecules that have beneficial effects on bone metabolism and against joint disorders. In the present study, we investigated the effect of CP supplementation on visceral fat mass and plasma lipid concentrations in high-fat diet (HFD)-induced obese mice. Male ddY mice were fed a normal diet or HFD for 3 wk, and assigned to N or NCP groups and to F or FCP groups, respectively. The NCP and FCP group mice were administered experimental diets containing 25 mg/g CPs for 3 wk further. During the experimental period, CP supplementation affected neither the food consumption nor the body weight of the mice. No significant differences in the plasma triglyceride, non-esterified fatty acid, and cholesterol concentrations were observed among all the groups. In contrast, the weight of testicular fat mass was significantly decreased in the FCP group as compared with that in the F group. The expression levels of leptin and tumor necrosis factor (TNF)-α genes in the adipose tissue correlated with the visceral fat mass, although these differences were not significant. These findings indicate that CPs may have a reducing effect on visceral fat content but are less effective in reducing body weight.

Effects of administration of glucosamine and chicken cartilage hydrolysate on rheumatoid arthritis in SKG mice.

Supplementation with cartilage constituents, such as glucosamine, chondroitin sulfate and collagen peptide, are believed to reduce pain associated with joint disorders, such as rheumatoid arthritis (RA). Here, we administered daily, 10 mg glucosamine or 100 mg chicken cartilage hydrolysate (CH) to SKG/Jcl mice, a model for spontaneous RA, for 5 weeks and evaluated their effects on RA development. In SKG mice, the administration of glucosamine had no reducing effect on RA score but suppressed the expression of Mmp13 and Col3a1 genes in articular cartilage. In contrast, administration of CH suppressed the RA score and levels of plasma interleukin-6 and interleukin-17 to half, although the differences were not significant. Mice administered with glucosamine also showed decreased bone strength of femur and these adverse effects could be eliminated when glucosamine was used in conjunction with CH. These results suggest that CH and glucosamine exert effects on different aspects in SKG mice.

Menaquinone-4 Amplified Glucose-Stimulated Insulin Secretion in Isolated Mouse Pancreatic Islets and INS-1 Rat Insulinoma Cells.

Vitamin K2 is indispensable for blood coagulation and bone metabolism. Menaquinone-4 (MK-4) is the predominant homolog of vitamin K2, which is present in large amounts in the pancreas, although its function is unclear. Meanwhile, ß-cell dysfunction following insulin secretion has been found to decrease in patients with type 2 diabetes mellitus. To elucidate the physiological function of MK-4 in pancreatic ß-cells, we studied the effects of MK-4 treatment on isolated mouse pancreatic islets and rat INS-1 cells. Glucose-stimulated insulin secretion significantly increased in isolated islets and INS-1 cells treated with MK-4. It was further clarified that MK-4 enhanced cAMP levels, accompanied by the regulation of the exchange protein directly activated by the cAMP 2 (Epac2)-dependent pathway but not the protein kinase A (PKA)-dependent pathway. A novel function of MK-4 on glucose-stimulated insulin secretion was found, suggesting that MK-4 might act as a potent amplifier of the incretin effect. This study therefore presents a novel potential therapeutic approach for impaired insulinotropic effects.

Biotin augments acetyl CoA carboxylase 2 gene expression in the hypothalamus, leading to the suppression of food intake in mice.

It is known that biotin prevents the development of diabetes by increasing the functions of pancreatic beta-cells and improving insulin sensitivity in the periphery. However, its anti-obesity effects such as anorectic effects remain to be clarified. Acetyl CoA carboxylase (ACC), a biotin-dependent enzyme, has two isoforms (ACC1 and ACC2) and serves to catalyze the reaction of acetyl CoA to malonyl CoA. In the hypothalamus, ACC2 increases the production of malonyl CoA, which acts as a satiety signal. In this study, we investigated whether biotin increases the gene expression of ACC2 in the hypothalamus and suppresses food intake in mice administered excessive biotin. Food intake was significantly decreased by biotin, but plasma regulators of appetite, including glucose, ghrelin, and leptin, were not affected. On the other hand, biotin notably accumulated in the hypothalamus and enhanced ACC2 gene expression there, but it did not change the gene expression of ACC1, malonyl CoA decarboxylase (a malonyl CoA-degrading enzyme), and AMP-activated protein kinase α-2 (an ACC-inhibitory enzyme). These findings strongly suggest that biotin potentiates the suppression of appetite by upregulating ACC2 gene expression in the hypothalamus. This effect of biotin may contribute to the prevention of diabetes by biotin treatment.

The Effects of Light and Temperature on Biotin Synthesis in Pea Sprouts.

Biotin is an essential micronutrient, and is a cofactor for several carboxylases that are involved in the metabolism of glucose, fatty acids, and amino acids. Because plant cells can synthesize their own biotin, a wide variety of plant-based foods contains significant amounts of biotin; however, the influence of environmental conditions on the biotin content in plants remains largely unclear. In the present study, we investigated the effects of different cultivation conditions on the biotin content and biotin synthesis in pea sprouts (Pisum sativum). In the experiment, the pea sprouts were removed from their cotyledons and cultivated by hydroponics under five different lighting and temperature conditions (control [25ºC, 12-h light/12-h dark cycle], low light [25ºC, 4-h light/20-h dark cycle], dark [25ºC, 24 h dark], low temperature [12ºC, 12-h light/12-h dark cycle], and cold [6ºC, 12-h light/12-h dark cycle]) for 10 d. Compared to the biotin content of pea sprouts under the control conditions, the biotin contents of pea sprouts under the low-light, dark, and cold conditions had significantly decreased. The dark group showed the lowest biotin content among the groups. Expression of the biotin synthase gene (bio2) was also significantly decreased under the dark and cold conditions compared to the control condition, in a manner similar to that observed for the biotin content. No significant differences in the adenosine triphosphate content were observed among the groups. These results indicate that environmental conditions such as light and temperature modulate the biotin content of pea plant tissues by regulating the expression of biotin synthase.

Sulfation of keratan sulfate proteoglycan reduces radiation-induced apoptosis in human Burkitt's lymphoma cell lines.

This study focuses on clarifying the contribution of sulfation to radiation-induced apoptosis in human Burkitt's lymphoma cell lines, using 3'-phosphoadenosine 5'-phosphosulfate transporters (PAPSTs). Overexpression of PAPST1 or PAPST2 reduced radiation-induced apoptosis in Namalwa cells, whereas the repression of PAPST1 expression enhanced apoptosis. Inhibition of PAPST slightly decreased keratan sulfate (KS) expression, so that depletion of KS significantly increased radiation-induced apoptosis. In addition, the repression of all three N-acetylglucosamine-6-O-sulfotransferases (CHST2, CHST6, and CHST7) increased apoptosis. In contrast, PAPST1 expression promoted the phosphorylation of p38 MAPK and Akt in irradiated Namalwa cells. These findings suggest that 6-O-sulfation of GlcNAc residues in KS reduces radiation-induced apoptosis of human Burkitt's lymphoma cells.

Administration of biotin prevents the development of insulin resistance in the skeletal muscles of Otsuka Long-Evans Tokushima Fatty rats.

Otsuka Long-Evans Tokushima Fatty (OLETF) rat is an animal model for type 2 diabetes mellitus. In the present study, we investigated whether pharmacologic doses of biotin have the potential to abate insulin resistance in the skeletal muscles of OLETF rats. OLETF rats (34 weeks of age) were divided into 2 groups and given distilled water (OLETF-control group) or distilled water containing 3.3 mg L(-1) of biotin (OLETF-biotin group) for 8 weeks. At the end of experimental period, the OLETF-control rats developed severe hyperglycemia and hyperinsulinemia, whereas the OLETF-biotin rats showed significantly smaller responses to oral glucose tolerance test than the OLETF-control rats. The glucose uptake in the hind limbs of the rats was significantly higher in the OLETF-biotin group than in the OLETF-control group. Biotin administration increased the glucose transporter type 4 (GLUT4) protein content in the total membrane fraction but had little effect on the GLUT4 content in the plasma membrane fraction. These results indicate that administration of a pharmacological dose of biotin prevents the development of insulin resistance in the skeletal muscles of OLETF rats presumably via an increase in GLUT4 protein expression but not via GLUT4 translocation.

Expression and the role of 3'-phosphoadenosine 5'-phosphosulfate transporters in human colorectal carcinoma.

Sulfation represents an essential modification for various molecules and regulates many biological processes. The sulfation of glycans requires a specific transporter for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the Golgi apparatus. This study investigated the expression of PAPS transporter genes in colorectal carcinomas and the significance of Golgi-specific sulfation in the proliferation of colorectal carcinoma cells. The relative amount of PAPST1 transcripts was found to be higher than those of PAPST2 in colorectal cancerous tissues. Immunohistochemically, the enhanced expression of PAPST1 was observed in fibroblasts in the vicinity of invasive cancer cells, whereas the expression of PAPST2 was decreased in the epithelial cells. RNA interference of either of the two PAPS transporter genes reduced the extent of sulfation of cellular proteins and cellular proliferation of DLD-1 human colorectal carcinoma cells. Silencing the PAPS transporter genes reduced fibroblast growth factor signaling in DLD-1 cells. These findings indicate that PAPS transporters play a role in the proliferation of colorectal carcinoma cells themselves and take part in a desmoplastic reaction to support cancer growth by controlling their sulfation status.

Two pathways for importing GDP-fucose into the endoplasmic reticulum lumen function redundantly in the O-fucosylation of Notch in Drosophila.

Notch is a transmembrane receptor that shares homology with proteins containing epidermal growth factor-like repeats and mediates the cell-cell interactions necessary for many cell fate decisions. In Drosophila, O-fucosyltransferase 1 catalyzes the O-fucosylation of these epidermal growth factor-like repeats. This O-fucose elongates, resulting in an O-linked tetrasaccharide that regulates the signaling activities of Notch. Fucosyltransferases utilize GDP-fucose, which is synthesized in the cytosol, but fucosylation occurs in the lumen of the endoplasmic reticulum (ER) and Golgi. Therefore, GDP-fucose uptake into the ER and Golgi is essential for fucosylation. However, although GDP-fucose biosynthesis is well understood, the mechanisms and intracellular routes of GDP-fucose transportation remain unclear. Our previous study on the Drosophila Golgi GDP-fucose transporter (Gfr), which specifically localizes to the Golgi, suggested that another GDP-fucose transporter(s) exists in Drosophila. Here, we identified Efr (ER GDP-fucose transporter), a GDP-fucose transporter that localizes specifically to the ER. Efr is a multifunctional nucleotide sugar transporter involved in the biosynthesis of heparan sulfate-glycosaminoglycan chains and the O-fucosylation of Notch. Comparison of the fucosylation defects in the N-glycans in Gfr and Efr mutants revealed that Gfr and Efr made distinct contributions to this modification; Gfr but not Efr was crucial for the fucosylation of N-glycans. We also found that Gfr and Efr function redundantly in the O-fucosylation of Notch, although they had different localizations and nucleotide sugar transportation specificities. These results indicate that two pathways for the nucleotide sugar supply, involving two nucleotide sugar transporters with distinct characteristics and distributions, contribute to the O-fucosylation of Notch.

Absorption and effectiveness of orally administered low molecular weight collagen hydrolysate in rats.

Collagen, a major extracellular matrix macromolecule, is widely used for biomedical purposes. We investigated the absorption mechanism of low molecular weight collagen hydrolysate (LMW-CH) and its effects on osteoporosis in rats. When administered to Wistar rats with either [(14)C]proline (Pro group) or glycyl-[(14)C]prolyl-hydroxyproline (CTp group), LMW-CH rapidly increased plasma radioactivity. LMW-CH was absorbed into the blood of Wistar rats in the peptide form. Glycyl-prolyl-hydroxyproline tripeptide remained in the plasma and accumulated in the kidney. In both groups, radioactivity was retained at a high level in the skin until 14 days after administration. Additionally, the administration of LMW-CH to ovariectomized stroke-prone spontaneously hypertensive rats increased the organic substance content and decreased the water content of the left femur. Our findings show that LMW-CH exerts a beneficial effect on osteoporosis by increasing the organic substance content of bone.

The ortholog of human solute carrier family 35 member B1 (UDP-galactose transporter-related protein 1) is involved in maintenance of ER homeostasis and essential for larval development in Caenorhabditis elegans.

Although the solute carrier 35B1 (SLC35B1) is evolutionarily conserved, its functions in metazoans remain unknown. To elucidate its function, we examined developmental roles of an SLC35B1 family gene (HUT-1: homolog of UDP-Gal transporter) in Caenorhabditis elegans. We isolated a deletion mutant of the gene and characterized phenotypes of the mutant and hut-1 RNAi-treated worms. GFP-HUT-1 reporter analysis was performed to examine gene expression patterns. We also tested whether several nucleotide sugar transporters can compensate for hut-1 deficiency. The hut-1 deletion mutant and RNAi worms showed larval growth defect and lethality with disrupted intestinal morphology. Inactivation of hut-1 induced chronic endoplasmic reticulum (ER) stress, and hut-1 showed genetic interactions with the atf-6, pek-1, and ire-1 genes involved in unfolded protein response signaling. ER ultrastructure and ER marker distribution in hut-1-deficient animals showed that HUT-1 is required for maintenance of ER structure. Reporter analysis revealed that HUT-1 is an ER protein ubiquitously expressed in tissues, including the intestine. Lethality and the ER stress phenotype of the mutant were rescued with the human hut-1 ortholog UGTrel1. These results indicate important roles for hut-1 in development and maintenance of ER homeostasis in C. elegans.

Antihypertensive effect of biotin in stroke-prone spontaneously hypertensive rats.

Biotin is a member of the vitamin B-complex family. Biotin deficiency has been associated with hyperglycaemia and insulin resistance in animals and humans. In the present study, we investigated the pharmacological effects of biotin on hypertension in the stroke-prone spontaneously hypertensive rat (SHRSP) strain. We observed that long-term administration of biotin decreased systolic blood pressure in the SHRSP strain; also, a single dose of biotin immediately decreased systolic blood pressure in this strain. Pretreatment with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazole [4,3-alpha]quinoxalin-1-one abolished the hypotensive action of biotin in the SHRSP strain, while pretreatment with the NO synthase inhibitor NG-nitro-l-arginine methyl ester had no effect on the action of biotin. Biotin reduced coronary arterial thickening and the incidence of stroke in the SHRSP strain. These results suggest that the pharmacological dose of biotin decreased the blood pressure of the SHRSP via an NO-independent direct activation of soluble guanylate cyclase. Our findings reveal the beneficial effects of biotin on hypertension and the incidence of stroke.

Identification and characterization of a novel Drosophila 3'-phosphoadenosine 5'-phosphosulfate transporter.

Sulfation of macromolecules requires the translocation of a high energy form of nucleotide sulfate, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), from the cytosol into the Golgi apparatus. In this study, we identified a novel Drosophila PAPS transporter gene dPAPST2 by conducting data base searches and screening the PAPS transport activity among the putative nucleotide sugar transporter genes in Drosophila. The amino acid sequence of dPAPST2 showed 50.5 and 21.5% homology to the human PAPST2 and SLALOM, respectively. The heterologous expression of dPAPST2 in yeast revealed that the dPAPST2 protein is a PAPS transporter with an apparent K(m) value of 2.3 microm. The RNA interference of dPAPST2 in cell line and flies showed that the dPAPST2 gene is essential for the sulfation of cellular proteins and the viability of the fly. In RNA interference flies, an analysis of the genetic interaction between dPAPST2 and genes that contribute to glycosaminoglycan synthesis suggested that dPAPST2 is involved in the glycosaminoglycan synthesis and the subsequent signaling. The dPAPST2 and sll genes showed a similar ubiquitous distribution. These results indicate that dPAPST2 may be involved in Hedgehog and Decapentaplegic signaling by controlling the sulfation of heparan sulfate.