Kazuyo Kamitori

Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats

A rare sugar, D-psicose has progressively been evaluated as a unique metabolic regulator of glucose and lipid metabolism, and thus represents a promising compound for the treatment of type 2 diabetes mellitus (T2DM). The present study was undertaken to examine the underlying effector organs of D-psicose in lowering blood glucose and abdominal fat by exploiting a T2DM rat model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Rats were fed 5% D-psicose or 5% D-glucose supplemented in drinking water, and only water in the control for 13 weeks and the protective effects were compared. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. After 13 weeks feeding, D-psicose treatment significantly reduced the increase in body weight and abdominal fat mass. Oral glucose tolerance test (OGTT) showed the reduced blood glucose and insulin levels suggesting the improvement of insulin resistance in OLETF rats. Oil-red-O staining elucidated that D-psicose significantly reduced lipid accumulation in the liver. Immunohistochemical analysis showed D-psicose induced glucokinase translocation from nucleus to cytoplasm of the liver which enhances glucokinase activity and subsequent synthesis of glycogen in the liver. D-psicose also protected the pathological change of the β-cells of pancreatic islets. These data demonstrate that D-psicose controls blood glucose levels by reducing lipotoxicity in liver and by preserving pancreatic β-cell function.

FOXO/TXNIP pathway is involved in the suppression of hepatocellular carcinoma growth by glutamate antagonist MK-801

BackgroundAccumulating evidence has suggested the importance of glutamate signaling in cancer growth, yet the signaling pathway has not been fully elucidated. N-methyl-D-aspartic acid (NMDA) receptor activates intracellular signaling pathways such as the extracellular-signal-regulated kinase (ERK) and forkhead box, class O (FOXO). Suppression of lung carcinoma growth by NMDA receptor antagonists via the ERK pathway has been reported. However, series of evidences suggested the importance of FOXO pathways for the regulation of normal and cancer cell growth. In the liver, FOXO1 play important roles for the cell proliferation such as hepatic stellate cells as well as liver metabolism. Our aim was to investigate the involvement of the FOXO pathway and the target genes in the growth inhibitory effects of NMDA receptor antagonist MK-801 in human hepatocellular carcinoma.MethodsExpression of NMDAR1 in cancer cell lines from different tissues was examined by Western blot. NMDA receptor subunits in HepG2, HuH-7, and HLF were examined by reverse transcriptase polymerase chain reaction (RT-PCR), and growth inhibition by MK-801 and NBQX was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of MK-801 on the cell cycle were examined by flow cytometry and Western blot analysis. Expression of thioredoxin-interacting protein (TXNIP) and p27 was determined by real-time PCR and Western blotting. Activation of the FOXO pathway and TXNIP induction were examined by Western blotting, fluorescence microscopy, Chromatin immunoprecipitation (ChIP) assay, and reporter gene assay. The effects of TXNIP on growth inhibition were examined using the gene silencing technique.ResultsNMDA receptor subunits were expressed in all cell lines examined, and MK-801, but not NBQX, inhibited cell growth of hepatocellular carcinomas. Cell cycle analysis showed that MK-801 induced G1 cell cycle arrest by down-regulating cyclin D1 and up-regulating p27. MK-801 dephosphorylated Thr24 in FOXO1 and induced its nuclear translocation, thus increasing transcription of TXNIP, a tumor suppressor gene. Knock-down of TXNIP ameliorated the growth inhibitory effects of MK-801.ConclusionsOur results indicate that functional NMDA receptors are expressed in hepatocellular carcinomas and that the FOXO pathway is involved in the growth inhibitory effects of MK-801. This mechanism could be common in hepatocellular carcinomas examined, but other mechanisms such as ERK pathway could exist in other cancer cells as reported in lung carcinoma cells. Altered expression levels of FOXO target genes including cyclin D1 and p27 may contribute to the inhibition of G1/S cell cycle transition. Induction of the tumor suppressor gene TXNIP plays an important role in the growth inhibition by MK-801. Our report provides new evidence that FOXO-TXNIP pathway play a role in the inhibition of the hepatocellular carcinoma growth by MK-801.

Hepatocyte Growth Factor-induced Differential Activation of Phospholipase Cγ1 and Phosphatidylinositol 3-Kinase Is Regulated by Tyrosine Phosphatase SHP-1 in Astrocytes

Hepatocyte growth factor (HGF) elicits pleiotropic effects on various types of cells through the c-Met receptor tyrosine kinase. However, the mechanisms underlying the diverse responses of cells remain unknown. We show here that HGF promoted chemokinesis of rat primary astrocytes through the activation of phosphatidylinositol 3 (PI3)-kinase without any influence on mitogenesis of the cells. Under the same condition, phospholipase Cγ1 (PLCγ1), which is another signal mediator of c-Met, was not tyrosine-phosphorylated during HGF stimulation. However, treatment of the cells with orthovanadate, a tyrosine phosphatase inhibitor, restored the HGF-induced tyrosine phosphorylation of PLCγ1. A tyrosine phosphatase, SHP-1, was associated with both PI3-kinase and PLCγ1 before HGF stimulation, but it was dissociated only from PI3-kinase after the stimulation. Furthermore, transfectants of catalytically inactive mutant of SHP-1 showed tyrosine phosphorylation of PLCγ1 and mitogenic responses to HGF, and the mitogenic response was blocked with U73122, an inhibitor of phosphatidylinositol-specific PLC, and calphostin C, an inhibitor of protein kinase C downstream of the PLCγ1. These results indicate that PLCγ1 is selectively prevented from being a signal mediator by constitutive association of SHP-1, and that this selective inhibition of PLCγ1 may determine the cellular response of astrocytes to HGF.

Eicosapentaenoic acid upregulates VEGF-A through both GPR120 and PPARγ mediated pathways in 3T3-L1 adipocytes.

Vascular endothelial growth factor-A (VEGF-A) released from adipocytes promotes angiogenesis; and thereby ameliorates the local hypoxia-induced adipose inflammation and insulin resistance. Here, we newly found that eicosapentaenoic acid (EPA) upregulated both mRNA expression and release of VEGF-A in mature 3T3-L1 adipocytes. Silencing mRNA of G-protein coupled receptor 120 (GPR120) and specific inhibition of peroxisome proliferator-activated receptor γ (PPARγ) by GW9662 respectively attenuated the EPA-induced augmentation of VEGF-A release by adipocytes. Furthermore, transfection of GPR120 gene alone and PPARγ gene alone to HEK293 cells respectively increased the promoter activity of VEGF-A as assessed by luciferase reporter assay, which was further augmented when both genes were co-transfected. Promoter deletion analysis and chromatin immunoprecipitation assay revealed that co-transfection of GPR120 enhanced EPA-induced PPARγ binding to PPAR-response element in VEGF-A promoter region. Thus, by the synchronized activation of a membrane receptor GRP120 and a nuclear receptor PPARγ, EPA enhances VEGF-A production in adipocytes.

Selective activation of phospholipase Cγ1 and distinct protein kinase C subspecies in intracellular signaling by Hepatocyte growth factor/scatter factor in primary cultured rat neocortical cells

Abstract: Hepatocyte growth factor/scatter factor (HGF) was recently reported to function as a neurotrophic factor in the CNS. To investigate the intracellular signal pathways after activation of the HGF receptor c‐Met in primary cultured rat neocortical cells, in vitro kinase assays were performed. HGF stimulation enhances the phosphorylation of endogenous 80‐ and 45‐kDa substrates. Studies with protein kinase inhibitors and phorbol 12‐myristate 13‐acetate showed that protein kinase C (PKC) is activated intracellularly. The 80‐kDa protein was identified to be the major PKC substrate MARCKS. Although four PKC subspecies, PKCα, PKCε, PKCγ, and PKCλ, were expressed in the cells, only PKCα, PKCε, and PKCγ were selectively translocated in the plasma membrane after HGF stimulation. As expected from these three PKC subspecies, phosphorylation of phospholipase Cγ1 (PLCγ1) but not phosphatidylinositol 3‐kinase was enhanced, although the stimulation of brain‐derived neurotrophic factor induced phosphorylation of phosphatidylinositol 3‐kinase. In contrast to the neocortical cells, HGF did not enhance phosphorylation of PLCγ1 in primary astrocytes. We also found that activated PKC(s) served as a major mitogen‐activated protein kinase activator in this pathway. These findings suggest that HGF exerts neurotrophic effects through selective phosphorylation of PLCγ1 and activation of distinct PKC subspecies in neocortical cells, most likely neurons.

Rare sugar D-psicose protects pancreas β-islets and thus improves insulin resistance in OLETF rats.

Rare sugar D-psicose has cropped up as a non-toxic and effective compound to protect and preserve pancreatic β-islets in the growing type 2 diabetes mellitus (T2DM) rats through the regulation of glucose and fat metabolism. The present study was undertaken to examine the effect of rare sugar D-psicose on the protection of pancreatic β-islets using Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a T2DM model. Treated rats were fed with 5% D-psicose or 5% D-glucose supplemented drinking water, and only water in the control for 13 weeks. A non-diabetic Long-Evans Tokushima Otsuka (LETO), fed with water served as a counter control of OLETF. D-Psicose significantly attenuated progressive β-islet fibrosis and preserved islets, evaluated by hematoxylin-eosin staining, Massons trichrome staining and immunostainings of insulin and α-smooth muscle actin (SMA). D-Psicose significantly reduced increase in body weight and abdominal fat deposition. Oral glucose tolerance test (OGTT) showed reduced blood glucose levels suggesting the improvement of insulin resistance. All these data suggests that D-psicose protected and preserved pancreatic β-islets through the maintenance of hyperglycemia and by the prevention of fat accumulation in OLETF rats.

Growth inhibition of head and neck carcinomas by D-allose.

An inhibitory effect of D‐allose, a rare sugar, on several cancer cell lines has been reported. This study aimed to investigate the growth inhibition of head and neck squamous cell carcinoma cells by D‐allose.

Rare sugar d-allose enhances anti-tumor effect of 5-fluorouracil on the human hepatocellular carcinoma cell line HuH-7

d-Allose is a novel anti-tumor monosaccharide that causes cell growth inhibition, specifically of the cancer cells, by inducing the tumor suppressor gene thioredoxin interacting protein (TXNIP). The commonly used anti-tumor drug, 5-fluorouracil (5-FU), blocks the cell cycle by inhibiting thymidylate synthase, and is also known to induce TXNIP gene expression. In this study, we examined the synergistic effect of d-allose and 5-FU and the role of TXNIP on cancer cell growth. The treatment of HuH-7 cells with d-allose or 5-FU inhibited the cell growth in a dose-dependent manner (75.2+/-2.7% with 50 mM d-allose and 66.1+/-2.7% with 0.5 mug/ml 5-FU) and d-allose enhanced the anti-tumor effect of 5-FU (55.3+/-1.1 %). TUNEL analysis did not show any evidence of apoptosis with either d-allose or 5-FU treatment. 5-FU suppressed the expression of p27(kip1), p53, and cyclin E, whereas d-allose induced p53 and reduced cyclins D, A, and E. The expression of p27(kip1) remained unchanged by d-allose at transcriptional level, but increased at the protein level suggesting an increase in protein stability by TXNIP. d-Allose and to a lesser extent 5-FU induced TXNIP expression significantly (808.4+/-122.9% and 186.8+/-32.9%, respectively) and the combination of both further enhanced TXNIP expression. As d-allose has no known side effects on normal cells, the combination of d-allose and 5-FU might be a potent candidate for cancer therapy.

Analysis of the inhibitory mechanism of D-allose on MOLT-4F leukemia cell proliferation

D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars found in nature. In the present study, we have elucidated for the first time that various leukemia cell lines have different susceptibility to anti-proliferative activity of D-allose, and that this difference is related to the difference in induction of thioredoxin interacting protein (TXNIP) expression. We examined 5 leukemia cell lines (MOLT-4F, IM-9, HL-60, BALL-1 and Daudi), and found that MOLT-4F (T-cell lymphoblastic leukemia) had the highest susceptibility to D-allose, and that Daudi (Burkitts lymphoma) had the lowest. D-Allose significantly slowed the cell cycle progression without causing apoptosis of MOLT-4F cells. Intracellular TXNIP expression was specifically and markedly enhanced in MOLT-4F cells by D-allose treatment, and subsequent increase of p27(kip1), a cell cycle inhibitor, was observed. On the other hand, D-allose did not increase TXNIP and p27(kip1) levels at all in Daudi cells. These results indicate that D-allose suppresses MOLT-4F cell proliferation possibly by the inhibition of cell cycle progression via induction of TXNIP expression.

Enzymatic synthesis of novel disaccharides using disaccharide phosphorylases.

A method for the synthesis of novel disaccharides was developed. It involved the following two steps. The first step consisted of two continuous reactions: the conversion of maltose to beta-D-glucose-1-phosphate and D-glucose by the phosphorolytic activity of maltose phosphorylase and the specific consumption of only D-glucose by incubation with glucose-consuming yeast cells. The second step involved the addition of an appropriate carbohydrate and its condensation with the remaining beta-D-glucose-1-phosphate by the synthetic activity of maltose phosphorylase or trehalose phosphorylase. Several factors affecting the yields of disaccharides were optimized. Using this method, five maltose-like derivatives and two trehalose-like derivatives were synthesized from maltose and the corresponding carbohydrates. Among these, 4-O-alpha-D-glucopyranosyl-L-fucopyranose (Glc(alpha1-4)Fuc) and alpha-d-glucopyranosyl alpha-D-fucopyranoside (Glc(alpha1-1alpha)Fuc) were purified, and identified by 1H NMR and 13C NMR.