Not only metformin, but also D-allulose, alleviates metabolic disturbance and cognitive decline in prediabetic rats.

BACKGROUND: Prediabetes can be characterized as obesity with metabolic disturbance, leading to cognitive decline and brain pathologies. D-allulose administration in obese animals decreased metabolic disturbance. However, the comparative effects of D-allulose and metformin on cognition and brain functions in the diet-induced prediabetic condition are unclear. We assume that both D-allulose and metformin equally restore cognition and brain functions in prediabetic rats to an equal extent. MATERIALS AND METHODS: Fifty-six rats were randomly divided into two groups: a control and diet-induced prediabetic group which had received a normal diet (ND) and a high-fat diet (HFD) for 24 weeks, respectively. After dietary protocol had been followed for 12 weeks, ND rats were given solely drinking water daily for 12 weeks. HFD-prediabetic rats randomly received drinking water with either D-allulose (1.9 g/kg/day of D-allulose) or metformin (300 mg/kg/day of metformin) for 12 weeks. Following this, cognition and brain parameters were determined. RESULTS: Brain oxidative stress, mitochondrial dysfunction, microglial hyper-activation, apoptosis, brain insulin insensitivity, hippocampal synaptic dysfunction, and cognitive decline were observed in prediabetic rats. D-allulose and metformin equally attenuated brain oxidative stress, brain mitochondrial ROS production, hippocampal apoptosis, brain insulin insensitivity, hippocampal synaptic dysfunction, resulting in improved learning process in prediabetic rats. Metformin conferred greater advantage on the amelioration of brain mitochondrial dysfunction and brain microglial hyper-activation than D-allulose, resulting in improvement in both learning and memory processes in prediabetic rats. CONCLUSIONS: Not only metformin, but also D-allulose, has beneficial effects on the enhancement of brain function and cognition in prediabetic condition.

Antiallergic activities of shallot (Allium ascalonicum L.) and its therapeutic effects in allergic rhinitis.

BACKGROUND: Onion has antiallergic activity but lack of evidence for shallot. OBJECTIVE: To determine whether shallot owns similar antiallergic activity to onion and its therapeutic effects in allergic rhinitis when added to standard treatment. METHODS: In-vitro ß-hexosaminidase inhibitory activities of shallot was compared with onion on RBL-2H3 cells. In clinical study, a randomized, double-blind, placebo-controlled trial was performed. Sixteen AR patients were randomized equally into the controls who received cetirizine 10 mg once daily and placebo capsules for 4 weeks, and the treatment who received 3g of oral shallot per day (equivalent to 1 ½ bulbs) and cetirizine. Visual analog scores of overall symptoms (VAS), total nasal and ocular symptom scores (TNSS and TOSS), nasal airway resistance (NAR), and adverse events were assessed. RESULTS: Shallot extract at 200 µg/mL had an average ß-hexosaminidase inhibition rate of 97% while onion extract had 73%. HPLC chromatograms (λ = 290nm) of both plants showed nearly identical patterns of quercetin compounds, such as quercetin 3,4'-diglucoside, quercetin 4'-glucoside, and quercetin. After 4-week of treatment, 62.5% of patients in shallot group and 37.5% of patients in control group showed improvement of post-treatment VAS. TNSS were significantly reduced in both groups, however no difference between groups (P = 0.18). TOSS were significantly improved only in the shallot group (P = 0.01). Adverse events from shallot were not different from placebo. CONCLUSIONS: Shallot had antiallergic activity and similar quercetin compounds to onion. The shallot oral supplement and cetirizine was shown to improve the overall AR symptoms more than cetirizine alone.

D-allulose provides cardioprotective effect by attenuating cardiac mitochondrial dysfunction in obesity-induced insulin-resistant rats.

PURPOSE: Obesity-induced insulin resistant is associated with cardiovascular diseases via impairing cardiac mitochondria. Recently, D-allulose could protect ß-islets and improve insulin resistance. However, the effects of D-allulose on the heart and cardiac mitochondrial function under obesity-induced insulin-resistant condition has not been investigated. In this study, we aimed to investigate the effects of D-allulose on metabolic parameters, cardiac function, heart rate variability (HRV), cardiac mitochondrial function, and apoptosis in the heart of obesity-induced insulin-resistant rats induced by chronic high fat diet consumption. METHODS: Male Wistar rats (n = 24) received a normal fat diet (ND) or high fat diet (HFD) for 12 weeks. Then, HFD group was randomly divided into three subgroups to receive (1) HFD with distilled water, (2) HFD with 3% D-allulose 1.9 g/ kg·BW/ day (HFR), and (3) HFD with metformin 300 mg/kg·BW/ day (HFM) by diluted in drinking water daily for 12 weeks. At week 24, proposed study parameters were investigated. RESULTS: Chronic HFD consumption induced obesity-induced insulin resistant in rats and high fat diet impaired cardiac function and HRV. HFR rats had improved insulin sensitivity as indicated by decreasing HOMA index, plasma insulin, whereas HFM decreased body weight, visceral fat, plasma cholesterol, and plasma LDL. HFR and HFM provided similar efficacy in improving HRV and attenuating cardiac mitochondrial dysfunction, leading to improved cardiac function. CONCLUSIONS: Even though this is the first investigation of the D-allulose impact on the heart with a relatively small sample size, it clearly demonstrated a beneficial effect on the heart. D-allulose exerted a therapeutic effect on metabolic parameters except for body weight and lipid profiles and provided cardioprotective effects similar to metformin via attenuating cardiac mitochondrial function in obesity-induced insulin-resistant rats.

D-Tagatose Effectively Reduces the Number of Streptococcus mutans and Oral Bacteria in Healthy Adult Subjects: A Chewing Gum Pilot Study and Randomized Clinical Trial.

We examined the effect of D-Tagatose on the growth of oral bacteria including Streptococcus mutans (S. mutans). Saliva collected from 10 healthy volunteers was plated on BHI medium (to culture total oral bacteria) and MBS medium (to culture S. mutans, specifically). Agar plates of BHI or MBS containing xylitol or D-Tagatose were cultured under aerobic or anaerobic conditions. We then counted the number of colonies. In BHI plates containing D-Tagatose, a complete and significant reduction of bacteria occurred under both aerobic and anaerobic conditions. In MSB medium, significant reduction of S. mutans was also observed. We then performed a doubleblind parallel randomized trial with 19 healthy volunteers. They chewed gum containing xylitol, D-Tagatose, or both for 4 weeks, and their saliva was collected weekly and plated on BHI and MSB media. These plates were cultured under anaerobic conditions. Total bacteria and S. mutans were not effectively reduced in either the D-Tagatose or xylitol gum group. However, S. mutans was significantly reduced in volunteers chewing gum containing both D-Tagatose and xylitol. Thus, D-Tagatose inhibited the growth of S. mutans and many types of oral bacteria, indicating that D-Tagatose intake may help prevent dental caries, periodontitis, and many oral diseases.

Naturally occurring rare sugars are free radical scavengers and can ameliorate endoplasmic reticulum stress.

Because of potential use of naturally occurring rare sugars as sweeteners, their effect on superoxide (SO), hydroxyl and peroxyl radicals and endoplasmic reticulum (ER) stress was examined in human coronary artery endothelial cells. SO generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence. Phycoerythrin fluorescence based assay was used to monitor scavenging activity of sugars in the presence of hydroxyl or peroxyl radical generators [CuSO4 and azobis (2 amidinopropane) hydrochloride respectively]. Measurements were made in relative light units (RLU). ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (SAP) assay and by Western blot analysis of the expression and phosphorylation of key proteins in the unfolded protein response, namely CHOP47, eIF2α and JNK1. D-Glucose (27.5 mM) increased SO generation (5536 ± 283 vs. 2963 ± 205 RLU in controls; p < 0.0007) and decreased SAP secretion (73411 ± 3971 vs. 101749 ± 7652 RLU in controls; p < 0.005) indicating ER stress. Treatment of cells with 5.5 or 27.5 mM of D-allulose, D-allose, D-sorbose and D-tagatose reduced SO generation (all p < 0.05). This could not be attributed to inhibition of cellular uptake of dextrose by the rare sugars tested. In a cell free system, all four rare sugars had significantly more SO, hydroxyl and peroxyl radical scavenging activity compared to dextrose (all p < 0.01). Treatment of cells with rare sugars reduced ER stress. However, unlike other three rare sugars, D-sorbose did not inhibit tunicamycin-induced eIF2α phosphorylation. Naturally occurring rare sugars are free radical scavengers and can reduce ER stress.

PPARγ activation mitigates glucocorticoid receptor-induced excessive lipolysis in adipocytes via homeostatic crosstalk.

Proper balance between lipolysis and lipogenesis in adipocytes determines the release of free fatty acids (FFA) and glycerol, which is crucial for whole body lipid homeostasis. Although, dysregulation of lipid homeostasis contributes to various metabolic complications such as insulin resistance, the regulatory mechanism remains elusive. This study clarified the individual and combined roles for glucocorticoid receptor (GCR) and peroxisome proliferator-activated receptor (PPAR)γ pathways in lipid metabolism of adipocytes. In mature 3T3-L1 adipocytes, GCR activation using dexamethasone upregulated adipose triglyceride lipase (ATGL) and downregulated phosphoenolpyruvate carboxykinase (PEPCK), resulting in enhanced glycerol release into the medium. In contrast, PPARγ ligand pioglitazone modestly upregulated ATGL and hormone sensitive lipase (HSL), but markedly enhanced PEPCK and glycerol kinase (GK), thereby suppressed glycerol release. Dexamethasone showed permissive like effect on PPARγ target genes including perilipin A and aP2, therefore co-administration of dexamethasone and pioglitazone demonstrated synergistic upregulation of these enzymes excepting PEPCK, of which downregulation by dexamethasone was abolished by pioglitazone to the level above control. Thus, the excessive glycerol release was prevented as the net outcome of the co-administration. Consistently, the bodipy stain demonstrated that dexamethasone reduced the amount of cytosolic lipid, which was preserved in co-treated adipocytes. Moreover, silencing of PPARγ suppressed the synergistic effects of co-treatment on the lipolytic and lipogenic genes, and therefore the GCR pathway indeed involves PPARγ. In conclusion, crosstalk between GCR and PPARγ is largely synergistic but counter-regulatory in lipogenic genes, of which enhancement prevents excessive glycerol and possibly FFA release by glucocorticoids into the circulation.

IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression.

Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, ß-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis.

GPR120 in adipocytes has differential roles in the production of pro-inflammatory adipocytokines.

How nutritional excess leads to inflammatory responses in metabolic syndrome is not well characterized. Here, we evaluated the effects of ω-3 polyunsaturated fatty acid specific G-protein coupled receptor 120 (GPR120) activation on inflammatory pathways in adipocytes, and the influence of this process on macrophage migration. Using 3T3-L1 adipocytes, we found that agonizing GPR120 using its synthetic ligand, GSK137647, attenuated both basal and lipopolysaccharide-induced production of interleukin-6 (IL-6) and C-C motif chemokine ligand 2 (CCL2). Moreover, the intervention reduced the phosphorylation of nuclear factor kappa B inhibitor alpha (IκBα) and nuclear translocation of nuclear factor kappa-B p65 subunit (p65). Furthermore, the silencing of GPR120 itself reduced IL-6 and CCL2 mRNA expression. Inhibition of protein kinase C (PKC) augmented the down-regulatory effect of GSK137647 on IL-6 and CCL2 mRNA. Using a luciferase assay to measure promoter activity of the IL-6 gene in mouse embryonic fibroblasts, we demonstrated that exogenous transfection of GPR120 alone reduced the promoter activity, which was augmented by GSK137647. Inhibition of PKC further reduced the promoter activity. Nevertheless, RAW 264.7 macrophages grown in conditioned medium collected from GSK137647-treated adipocytes attenuated the expressions of matrix metalloproteinases-9 and -3, and tissue inhibitor of metalloproteinase-1. Conditioned medium also inhibited the lipopolysaccharide-induced migration of these macrophages. Taken together, these findings provide critical evidence that although GPR120 is associated with a PKC-mediated pro-inflammatory pathway, the direct inhibitory effects of GPR120 on the nuclear factor kappa B pathway are anti-inflammatory. Moreover, GPR120 activity can attenuate the adipocyte-mediated enhanced production of extracellular matrix-modulating factors in macrophages and can reduce their migration by a paracrine mechanism.

Increase in tumor suppressor Arf compensates gene dysregulation in in vitro aged adipocytes.

Deterioration of adipocyte function due to increased oxidative stress predisposes patients to metabolic disorders in advanced age. However, the roles of tumor suppressors in such conditions remain largely unknown. Therefore, we aimed to address their dynamics in aged adipocytes using a long-term culture model. We compared 3T3-L1 adipocytes at 17-19 days (long-term) with those at 8-10 days (short-term) after initiation of adipogenic induction for mimicking 'aged' and 'young' adipocytes, respectively. H2O2 release and dihydroethidium (DHE) staining was increased, while superoxide dismutase (SOD) activity was reduced in long-term cultured adipocytes, which is suggestive of enhanced oxidative stress in this group. Moreover, qRT-PCR revealed increased mRNAs of Nox4 (a subunit of NADPH oxidase complex), Ccl2 (a proinflammatory chemokine) and Il6 [a marker of senescence-associated secretory phenotype (SASP)] along with decreased levels of Pparγ, Adipoq and Slc2a4 (genes related to glucose metabolism). These alterations were associated with increased expression of the tumor suppressors alternate-reading-frame protein p19Arf (Arf) and p16Ink4a. However, silencing of Arf reduced mRNAs of Adipoq and Slc2a4 and enhanced release of Il6. The effect was opposite in Arf overexpressing adipocytes, which showed reduced superoxide production as assessed with DHE staining and SOD activity. Western blots showed that Arf negatively regulates the phosphorylation of Akt. Luciferase assay in Hela cells additionally suggested that Arf negatively regulates Il6 transcriptional activity through a PI3 K/Akt mediated pathway. These findings strongly suggest that the enhanced Arf expression in oxidative stress plays compensatory protective roles against aging-related dysregulation of gene expression in adipocytes.

D-Allose Inhibits Cancer Cell Growth by Reducing GLUT1 Expression.

Glucose is a major energy source for mammalian cells and is transported into cells via cell-specific expression of various glucose transporters (GLUTs). Especially, cancer cells require massive amounts of glucose as an energy source for their dysregulated growth and thus over-express GLUTs. d-allose, a C-3 epimer of d-glucose, is one of rare sugars that exist in small quantities in nature. We have shown that d-allose induces the tumor suppressor gene coding for thioredoxin interacting protein (TXNIP) and inhibits cancer cell growth by G1 cell cycle arrest. It has also been reported that GLUTs including GLUT1 are over-expressed in many cancer cell lines, which may contribute to larger glucose utilization. Since d-allose suppresses the growth of cancer cells through the upregulation of TXNIP expression, our present study focused on whether d-allose down-regulates GLUT1 expression via TXNIP expression and thus suppresses cancer cell growth. Western blot and real-time PCR analyses revealed that d-allose significantly induced TXNIP expression and inhibited GLUT1 expression in a dose-dependent manner in three human cancer cell lines: hepatocellular carcinoma (HuH-7), Caucasian breast adenocarcinoma (MDA-MB-231), and neuroblastoma (SH-SY5Y). In these cell lines, d-allose treatment inhibited cell growth. Importantly, d-allose treatment decreased glucose uptake, as measured by the uptake of 2-deoxy d-glucose. Moreover, the reporter assays showed that d-allose decreased the expression of luciferase through the hypoxia response element present in the tested promoter region. These results suggest that d-allose may cause the inhibition of cancer growth by reducing both GLUT1 expression and glucose uptake.

Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.

d-Allose Attenuates Overexpression of Inflammatory Cytokines after Cerebral Ischemia/Reperfusion Injury in Gerbil.

BACKGROUND: The present study investigates the effects of d-allose, a rare sugar, on the inflammatory response after transient forebrain ischemia in the gerbil and whether it reduces oxidative stress (8-hydroxyl-2'-deoxyguanosine levels) and behavioral deficits. METHODS: Transient forebrain ischemia was induced by occlusion of the bilateral common carotid arteries for 5 minutes. d-Allose was intraperitoneally injected immediately after ischemia (400 mg/kg). Inflammatory cytokines and oxidative damage in the hippocampus and behavioral deficits were examined 3 days after ischemia. RESULTS: d-Allose administration reduced ischemia-induced cytokine production, oxidative stress, and behavioral deficits (motor and memory related). CONCLUSIONS: The present results suggest that d-allose reduces brain injury after transient global ischemia by suppressing inflammation as well as by inhibiting oxidative stress.

HIGH RATE OF HEPATITIS B VIRUS MOTHER-TO-CHILD TRANSMISSION IN LAO PEOPLE'S DEMOCRATIC REPUBLIC.

Hepatitis B virus infection is endemic in Lao People's Democratic Republic (PDR). Among 3,000 pregnant women attending an antenatal clinic at Mother and Child Hospital in Vientiane, Lao PDR, 5.8% were HBsAg positive by a rapid test. Among serum samples of 47 infants aged 9-12 months born to HBsAg-positive mothers, 38% were anti-HBs negative. Percent anti-HBs negative children is significantly higher in those born to HBeAg positive mothers than in those born to HBeAg negative mothers (60% vs 25%, p < 0.05). Out of 47 HBsAg-positive mothers, 10 had infants who were HBsAg positive. None of the infants born to HBsAg negative mothers became HBsAg positive but 10/19 (52.6%) of infants born to HBeAg positive mothers became HBsAg positive. This high rate of mother-to-child transmission of HBV in an endemic country is of concern and indicates that routine vaccination program for Lao infants needs strengthening.

Potent inhibitory effects of D-tagatose on the acid production and water-insoluble glucan synthesis of Streptococcus mutans GS5 in the presence of sucrose.

We examined and compared the inhibitory effects of D-tagatose on the growth, acid production, and water-insoluble glucan synthesis of GS5, a bacterial strain of Streptococcus mutans, with those of xylitol, D-psicose, L-psicose and L-tagatose. GS5 was cultured for 12h in a medium containing 10% (w/v) of xylitol, D-psicose, L-psicose, D-tagatose or L-tagatose, and the inhibitory effect of GS5 growth was assessed. Each sugar showed different inhibitory effects on GS5. Both D-tagatose and xylitol significantly inhibited the acid production and water-insoluble glucan synthesis of GS5 in the presence of 1% (w/v) sucrose. However, the inhibitory effect of acid production by D-tagatose was significantly stronger than that of xylitol in presence of sucrose.

Comparing the cultivated cochlear cells derived from neonatal and adult mouse.

BACKGROUND: Previous reports showed the presence of limited numbers of stem cells in neonatal murine cochlear sensory epithelia and these cells are progressively lost during the postnatal development. The goal of this study was to investigate whether stem cells can be derived from mature mouse cochleae under suspension culture conditions, and to analyze the expression of the stem cell and inner ear progenitor cell markers in cells dissociated from neonatal and adult mouse organs of Corti. METHODS: Organs of Corti were dissected from postnatal day 1 (P1) or postnatal day 60 (P60) mouse. The dissociated cells were cultivated under suspension cultures conditions. Reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were conducted for phenotype characterization. RESULTS: The number of cochlear stem cells (otospheres) yielded from P1 organ of Corti was significantly higher than that of the P60 organ of Corti. RT-PCR analyses showed that the stem markers, such as nanog, sox2, klf4, and nestin can be found to be distributed similarly in the cells derived from both of organisms, but the inner ear developmental/progenitor cell markers showed lower expression in P60 organ of Corti compared to P1. Immunocytochemistry results also revealed the evidence that P60 otospheres lacking of differentiation potential in vitro, which opposed to the strong differentiation potential of otospheres at P1 stage. CONCLUSIONS: Our findings suggest that the loss of numbers and features of stem cells in the adult organ of Corti is associated with the substantial down-regulation of inner ear progenitor key-markers during maturation of the cells in organ of Corti.

Effects of D-Allulose with Sucrose Beverage on Glucose Tolerance and Insulin Levels among Thai Healthy Volunteers.

D-Allulose has blood glucose suppression effects in both animal and clinical studies. The mechanism mediating glucose suppression in animals is controlled by several actions including the inhibition of sucrase. To investigate the dose-response effects of D-allulose with a sucrose beverage on glucose tolerance and insulin levels using Thai volunteers. This was a prospective, randomized, double-blinded, crossover study. Subjects had five oral sucrose tolerance tests (OSTT) with escalating doses of D-allulose (0, 2.5, 5, 7.5 or 10 g) with a 50 g sucrose beverage in a random order once a week for five consecutive weeks. The five drinks were consumed in a random order; the order being blinded for both subjects and investigators. Blood samples were drawn immediately before consumption and at 30, 60, 90 and 120 min after consumption of the study product for measurement of plasma glucose and insulin levels. Thirty healthy subjects (11 men and 19 women) completed the study. The peak postprandial glucose (PePPG) and insulin levels (PePPI) were lower when D-allulose was added in a dose-dependent manner. The lowest plasma glucose and insulin levels occurred at 120 min after OSTT in all five products and they were raised when D-allulose was added in a dose-dependent manner. D-Allulose has a suppression response on glucose and insulin shown by the decrease in postprandial plasma glucose and insulin levels following the addition of D-allulose to sucrose in a dose-dependent manner. The more D-allulose added, the less marked the glucose and insulin response occurred.

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation.

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.

Evaluation of 3'-deoxy-3'-[18F]-fluorothymidine (18F-FLT) kinetics correlated with thymidine kinase-1 expression and cell proliferation in newly diagnosed gliomas.

PURPOSE: The thymidine analog 3'-deoxy-3'-[(18)F]fluorothymidine ((18)F-FLT) has been developed as a positron emission tomography (PET) tracer to assess the proliferation activity of tumors in vivo. The present study investigated the relationship between the kinetic parameters of (18)F-FLT in vivo and thymidine kinase-1 (TK-1) expression and cell proliferation rate in vitro, and blood-brain barrier (BBB) breakdown in human brain gliomas. METHODS: A total of 21 patients with newly diagnosed gliomas were examined by (18)F-FLT PET kinetic analysis. Maximum standardized uptake value (SUVmax) and tumor-to-normal (T/N) ratio of (18)F-FLT in the tumor and (18)F-FLT kinetic parameters in the corresponding contralateral region were determined. The expression levels of TK-1 protein and mRNA were determined by immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR), respectively, using surgical specimens. The cell proliferation rate of the tumor was determined in terms of the Ki-67 labeling index. BBB breakdown was evaluated on MR images with contrast enhancement. RESULTS: (18)F-FLT SUVmax and T/N ratio were significantly correlated with the influx rate constant (K (1); P = 0.001 and P < 0.001, respectively), but not with the phosphorylation rate constant (k (3)). IHC and real-time PCR studies demonstrated a significant correlation between K (1) and TK-1 mRNA expression (P = 0.001), but not between k (3) and TK-1 protein and mRNA expression. Linear regression analysis revealed a significant correlation between K (1) and the Ki-67 index (P = 0.003), but not between k (3) and the Ki-67 index. TK-1 mRNA expression was significantly correlated with the Ki-67 index (P = 0.009). (18)F-FLT SUVmax and T/N ratio were significantly correlated with BBB breakdown evaluated by contrast enhancement in MR images (P = 0.003 and P = 0.011, respectively). CONCLUSION: These results indicate that (18)F-FLT uptake in the tumor is significantly related to transport through the disrupted BBB, but not through phosphorylation activity. Although the tissue TK-1 expression reflects tumor proliferation activity, the phosphorylation rate constant k (3) determined by (18)F-FLT PET kinetic analysis does not accurately reflect TK-1 expression in the tissue and should not be used as a surrogate biomarker of cell proliferation activity in human brain gliomas.

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

BACKGROUND: Accumulating 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. METHODS: Expression 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. RESULTS: NMDA 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. CONCLUSIONS: Our 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.