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In the pursuit of bioactive phytochemicals as a therapeutic strategy to manage metabolic risk factors for type 2 diabetes (T2D), aspalathin, C-glucosyl dihydrochalcone from rooibos (Aspalathus linearis), has received much attention, along with its C-glucosyl flavone derivatives and phlorizin, the apple O-glucosyl dihydrochalcone well-known for its antidiabetic properties. We provided context for dietary exposure by highlighting dietary sources, compound stability during processing, bioavailability and microbial biotransformation. The review covered the role of these compounds in attenuating insulin resistance and enhancing glucose metabolism, alleviating gut dysbiosis and associated oxidative stress and inflammation, and hyperuricemia associated with T2D, focusing largely on the literature of the past 5 years. A key focus of this review was on emerging targets in the management of T2D, as highlighted in the recent literature, including enhancing of the insulin receptor and insulin receptor substrate 1 signaling via protein tyrosine phosphatase inhibition, increasing glycolysis with suppression of gluconeogenesis by sirtuin modulation, and reducing renal glucose reabsorption via sodium-glucose co-transporter 2. We conclude that biotransformation in the gut is most likely responsible for enhancing therapeutic effects observed for the C-glycosyl parent compounds, including aspalathin, and that these compounds and their derivatives have the potential to regulate multiple factors associated with the development and progression of T2D.

Muscle is the largest tissue in our body and plays an important role in glucose homeostasis and hence diabetes. In the present study, we examined the effects of taxifolin (TXF) on glucose metabolism in cultured L6 muscle cells (myotubes) and in type 2 diabetic (T2D) model KK-Ay/Ta mice. TXF dose-dependently increased glucose uptake (GU) in L6 myotubes under the condition of insulin absence. This increase in GU was partially, but significantly canceled by TXF treatment in combination with either LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), which phosphorylates protein kinase B (Akt) or Compound C, an inhibitor of 5’-adenosine monophosphate-activated protein kinase (AMPK). Furthermore, TXF was demonstrated to activate (=phosphorylate) both Akt and AMPK, and promote glucose transporter 4 (GLUT4) translocation to the plasma membrane from cytosol of L6 myotubes via both PI3K/Akt and AMPK signaling pathways. Based on these in vitro findings, we conducted an in vivo experiment in KK-Ay/Ta mice with hyperglycemia and hyperuricemia. Fasting plasma glucose, insulin, uric acid levels and an index of insulin resistance (HOMA-IR) increased significantly in the T2D model mice compared with normal ones. Such rises in the T2D state were significantly suppressed by oral administration of TXF for four weeks. These results suggest that TXF is a potent antihyperglycemic and antihyperuricemic phytochemical in the T2D state.

The recovery of useful components such as antioxidants from strawberry leaves would be desirable because this byproduct is usually disposed of as agricultural waste. In this study, strawberry leaves were extracted with supercritical carbon dioxide (sc-CO2) and the effects of various entrainers were evaluated under the following conditions: temperature from 308 to 333 K, pressure from 15 to 30 MPa, and a molar ratio of entrainer to CO2 of 0.1. The use of ethanol, acetone, and water improved the extraction yield, antioxidant capacity and total phenolic content of the extract. In sc-CO2 + ethanol, the antioxidant capacity and total phenolic content exhibited maximum values for a given temperature at 20 MPa. The antioxidant capacity was almost independent of pressure and total phenolic content had a maximum at 308 K. The solvent density, solubility of organics, and vapor pressure of compounds appeared to influence the antioxidant capacity and total phenolic content. Furthermore, the strawberry leaf extract obtained using sc-CO2 + ethanol significantly and dose-dependently suppressed uric acid production in cultured hepatocytes.

Hyperuricemia is an important risk factor for gout. Isorhamnetin (3'-O-methylquercetin) is an O-methylated flavonol, which occurs in onion, almond and sea buckthorn. It is also one of the metabolites of quercetin in mammals. In the present study, we investigated anti-hyperuricemic effect of isorhamnetin adopting both cultured hepatocytes and mice with hyperuricemia induced by purine bodies. In cultured hepatocytes, isorhamnetin as well as quercetin significantly and dose-dependently inhibited uric acid (UA) production. We also examined the inhibitory effects on UA production of other mono-methylquercetins, i.e., tamarixetin, 3-O-methylquercetin, azaleatin, and rhamnetin in addition to isorhamnetin for studying their structure-activity relationships. From the results obtained, hydroxyl groups at C-3, C-5, and especially C-7, but not C-3' and C-4' of quercetin are demonstrated to play a critical role in suppressing UA production in the AML12 hepatocytes. Oral administration of isorhamnetin significantly reduced plasma and hepatic UA levels in the hyperuricemic model mice. Isorhamnetin also decreased hepatic xanthine oxidase (XO) activity without changes in XO protein expression, indicating that anti-hyperuricemic effect of isorhamnetin could be, at least partly, attributable to suppression of UA production by directly inhibiting XO activity in the liver. These findings demonstrate that isorhamnetin has a potent anti-hyperuricemic effect and may be a potential candidate for prevention and remediation of hyperuricemia.

Hyperuricemia is defined as a disease with high uric acid (UA) levels in the blood and a strong risk factor for gout. Urolithin A (UroA) is a main microbial metabolite derived from ellagic acid (EA), which occurs in strawberries and pomegranates. In this study, we evaluated antihyperuricemic effect of UroA in both cultured hepatocytes and hyperuricemic model mice. In cultured hepatocytes, UroA significantly and dose-dependently reduced UA production. In model mice with purine bodies-induced hyperuricemia, oral administration of UroA significantly inhibited the increase in plasma UA levels and hepatic xanthine oxidase (XO) activity. In addition, DNA microarray results exhibited that UroA, as well as allopurinol, a strong XO inhibitor, induced downregulation of the expression of genes associated with hepatic purine metabolism. Thus, hypouricemic effect of UroA could be, at least partly, attributed to inhibition of purine metabolism and UA production by suppressing XO activity in the liver. These results indicate UroA possesses a potent antihyperuricemic effect and it could be a potential candidate for a molecule capable of preventing and improving hyperuricemia and gout.

Hyperuricemia, the high uric acid (UA) state in blood, has been accepted as an important risk factor for gout. The liver is a main factory of UA production. In the present study, we have examined the effects of three kinds of flavonol and flavones as typical aglycons, i.e., quercetin, luteolin, apigenin, their glycosides and related compounds, on UA productivity in cultured hepatocytes, adopting allopurinol as the positive control drug. Quercetin, luteolin, diosmetin (4′-O-methylluteolin) and apigenin at 10, 30 and 100 μM as well as allopurinol at 0.1, 0.3 and 1 μM dose-dependently and significantly decreased UA production in the hepatocytes, when compared with 0 μM (control). Both rutin (quercetin-3-O-rutinoside) and quercitrin (quercetin-3-O-ramnoside) significantly reduced UA production in the hepatocytes at 100 μM. Luteolin glycosides such as orientin (luteolin-8-C-glucoside) and isoorientin (luteolin-6-C-glucoside) exerted no influences on it even at 100 μM. Likewise, apigenin glycosides such as vitexin (apigenin-8-C-glucoside) and isovitexin (apigenin-6-C-glucoside) showed no inhibitory effect on it, while apigetrin (apigenin-7-O-glucoside) significantly reduced it at 100 μM. In model mice with purine bodies-induced hyperuricemia, allopurinol completely suppressed the hyperuricemia at a dose of 10 mg/kg body weight. Rutin suppressed significantly the hyperuricemia at a dose of 300 mg/kg body weight, while vitexin showed no significant effect up to 300 mg/kg body weight. Thus, rutin (O-glycoside) is demonstrated to be hypouricemic in both cultured hepatocytes and model mice with recently contrived purine bodies-induced hyperuricemia.

Cases of autochthonous infections of dengue virus type 1 (DENV-1) were detected in Japan after a 70-year period devoid of dengue outbreaks. We previously showed that E gene sequences are identical in 11 of the 12 DENV-1 strains autochthonous to Japan. However, the E sequence represents only 14% of the DENV-1 genome. In the present study, we have sequenced the entire genome of 6 autochthonous DENV-1 strains that were isolated from patients during the 2014 outbreak. Sequencing of 5 Yoyogi group strains with identical E sequences and 1 Shizuoka strain with a different E sequence revealed that the first Yoyogi group strain differed from the Shizuoka strain by 18 amino acid residues. Furthermore, 2 Yoyogi group strains had different genomic sequences while the other 3 had identical genomes. Phylogenetic analyses indicated that the Hyogo strain, a Yoyogi group strain, was the first to diverge from the other 4 Yoyogi group strains. The E gene sequence of the Yoyogi group strains exhibits the highest homology to those of the strains isolated in Malaysia and Singapore between 2013 and 2014. The patient infected with the Hyogo strain visited Malaysia before the onset of dengue fever, suggesting that this was a case of dengue infection imported from Malaysia.

Dengue virus (DENV) is the causative agent of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) and continues to be a public health problem in the tropical and subtropical areas. However, there is currently no antiviral treatment for DENV infection. In this study, our aim was to develop a stable reporter replicon cell system that supports constant viral RNA replication in cultured cells. The isolated replicon cells exhibited high levels of luciferase activity in the culture supernatant concomitant with expression of virus-encoded NS1, NS3 and NS5 proteins in the cells. The NS1, NS3 proteins and dsRNA were detected in the replicon cells by immunofluorescence analysis. Furthermore, the anti-DENV inhibitors ribavirin and bromocriptine significantly reduced the luciferase activity in a dose-dependent manner. High-throughput screening with a compound library using the stably-transfected replicon cells showed a Z' factor value of 0.57. Our screening yielded several candidates including one compound that has already shown anti-DENV activity. Taken together, our results demonstrate that this DENV subgenomic replicon cell system expressing a secretory luciferase gene can be useful for the high-throughput screening of anti-DENV compounds and the analysis of the replication mechanism of the DENV RNA.

Skeletal muscles are the largest tissue in our body and play an important role in maintaining glucose homeostasis. After contriving a simple and rapid glucose uptake assay system by adopting cultured L6 myotubes, phytochemicals that promote glucose uptake under the condition of insulin absence were screened. All the screened phytochemicals [(resveratrol, piceatannol, aspalathin, nepodin, gingerol, genistein, daidzein, equol, enterolactone (ENL)] activated 5′-adenosine monophosphate-activated protein kinase (AMPK) and hence promoted translocation of glucose transporter 4 to plasma membrane. These phytochemicals suppressed the rises in blood glucose levels and improved the glucose intolerance in type 2 diabetes (T2D) model mice (db/db, ob/ob, KK-Ay). Daidzein and nepodin also activated AMPK in gastrocnemius muscle of db/db mice. Genistein and daidzein reduced urinary glucose excretion in KK-Ay mice. Some microbial metabolites in the intestine such as equol from daidzein and ENL from lignans promoted glucose uptake in vitro, suppressed the rises in blood glucose levels, and improved glucose intolerance in vivo at low doses. Phytochemicals that have potential to stimulate glucose uptake in muscle cells are suggested to be antidiabetic. Thus, studies on novel compounds that activate skeletal muscle glucose uptake and AMPK would be useful for the development of new treatment of insulin resistance and T2D.

Urolithin A, a gut microbial metabolite of ellagic acid, is reported to exert anti-inflammatory effects in vitro and in vivo. However, complete mechanisms underlying the regulation of inflammatory responses by urolithin A remain unclear. This study aimed to evaluate the anti-inflammatory potential of urolithin A and its underlying mechanisms in lipopolysaccharide (LPS)-stimulated RAW264 macrophages. Urolithin A significantly attenuated the pro-inflammatory mediator production in LPS-stimulated RAW264 and mouse peritoneal macrophages. This compound significantly suppressed the LPS-elicited nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activation. The phosphorylation of Akt and c-Jun N-terminal kinase (JNK) was also inhibited by the treatment with urolithin A. Through experiments using kinase inhibitors, urolithin A abolished the LPS-induced phosphatidylinositol 3-kinase (PI3-K)/Akt/NF-κB and JNK/AP-1 signaling pathways, resulting in suppression of pro-inflammatory mediator production. Furthermore, treatment with this compound significantly reduced the intracellular accumulation of reactive oxygen species, which are known to act as secondary messengers in the activation of redox-sensitive transcription factors NF-κB and AP-1. Urolithin A treatment also diminished the LPS-evoked activation of NADPH oxidase (NOX), which is the main source of reactive oxygen species in activated macrophages. The inhibition of this activity by urolithin A led to the prevention of LPS-elicited NF-κB and AP-1 activation as well as Akt and JNK phosphorylation, resulting in the reduction of pro-inflammatory mediator production. Collectively, these results indicate that urolithin A treatment attenuates pro-inflammatory mediator production by suppressing NOX-derived reactive oxygen species-mediated PI3-K/Akt/NF-κB and JNK/AP-1 signaling pathways in LPS-stimulated macrophages.

Previous studies have demonstrated antidiabetic effects for rooibos (Aspalathus linearis) and aspalathin (ASP), one of its main polyphenols. Rooibos, an endemic plant of South Africa, is well-known for its use as herbal tea. Green ('unfermented') rooibos has been shown to contain more ASP than 'fermented' rooibos tea, currently the major product. In the present study, we investigated the antidiabetic effect of green rooibos extract (GRE) through studies on glucose uptake in L6 myotubes and on pancreatic β-cell protective ability from reactive oxygen species (ROS) in RIN-5F cells. Its in vivo effect was also examined using obese diabetic KK-A(y) mice. GRE increased glucose uptake under insulin absent condition and induced phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK) in L6 myotubes as previously demonstrated for ASP. In addition to AMPK, GRE also promoted phosphorylation of Akt, another promoter of glucose transporter 4 (GLUT4) translocation, in L6 myotubes unlike ASP, suggesting an involvement of GRE component(s) other than ASP in Akt phosphorylation. Promotion of GLUT4 translocation to the plasma membrane by GRE in L6 myotubes was demonstrated by Western blotting analysis. GRE suppressed the advanced glycation end products (AGEs)-induced increase in ROS levels in RIN-5F pancreatic β-cells. Subchronic feeding with GRE suppressed the increase in fasting blood glucose levels in type 2 diabetic model KK-A(y) mice. These in vitro and in vivo results strongly suggest that GRE has antidiabetic potential through multiple modes of action.

There have been studies on health beneficial effects of ginger and its components. However, there still remain certain aspects that are not well defined in their anti-hyperglycemic effects. Our aims were to find evidence of possible mechanisms for antidiabetic action of [6]-gingerol, a pungent component of ginger, employing a rat skeletal muscle-derived cell line, a rat-derived pancreatic β-cell line, and type 2 diabetic model animals. The antidiabetic effect of [6]-gingerol was investigated through studies on glucose uptake in L6 myocytes and on pancreatic β-cell protective ability from reactive oxygen species (ROS) in RIN-5F cells. Its in vivo effect was also examined using obese diabetic db/db mice. [6]-Gingerol increased glucose uptake under insulin absent condition and induced 5' adenosine monophosphate-activated protein kinase phosphorylation in L6 myotubes. Promotion by [6]-gingerol of glucose transporter 4 (GLUT4) translocation to plasma membrane was visually demonstrated by immunocytochemistry in L6 myoblasts transfected with glut4 cDNA-coding vector. [6]-Gingerol suppressed advanced glycation end product-induced rise of ROS levels in RIN-5F pancreatic β-cells. [6]-Gingerol feeding suppressed the increases in fasting blood glucose levels and improved glucose intolerance in db/db mice. [6]-Gingerol regulated hepatic gene expression of enzymes related to glucose metabolism toward decreases in gluconeogenesis and glycogenolysis as well as an increase in glycogenesis, thereby contributing to reductions in hepatic glucose production and hence blood glucose concentrations. These in vitro and in vivo results strongly suggest that [6]-gingerol has antidiabetic potential through multiple mechanisms.

The effect of dietary milk-derived sphingomyelin concentrate (SPC) on atopic-like dermatitis was investigated. Atopic-like dermatitis was induced by sensitization to and repeated challenges with picryl chloride in male NC/Nga mice. Mice were fed an SPC-containing diet for 12 weeks, beginning 2 weeks before sensitization. The effect of SPC was assessed by monitoring clinical scores, serum immunoglobulin E levels, scratching behaviours, and in vitro cytokine productivities by splenocytes and mesenteric lymph node cells. Skin lesions were histologically investigated. Scratching behaviours were improved in the SPC group compared with the control group. The clinical score was ameliorated, and interleukin-6 productivity by splenocytes was significantly reduced in the SPC group. The numbers of infiltrated and degranulated mast cells in skin lesions were significantly lower in the SPC group than in the control group. These results indicate that dietary SPC significantly ameliorates atopic-like dermatitis in NC/Nga mice by suppressing systemic and peripheral inflammation.

Many active components derived from edible natural resources such as plant extracts have recently attracted attention for their potential use as functional foods or drugs for preventing and treating metabolic diseases such as diabetes. To obtain a novel modulator of glucose metabolism, we conducted screening of a small compound library in cultured L6 myotubes. We identified nepodin that stimulated glucose uptake dose-dependently in differentiated L6 myotubes. The stimulatory effect of nepodin on glucose uptake was abrogated by a 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibitor. In addition, nepodin stimulated the phosphorylation of AMPK. Nepodin also stimulated the translocation of GLUT4 to the plasma membrane in L6 myoblasts transfected with a Glut4 cDNA-coding vector and in differentiated L6 myotubes. In in vivo study, nepodin suppressed the increases in fasting blood glucose levels and improved the glucose intolerance of C57BL/KsJ-db/db mice, a type 2 diabetic animal model. Nepodin rescued the impaired phosphorylation of AMPK in the skeletal muscle of db/db mice. These results suggest that nepodin has an antidiabetic effect, which is at least partly mediated by stimulation of GLUT4 translocation via AMPK activation by nepodin.

Skeletal muscles are the largest tissue in our body and play an important role in maintaining glucose homeostasis. Cultured L6 myotubes and C2C12 myotubes are useful to construct simple glucose uptake assay systems, to screen various phytochemicals that promote glucose uptake, and to clarify their modes of actions. In skeletal muscles, insulin promotes glucose uptake by activating phosphatidylinositol-3 kinase (PI3K) and Akt, leading to increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Another GLUT4 translocation promoter is 5′ adenosine monophosphate-activated protein kinase (AMPK). In mammalian cells, AMPK activated by an increase in AMP/ATP ratio acts as an energy sensor. AMPK is activated by exercise/contraction in muscle cells and compound such as metformin, this resulting in stimulation of GLUT4 translocation to plasma membrane and hence glucose uptake in skeletal muscles. Thus, studies on novel compounds that activate skeletal muscle glucose uptake and AMPK would be useful for the development of new treatment of insulin resistance and type 2 diabetes (T2D). Here we review the current knowledge of phytochemicals in foods and beverages that promote GLUT4 translocation via AMPK signaling in muscle cells, and their effects on glucose metabolism in mainly T2D model mice. Phytochemicals that have potential to stimulate glucose uptake in muscle cells are suggested to be anti-diabetic.

The effect of natural IgG antibody recognizing β-galactosyl epitope on hepatoma cell invasion was investigated. Anti-β-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-α-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-β-galactosyl antibody was completely canceled by adding lactose (galactose-β-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with β-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48 h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-β-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-β-galactosyl specificity may recognize the β-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.

Although several researches have demonstrated that rooibos extract has hypoglycemic effect, the role of aspalathin, a main polyphenol in the extract, remains unclear. Our aims were to find specific mechanisms for anti-diabetic action of aspalathin employing a rat skeletal muscle-derived cell line (L6 myocytes) and a rat-derived pancreatic β-cell line (RIN-5F cells) and to investigate its effect in type 2 diabetic model ob/ob mice. We investigated in vitro the effect of aspalathin on the glucose metabolism through the studies on molecular mechanisms of glucose uptake using cultured L6 myotubes. We also measured the antioxidative ability of aspalathin against reactive oxygen species (ROS) generated by artificial advanced glycation end product (AGE) in RIN-5F cells. In vivo, ob/ob mice were fed 0.1 % aspalathin-containing diet for 5 weeks, and the effect of aspalathin on fasting blood glucose level, glucose intolerance, and hepatic gene expression was studied. Aspalathin dose dependently increased glucose uptake by L6 myotubes and promoted AMP-activated protein kinase (AMPK) phosphorylation. Aspalathin enhanced GLUT4 translocation to plasma membrane in L6 myoblasts and myotubes. In RIN-5F cells, aspalathin suppressed AGE-induced rises in ROS. In vivo, aspalathin significantly suppressed the increase in fasting blood glucose levels and improved glucose intolerance. Furthermore, aspalathin decreased expression of hepatic genes related to gluconeogenesis and lipogenesis. Hypoglycemic effect of aspalathin is related to increased GLUT4 translocation to plasma membrane via AMPK activation. In addition, aspalathin reduces the gene expression of hepatic enzymes related to glucose production and lipogenesis. These results strongly suggest that aspalathin has anti-diabetic potential.

Hyperuricemia is recognized as an important risk factor for gout. High dietary intake of purine-rich foods such as meats and sea foods increases uric acid (UA) levels in the blood. Taxifolin present in Siberian larch and strawberries has been reported to possess health promoting activities including anti-oxidant effect. In this study, we examined anti-hyperuricemic effect of taxifolin in both cultured hepatocytes and hyperuricemic model mice. In cultured AML12 hepatocytes, taxifolin significantly suppressed UA production dose- and time-dependently. In mice with hyperuricemia induced by concurrent administration of guanosine-5'-monophosphate and inosine-5'-monophosphate, oral administration of taxifolin suppressed the increases in plasma and liver UA levels. In addition, it also suppressed hepatic xanthine oxidase (XO) activity. Thus, anti-hyperuricemic effect of taxifolin could be explained, at least partly, by suppressing UA production via inhibition of XO activity in the liver. These results suggest that taxifolin possesses a potent hypouricemic effect and it could be a potential candidate for an anti-hyperuricemic phytochemical.

Risk factors of type 2 diabetes mellitus (T2D) and cardiovascular disease (CVD) cluster together and are termed the metabolic syndrome. Key factors driving the metabolic syndrome are inflammation, oxidative stress, insulin resistance (IR), and obesity. IR is defined as the impairment of insulin to achieve its physiological effects, resulting in glucose and lipid metabolic dysfunction in tissues such as muscle, fat, kidney, liver, and pancreatic β-cells. The potential of rooibos extract and its major C-glucosyl flavonoids, in particular aspalathin, a C-glucoside dihydrochalcone, as well as the phenolic precursor, Z-2-(β-D-glucopyranosyloxy)-3-phenylpropenoic acid, to prevent the metabolic syndrome, will be highlighted. The mechanisms whereby these phenolic compounds elicit positive effects on inflammation, cellular oxidative stress and transcription factors that regulate the expression of genes involved in glucose and lipid metabolism will be discussed in terms of their potential in ameliorating features of the metabolic syndrome and the development of serious metabolic disease. An overview of the phenolic composition of rooibos and the changes during processing will provide relevant background on this herbal tea, while a discussion of the bioavailability of the major rooibos C-glucosyl flavonoids will give insight into a key aspect of the bioefficacy of rooibos.

(1992). Effect of Dietary Lysine on Endogenous Hyperlipidemia in Nephrotic Rats. Bioscience, Biotechnology, and Biochemistry: Vol. 56, No. 6, pp. 980-982.

Hyperuricemia is characterized by the high uric acid (UA) level in serum (or plasma) and has been considered to be an important risk factor for gout. In the present study, we have attempted to construct an assay system for UA production in vitro employing cultured AML12 hepatocytes. UA levels in balanced salt solution (BSS) in the presence of UA precursor nucleosides, adenosine, inosine, guanosine and xanthine, at 12.5, 25, and 100 µM were significantly higher than BSS alone and their effects were dose-dependent, while all the UA precursors did not significantly increase intracellular UA levels. Hence, UA levels in BSS were thereafter adopted as an index of UA production. UA production from nucleosides was significantly higher than that from nucleotides (GMP, IMP and AMP). UA production from guanosine and inosine in combination (GI mixture) as well as nucleosides increased time-dependently and almost linearly up to 2 h. Selecting GI mixture, effects of allopurinol, a widely used anti-hyperuricemic agent, and quercetin, a well-known polyphenol in onion and strawberry, on UA production were examined. Both allopurinol and quercetin dose-dependently (0.1, 0.3 and 1 μM for allopurinol and 10, 30, and 100 μM for quercetin) and significantly reduced UA production in the hepatocytes. They also significantly reduced hyperuricemia induced by intraperitoneal injection of UA precursor purine bodies to mice at a single oral dose of 10 (allopurinol) or 200 (quercetin) mg/kg body weight. This assay system for UA production in cultured hepatocytes is considered to be useful to search for novel anti-hyperuricemic compounds in foods and natural resources with possibility to have human health benefits.

The skeletal muscle cells are one of the main sites of glucose uptake through glucose transporter 4 (GLUT4) in response to insulin. In muscle cells, 5' adenosine monophosphate-activated protein kinase (AMPK) is known as another GLUT4 translocation promoter. Natural compounds that activate AMPK have a possibility to overcome insulin resistance in the diabetic state. Piceatannol is a natural analog and a metabolite of resveratrol, a known AMPK activator. In this study, we investigate the in vitro effect of piceatannol on glucose uptake, AMPK phosphorylation and GLUT4 translocation to plasma membrane in L6 myocytes, and its in vivo effect on blood glucose levels in type 2 diabetic model db/db mice. Piceatannol was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for type 2 diabetes and become an antidiabetic phytochemical.

Genistein, an isoflavone, is known to possess diverse biological functions such as antioxidative and anti-inflammatory actions. It also acts like estrogen and inhibits several tyrosine kinases. Genistein was reported to suppress insulin-mediated glucose uptake in adipocytes. In this study, we investigated the effects of genistein on glucose uptake in vitro and in vivo as well as the mechanisms associated with the glucose uptake. We found that genistein decreased nonfasting blood glucose levels in KK-Ay/Ta Jcl mice, a type 2 diabetic animal model. It also dose-dependently induced insulin secretion by Rin-5F cells. In L6 myotubes, it directly stimulated glucose uptake independently of insulin under normal and high glucose conditions in dose-dependent manners. It promoted the translocation of glucose transporter 4 to the cell membrane under both glucose conditions. Based on studies using inhibitors of signaling molecules related to glucose uptake, the stimulatory effect of genistein on glucose uptake appeared to be dependent on the phosphatidylinositol 3-kinase, mammalian target of rapamycin, protein kinase C and 5'-adenosine-monophosphate-activated protein kinase pathway under both glucose conditions. In addition, O-GlcNAcylation by O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamate, an inhibitor of N-acetylglucosaminidase, reduced the stimulatory effect of genistein on glucose uptake under both glucose conditions. Taken together, genistein may regulate glucose uptake by increasing the phosphorylation and decreasing the O-GlcNAcylation of proteins related to glucose homeostasis.

We used a ProteoChip coated with a calixcrown derivative protein linker to measure adiponectin and monocyte chemoattractant protein-1 (MCP-1) levels and compared the results with commercial enzyme-linked immunosorbent assay (ELISA) kits. Adiponectin and MCP-1 levels in normal human serum and RAW264 cell supernatants, respectively, were measured. The ProteoChip quantification results correlated with those from the ELISA kits; however, the ProteoChip required less sample volume, exhibited higher sensitivity, and had a wider detection range. The ProteoChip was capable of detecting and quantifying small amounts of protein, possibly replacing ELISA kits in evaluating the levels of adiponectin and MCP-1.

The mass and function of bones depend on the maintenance of a complicated balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An inhibitor of osteoclast differentiation and/or function is expected to be useful for treatment of bone lytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis into bone. Biselyngbyaside is a recently isolated macrolide compound from marine cyanobacteria Lyngbya sp. that shows wide-spectrum cytotoxicity toward human tumor cell lines. In this study, we investigated the effects of biselyngbyaside on osteoclast differentiation and function. Biselyngbyaside inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse monocytic RAW264 cells and primary bone marrow-derived macrophages at a low concentration. Similarly, biselyngbyaside suppressed osteoblastic cell-mediated osteoclast differentiation in cocultures. In the RANKL-induced signaling pathway, biselyngbyaside inhibited the expression of c-Fos and NFATc1, which are important transcription factors in osteoclast differentiation. In mature osteoclasts, biselyngbyaside decreased resorption-pit formation. Biselyngbyaside also induced apoptosis accompanied by the induction of caspase-3 activation and nuclear condensation, and these effects were negated by the pancaspase inhibitor z-VAD-FMK. Taken together, the present findings indicate that biselyngbyaside suppresses bone resorption via inhibition of osteoclastogenesis and induction of apoptosis. Thus, biselyngbyaside may be useful for the prevention of bone lytic diseases.

Enterolactone (ENL) is formed by the conversion of dietary precursors like strawberry lignans via the gut microbiota. Urinary concentrations of lignan metabolites are reported to be significantly associated with a lower risk of Type 2 diabetes (T2D). In the present study, antidiabetic effect of ENL and its modes of action were studied in vitro and in vivo employing a rat skeletal muscle-derived cell line, L6 myocytes in culture, and T2D model db/db mice. ENL dose-dependently increased glucose uptake in L6 myotubes under insulin absent condition. This increase by ENL was canceled by compound C, an inhibitor of 5′-adenosine monophosphate-activated protein kinase (APMK). Activation (=phosphorylation) of AMPK and translocation of glucose transporter 4 (GLUT4) to plasma membrane in L6 myotubes were demonstrated by Western blotting analyses. Promotion by ENL of GLUT4 translocation to plasma membrane was also visually demonstrated by immunocytochemistry in L6 myoblasts that were transfected with glut4 cDNA-coding vector. T2D model db/db mice were fed the basal 20 % casein diet (20C) or 20C supplemented with ENL (0.001 or 0.01 %) for 6 weeks. Fasting blood glucose (FBG) levels were measured every week and intraperitoneal glucose tolerance test (IPGTT) was conducted. ENL at a higher dose (0.01 % in 20C) suppressed the increases in FBG levels. ENL was also demonstrated to improve the index of insulin resistance (HOMA-IR) and glucose intolerance by IPGTT in db/db mice. From these results, ENL is suggested to be an antidiabetic chemical entity converted from dietary lignans by gut microbiota.

Bone mass is regulated by osteoblast-mediated bone formation and osteoclast-mediated bone resorption. We previously reported that harmine, a β-carboline alkaloid, inhibits osteoclast differentiation and bone resorption in vitro and in vivo. In this study, we investigated the effects of harmine on osteoblast proliferation, differentiation and mineralization. Harmine promoted alkaline phosphatase (ALP) activity in MC3T3-E1 cells without affecting their proliferation. Harmine also increased the mRNA expressions of the osteoblast marker genes ALP and Osteocalcin. Furthermore, the mineralization of MC3T3-E1 cells was enhanced by treatment with harmine. Harmine also induced osteoblast differentiation in primary calvarial osteoblasts and mesenchymal stem cell line C3H10T1/2 cells. Structure-activity relationship studies using harmine-related β-carboline alkaloids revealed that the C3-C4 double bond and 7-hydroxy or 7-methoxy group of harmine were important for its osteogenic activity. The bone morphogenetic protein (BMP) antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated harmine-promoted ALP activity. In addition, harmine increased the mRNA expressions of Bmp-2, Bmp-4, Bmp-6, Bmp-7 and its target gene Id1. Harmine also enhanced the mRNA expressions of Runx2 and Osterix, which are key transcription factors in osteoblast differentiation. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by harmine treatment. Taken together, these results indicate that harmine enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and Runx2 pathways. Our findings suggest that harmine has bone anabolic effects and may be useful for the treatment of bone-decreasing diseases and bone regeneration as a lead compound.

Osteogenic activity of six diarylheptanoids, acerogenin A (1), (R)-acerogenin B (2), aceroside I (3), aceroside B(1) (4), aceroside III (5) and (-)-centrolobol (6) and two phenolic compounds; (+)-rhododendrol (7) and (+)-cathechin (8), isolated from the stem bark of Acer nikoense (Nikko maple) was evaluated using alkaline phosphatase (ALP) activity as a marker for early osteoblast differentiation. We found that the diphenyl ether-type cyclic diarylheptanoids 1-5 promoted ALP activity in mouse preosteoblastic MC3T3-E1 cells without affecting cell proliferation, but linear-type diarylheptanoid 6 and phenolic compounds 7 and 8 did not. Diphenyl ether-type cyclic diarylheptanoids 1-4 also increased protein production of osteocalcin, a late stage maker for osteoblast differentiation, and induced osteoblastic mineralization. Structure-activity relationships of these compounds demonstrated that the stimulative efficacy of aglycones was higher than that of its glycosides. Taken together, diphenyl ether-type cyclic diarylheptanoids promote early- and late-stage osteoblastogenesis, which may open the possibility for the development of novel osteogenic agents.

Effects of α-, β-, γ- and δ-tocopherols on the proliferation and invasion of AH109A hepatoma cells and their modes of action were investigated. Four tocopherols inhibited the invasion as well as the proliferation of AH109A cells. Their inhibitory effects were more prominent on the invasion than on the proliferation. At 1 µM, α-tocopherol showed most potent anti-invasive activity without any influence on the proliferation. We have previously demonstrated that reactive oxygen species increase the invasion of AH109A cells. α-Tocopherol suppressed the reactive oxygen species-induced invasion but failed to suppress the reactive oxygen species-induced rises in intracellular peroxide level. GF 109203X, a protein kinase C inhibitor, decreased the invasive activity of AH109A cells. In contrast, phorbol-12-myristate-13-acetate, a protein kinase C activator, increased the invasive capacity of AH109A cells. α-Tocopherol suppressed the phorbol-12-myristate-13-acetate-induced increase in the invasion, and canceled the phorbol-12-myristate-13-acetate-induced rises in protein kinase C activity and phosphorylation of extracellular signal-regulated kinase. These results suggest that tocopherols, especially α-tocopherol, possess inhibitory effect more strongly on the invasion of AH109A cells than on the proliferation. They also suggest that the anti-invasive activity of α-tocopherol is raised through suppression of PKC/ERK signaling.

The effects of dietary supplementation with methionine and cystine on lipid metabolism, including the serum lipid concentration, were studied in Donryu rats subcutaneously implanted with an ascites hepatoma cell line (AH109A) for comparison with normal rats. A diet supplemented with 1.2% L-methionine or L-cystine to 20% casein was found to suppress the hepatoma-induced increases in serum triglyceride and total cholesterol concentration. The lipoprotein lipase activity in tissues was enhanced by dietary methionine and cystine, with no change in the mRNA level. Dietary methionine and cystine increased bile acid excretion into the feces with enhanced hepatic cholesterol 7alpha-hydroxylase activity. Dietary methionine and cystine affected the lipid metabolism differently in normal rats from hepatoma-bearing rats. These results suggest that dietary methionine and cystine each had a hypolipidemic effect against cancer-induced hyperlipidemia, and that the different actions observed in the hepatoma-bearing and normal rats may have been due to a metabolic abnormality caused by the cancer.

Honokiol, a neolignan, is a physiologically active component of kouboku (Magnolia obovata), a herb used in traditional Chinese medicine. This study investigated the effects of honokiol on the differentiation and function of osteoclasts induced by receptor activator of nuclear factor-kappaB ligand (RANKL). Honokiol markedly inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and the formation of TRAP-positive multinucleated cells in both bone marrow-derived monocytes and RAW264 cells. In experiments to elucidate its mechanism of action, honokiol was found to suppress RANKL-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). The RANKL-induced expressions of c-Fos and nuclear factor of activated T cells-c1 (NFATc1), which are crucial transcriptional factors for osteoclastogenesis, were also reduced by treatment with honokiol. Furthermore, honokiol induced disruption of the actin rings in mature osteoclasts (mOCs) without affecting the cell viability and suppressed osteoclastic pit formation on dentin slices. Taken together, these results suggest that honokiol inhibits osteoclast differentiation by suppressing the activation of MAPKs (p38 MAPK, ERK and JNK), decreasing the expressions of c-Fos and NFATc1, and attenuates bone resorption by disrupting the actin rings in mOCs. Therefore, honokiol could prove useful for the treatment of bone diseases associated with excessive bone resorption.

Effects of aspalathin, a green rooibos tea component, on glucose metabolism were studied in vitro and in vivo. We first examined the effect of aspalathin on glucose uptake by cultured L6 myotubes and on insulin secretion from cultured RIN-5F pancreatic beta-cells in vitro, and then investigated the effect of dietary aspalathin on fasting blood glucose level and conducted an intraperitoneal glucose tolerance test (IPGTT) using type 2 diabetes model mice in vivo. Aspalathin dose-dependently and significantly increased glucose uptake by L6 myotubes at concentrations 1-100 microM. It also significantly increased insulin secretion from cultured RIN-5F cells at 100 microM. Dietary aspalathin (0.1-0.2%) suppressed the increase in fasting blood glucose levels of db/db mice for 5 weeks. In IPGTT, aspalathin improved impaired glucose tolerance at 30, 60, 90, and 120 min in db/db mice. These results suggest that aspalathin has beneficial effects on glucose homeostasis in type 2 diabetes through stimulating glucose uptake in muscle tissues and insulin secretion from pancreatic beta-cells.

The aim of this study is to investigate the effects of theanine, a tea characteristic amino acid, on human lung cancer and leukemia cells. In the present study, we have demonstrated that theanine suppressed the in vitro and ex vivo growth of human non-small cell lung cancer A549 and leukemia K562 cell lines in dose- and time-dependant manners. In addition, theanine displayed the inhibitory effect on the migration of A549 cells. More importantly, theanine enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor), berbamine and norcantharidin (the anticancer drugs in China) by strongly reducing the viability and/or migration rate in A549 cells. In addition, theanine significantly suppressed A549 cell invasion. Suppression of A549 cell migration may be one of the important mechanisms of action of theanine against the A549 cell invasion. Our present results suggest that theanine may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human lung cancer and leukemia.

Matrine has shown therapeutic and/or adjuvant therapeutic effects on the treatment of some patients with breast cancer. However, its mechanisms of action are largely unknown. To disclose the mechanisms, we investigated in vitro and ex vivo effects of matrine on the cancer cells. Our results confirmed that matrine significantly suppressed the proliferation of highly-metastatic human breast cancer MDA-MB-231 cell line. Matrine displayed synergistic effects with existing anticancer agents celecoxib (the inhibitor of cyclooxygenase-2), trichostatin A (the histone deacetylase inhibitor) and rosiglitazone against the proliferation and VEGF excretions in MDA-MB-231 cells. Matrine induced the apoptosis and cell cycle arrest by reducing the ratios of Bcl-2/Bax protein and mRNA levels in the cancer cells. Matrine significantly reduced the invasion, MMP-9/MMP-2 activation, Akt phosphorylation, nuclear factor kappaB p-65 expression and DNA binding activity, and mRNA levels of MMP-9, MMP-2, EGF and VEGFR1 in MDA-MB-231 cells. Collectively, our results suggest that matrine inhibits the cancer cell proliferation and invasion via EGF/VEGF-VEGFR1-Akt-NF-kappaB signaling pathway.

Effect of [6]-gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [(3)H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. [6]-Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25-200 muM (proliferation) and 50-200 muM (invasion). [6]-Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. [6]-Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with [6]-gingerol, HX and XO or with [6]-gingerol and hydrogen peroxide. Furthermore, [6]-gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of [6]-gingerol may be involved in its anti-invasive activity of hepatoma cells.

糸球体腎炎と肝がん (AH109A) に付随して発生する続発性脂質異常症モデルラットを用いてその脂質代謝異常に対する栄養素, 非栄養素の作用と機構について解析を行った。がんモデルについては, 無限増殖性と転移性というがん細胞の有する二大生物学的特性を細胞培養系で再現する系を構築し, がんの顕在化を防ぎ二次予防に有用な食品成分の探索と作用機構の解析を行った。糸球体腎炎モデルラットにアミノ酸補足低タンパク質食たとえば低 (8.5%) カゼイン食 (8.5C) に少量のメチオニンとスレオニンを添加したアミノ酸補足食 (8.5CMT) を摂取させると, 著しいタンパク質栄養障害を伴うことなく, 脂質異常症はもちろんタンパク尿症, ときに低アルブミン血症を軽減化することを見出した。腎炎時には, 細胞間基質を蓄積させ糸球体硬化の原因となるTGF-βが腎で過剰発現し, 一方, 赤血球産生を促進するエリスロポエチン (EPO) の産生能は低下した。8.5 CMT食はTGF-βの過剰発現を抑制し, 血清EPO濃度を高めることが明らかとなった。低 (8.5%) 分離大豆タンパク質の場合も少量のメチオニンを補足するのみで, 成長遅延が是正されるとともに糸球体腎炎時の諸症状が軽減化されアミノ酸の栄養学的重要性が明らかとなった。また, 魚油や一部のポリフェノールも糸球体腎炎時の脂質異常症を改善しうることを見出した。肝がん移植ラットでは固形がんの形成とともに, 血清リポタンパク質像に異常が生じ, 高コレステロール血症と高トリグリセリド血症が発生する。このがん性脂質異常症の発症機構を解明するとともに, アミノ酸, システイン誘導体, 魚油などの有効性とその作用機構を解明した。培養肝がん細胞の増殖と浸潤 (がん転移の重要かつ特徴的段階) を抑制する食品成分の探索を行った。いくつかの成分については個体レベルでの効果も検証した。肝がん細胞の増殖抑制はアポトーシス誘導と細胞周期アレストによるもののほか, 固形がん内マクロファージのTNF産生を賦活化してがんを壊死させるものを見出した。浸潤抑制食品成分としてはレスベラトロール, テアニンなどを見出した。レスベラトロールはその抗酸化機能により活性酸素種 (ROS) を補足し, 細胞運動因子としても作動する肝細胞成長因子の発現を低下させ, その結果, 肝がん細胞浸潤を抑制することを明らかにした。一方, プロスタグランジン (PG) とくにPGE2は肝がん細胞浸潤亢進作用を示すことが明らかとなったので, レスベラトロールの別の浸潤抑制機構としてPG合成阻害作用も考えられた。緑茶中の旨味アミノ酸であるテアニンは, グルタミン酸受容体を介して浸潤抑制作用を発揮するものと考えられた。これらの知見から, ある種の食事処方や食品因子は疾病によっては予防的に作用するばかりではなく, 軽症化や治療的にも作用することが示唆された。

Objective Total parenteral nutrition containing a lipid emulsion is often employed after surgical tumor resection. This study investigated the effects of a fish oil–based infusion on rat hepatoma cell invasion. Methods Rat ascites hepatoma cell line AH109A was precultured with a fish oil–based or safflower oil–based emulsion for 48 h. Changes in membranous fatty acid composition were evaluated by gas chromatography. The invasiveness of hepatoma cells was assessed by coculturing with mesentery-derived mesothelial cells. To examine ex vivo effects of the fish oil–based infusion on hepatoma invasion, sera were prepared from rats infused with fish oil– or safflower oil–based emulsion and the effects of these sera were assessed. To clarify the mechanism of inhibition of invasion by the fish oil–based emulsion, the effects of prostaglandin (PG) E 2 and PGE 3 on invasion were examined. Results Pretreatment with the fish oil–based emulsion reduced invasiveness without affecting growth compared with the safflower oil–based emulsion. Pretreatment with the sera from rats infused with the fish oil–based emulsion also reduced invasiveness compared with the sera from rats infused with the safflower oil–based emulsion. The addition of PGE 2 eliminated the inhibitory effect of the fish oil–based emulsion, and the addition of PGE 3 reduced the invasiveness of hepatoma cells pretreated with the safflower oil–based emulsion. Conclusion These results suggest that the fish oil–based emulsion may have anti-invasive effects. Changes in the membranous fatty acid composition and consequent changes in the prostaglandins produced may be involved in this inhibitory effect.

Both epidemiological and experimental evidence is accumulating to show that a lignan-rich diet may reduce the risk of human breast cancer. Possible anti-cancer effects of dietary lignans on hepatomas or hepatoma cells have not been the topic of earlier studies. In the present study, we examined the effect of 7-hydroxymatairesinol (HMR) and its mammalian metabolite, enterolactone (ENL), on AH109A hepatoma cell proliferation and invasion in vitro. HMR and ENL inhibited the proliferation and invasion of AH109A hepatoma cells in vitro. The 50% inhibitory concentration (IC50) of hepatoma cell proliferation was lower for ENL (10 microM) than HMR (200 microM). Likewise, IC50 of hepatoma cell invasion was lower for ENL (9 microM) than HMR (144 microM). ENL suppressed hepatoma cell proliferation by accumulating cells in G1 phase and elongating doubling time of these cells, and by increasing the rate of apoptosis. Subsequently, we investigated in vivo the effect of dietary HMR and ENL on growth and metastasis of AH109A hepatomas in rats. Both of these compounds reduced the growth and metastasis of solid AH109A hepatomas in rats. These in vitro and in vivo findings suggest that HMR has inhibitory activities on tumor growth and metastasis in the hepatoma-bearing rats, and that this anti-tumor effect is mediated at least partially by ENL, a metabolite of HMR.

In late August 2014, dengue cases were reported in Japan, and a total of 162 cases were confirmed. In the present study, the envelope (E) gene sequences of 12 specimens from the dengue patients were determined. A dengue virus serotype 1 (DENV1) strain (D1/Hu/Shizuoka/NIID181/2014), which was clearly different from the first reported strain (D1/Hu/Saitama/NIID100/2014), was identified, although the other 11 specimens showed the same nucleotide sequences as D1/Hu/Saitama/NIID100/2014. The E gene sequences of two different strains were compared with those of nine DENV1 strains of imported cases in Japan in 2014. Phylogenetic analysis based on the E gene sequences showed that the D1/Hu/Saitama/NIID100/2014 strain was closely related to a strain isolated from an imported case from Singapore. Although no strain closely related to D1/Hu/Shizuoka/NIID181/2014 was found in these imported strains, the strain was closely related to isolates in Thailand and Taiwan in 2009. These data indicate that the dengue cases in Japan were caused by two different dengue virus strains that entered Japan through different means.

The characteristics of genotype V Japanese encephalitis virus (GV JEV) remain poorly understood as only two strains have been isolated to date. In this study, we examined the effects of the GV JEV Muar strain on in vitro growth and pathogenicity in mice; we also evaluated the efficacy of inactivated JEV vaccines against the Muar strain. Although growth of the Muar strain in mouse neuroblastoma N18 cells was clearly worse than that of the GIII Beijing-1 and GI Mie/41/2002 strains, neuroinvasiveness of the Muar strain was similar to that of the Beijing-1 strain and significantly higher than that of the Mie/41/2002 strain. The results of a plaque reduction neutralization test suggested that the neutralization ability of the JEV vaccines against the Muar strain was reduced compared with the GI and GIII strains. However, the protection potency of the JEV vaccine against the Muar strain was similar to that for the Beijing-1 strain in mice. Our data indicate that GV JEV has unique growth, virulence and antigenicity features.

L-Leucine is known to stimulate protein synthesis in L6 myotubes. In the present study, we examined the possible involvement of calcium signaling pathways in the stimulation of protein synthesis induced by L-leucine in L6 myotubes. After 16 h of treatment with L-leucine-depleted medium, the re-addition of L-leucine for 4 h augmented protein synthesis by about 50% as compared with an L-leucine-depleted control. Ryanodine receptor antagonists almost completely abolished the stimulatory effect of L-leucine, while IP(3) receptor antagonists showed partial inhibition when added simultaneously with L-leucine. These results suggest the possibility that calcium signaling pathways are involved in L-leucine-stimulated protein synthesis.