Effects of angiotensin II AT1 receptor inhibition and exercise training on insulin action in rats on high-fat diet

AimThis study was to determine whether combination of the angiotensin II AT1 receptor blocker (ARB), candesartan cilexetil, and exercise training can prevent the development of high-fat diet-induced insulin resistance.Main methodsF344/NSlc rats were fed normal chow diet or high-fat (HF) diet for 7 weeks. The HF-fed rats were either administered candesartan cilexetil (5 mg·kg^? 1·day^? 1), exercise-trained, or received a combination of these 2 treatments.Key findingsOral glucose tolerance tests (OGTT) showed that combined treatment with candesartan cilexetil and exercise increased glucose tolerance as compared with each treatment alone in HF-fed rats. Moreover, euglycemic–hyperinsulinemic clamp analysis showed improvement in glucose infusion rate with exercise training or candesartan cilexetil treatment alone, and further improvement was observed with the combination treatment. Systolic blood pressure improved with candesartan cilexetil but not with exercise alone. Finally, Glut-4 protein expression in soleus muscle was decreased with HF diet, and the expression was increased by exercise and not candesartan cilexetil treatment.SignificanceThese results suggest that the combination of candesartan cilexetil and exercise training improves insulin resistance as compared with each treatment alone.     

Discrimination of genotoxic and non-genotoxic hepatocarcinogens by statistical analysis based on gene expression profiling in the mouse liver as determined by quantitative real-time PCR

The general aim of the present study is to discriminate between mouse genotoxic and non-genotoxic hepatocarcinogens via selected gene expression patterns in the liver as analyzed by quantitative real-time PCR (qPCR) and statistical analysis. qPCR was conducted on liver samples from groups of 5 male, 9-week-old B6C3F(1) mice, at 4 and 48h following a single intraperitoneal administration of chemicals. We quantified 35 genes selected from our previous DNA microarray studies using 12 different chemicals: 8 genotoxic hepatocarcinogens (2-acetylaminofluorene, 2,4-diaminotoluene, diisopropanolnitrosamine, 4-dimethylaminoazobenzene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosomorpholine, quinoline and urethane) and 4 non-genotoxic hepatocarcinogens (1,4-dichlorobenzene, dichlorodiphenyltrichloroethane, di(2-ethylhexyl)phthalate and furan). A considerable number of genes exhibited significant changes in their gene expression ratios (experimental group/control group) analyzed statistically by the Dunnett's test and Welch's t-test. Finally, we distinguished between the genotoxic and non-genotoxic hepatocarcinogens by statistical analysis using principal component analysis (PCA) of the gene expression profiles for 7 genes (Btg2, Ccnf, Ccng1, Lpr1, Mbd1, Phlda3 and Tubb2c) at 4h and for 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2 and Tubb2c) at 48h. Seven major biological processes were extracted from the gene ontology analysis: apoptosis, the cell cycle, cell proliferation, DNA damage, DNA repair, oncogenes and tumor suppression. The major, biologically relevant gene pathway suggested was the DNA damage response pathway, resulting from signal transduction by a p53-class mediator leading to the induction of apoptosis. Eight genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Phlda3 and Plk2) that are directly associated with Trp53 contributed to the PCA. The current findings demonstrate a successful discrimination between genotoxic and non-genotoxic hepatocarcinogens, using qPCR and PCA, on 12 genes associated with a Trp53-mediated signaling pathway for DNA damage response at 4 and 48 h after a single administration of chemicals.     

Design and synthesis of a metabolically stable and potent antitussive agent, a novel delta opioid receptor antagonist, TRK-851

We have previously reported on antitussive effect of (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-5',6'-dihydro-3-methoxy-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-14-ol(1b) methanesulfonate (TRK-850), a selective delta opioid receptor antagonist which markedly reduced the number of coughs in a rat cough model. We designed TRK-850 based on naltrindole (NTI), a typical delta opioid receptor antagonist, to improve its permeability through the blood-brain barrier by introducing hydrophobic moieties to NTI. The ED(50) values of NTI and compound 1b by intraperitoneal injections were 104 microg/kg and 2.07 microg/kg, respectively. This increased antitussive potency probably resulted from the improved brain exposure of compound 1b. However, 1b was extremely unstable toward metabolism by cytochrome P450. In this study, we designed and synthesized compound 1b derivatives to improve the metabolic instability, which resulted in affording highly potent and metabolically stable oral antitussive agent (5R,9R,13S,14S)-17-cyclopropylmethyl-6,7-didehydro-4,5-epoxy-8'-fluoro-5',6'-dihydro-4'H-pyrrolo[3,2,1-ij]quinolino[2',1':6,7]morphinan-3,14-diol (1c) methanesulfonate (TRK-851).     

Effects of rhinovirus infection on histamine and cytokine production by cell lines from human mast cells and basophils

To understand the biochemical events that occur in the airways after rhinovirus (RV) infection, we developed for the first time a model in which the cell lines from human mast cells (HMC-1) and basophils (KU812) can be infected with RV14, a major group RV. Viral infection was confirmed by demonstrating that viral titers in culture supernatants, and RV RNA increased with time. RV14 infection alone and a combination of PMA plus calcium ionophore A23187, did not increase histamine production by these cells, although IgE plus anti-IgE increased the histamine production. However, histamine content in the supernatants increased in response to PMA plus A23187, or IgE plus anti-IgE after RV14 infection. PMA plus A23187 or IgE plus anti-IgE induced the production of IL-8 and GM-CSF in supernatants of HMC-1 cells and IL-4 and IL-6 in supernatants of KU812 cells. RV14 infection further increased the production of the cytokines, whereas RV14 infection alone did not alter the production of the cytokines by these cells. An Ab to ICAM-1 inhibited RV14 infection of the cells and decreased the production of cytokines and histamine after RV14 infection. RV14 infection enhanced the increases in intracellular calcium concentration and activation of NF-kappaB by PMA plus A23187 in the cells. These findings suggest that RV14 infection may prime the cytokine and histamine production from mast cells and basophils and may cause airway inflammation in asthma.     

Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF- were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF- exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF- exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.     

Proteomic analysis of human brain identifies alpha-enolase as a novel autoantigen in Hashimoto's encephalopathy

Hashimoto's encephalopathy (HE) is a rare autoimmune disease associated with Hashimoto's thyroiditis (HT). To identify the HE-related autoantigens, we developed a human brain proteome map using two-dimensional electrophoresis and applied it to the immuno-screening of brain proteins that react with autoantibodies in HE patients. After sequential MALDI-TOF-MASS analysis, immuno-positive spots of 48 kDa (pI 7.3-7.8) detected from HE patient sera were identified as a novel autoimmuno-antigen, alpha-enolase, harboring several modifications. Specific high reactivities against human alpha-enolase were significant in HE patients with excellent corticosteroid sensitivity, whereas the patients with fair or poor sensitivity to the corticosteroid treatment showed less reactivities than cut-off level. Although a few HT patients showed faint reactions to alpha-enolase, 95% of HT patients, patients with other neurological disorders, and healthy subjects tested were all negative. These results suggest that the detection of anti-alpha-enolase antibody is useful for defining HE-related pathology, and this proteomic strategy is a powerful method for identifying autoantigens of various central nervous system diseases with unknown autoimmune etiologies.     

Structures of (-)-epicatechin glucuronide identified from plasma and urine after oral ingestion of (-)-epicatechin: differences between human and rat

(-)-epicatechin is one of the most potent antioxidants present in the human diet. Particularly high levels are found in black tea, apples, and chocolate. High intake of catechins has been associated with reduced risk of cardiovascular diseases. There have been several reports concerning the bioavailability of catechins, however, the chemical structure of (-)-epicatechin metabolites in blood, tissues, and urine remains unclear. In the present study, we purified and elucidated the chemical structure of (-)-epicatechin metabolites in human and rat urine after oral administration. Three metabolites were purified from human urine including (-)-epicatechin-3'-O-glucuronide, 4'-O-methyl-(-)-epicatechin-3'-O-glucuronide, and 4'-O-methyl-(-)-epicatechin-5 or 7-O-glucuronide, according to 1H- and 13C-NMR, HMBC, and LC-MS analyses. The metabolites purified from rat urine were 3'-O-methyl-(-)-epicatechin, (-)-epicatechin-7-O-glucuronide, and 3'-O-methyl-(-)-epicatechin-7-O-glucuronide. These compounds were also detected in the blood of humans and rats by LC-MS. The presence of these metabolites in blood and urine suggests that catechins are metabolized and circulated in the body after administration of catechin-containing foods.     

Identification of the ligand-binding site of the BMP type IA receptor for BMP-4

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of multifunctional cytokines. BMP induces its signal to regulate growth, differentiation, and apoptosis of various cells upon trimeric complex formation with two distinct type I and type II receptors on the cell surface: both are single-transmembrane serine/threonine kinase receptors. To identify the amino acid residues on BMP type I receptor responsible for its ligand binding, the structure-activity relationship of the extracellular ligand-binding domain of the BMP type IA receptor (sBMPR-IA) was investigated by alanine-scanning mutagenesis. The mutant receptors, as well as sBMPR-IA, were expressed as fusion proteins with thioredoxin in Escherichia coli, and purified using reverse phase high performance liquid chromatography (RP-HPLC) after digestion with enterokinase. Structural analysis of the parent protein and representative mutants in solution by CD showed no detectable differences in their folding structures. The binding affinity of the mutants to BMP-4 was determined by surface plasmon resonance biosensor. All the mutant receptors examined, with the exception of Y70A, displayed reduced affinities to BMP-4 with the rank order of decreases: I52A (17-fold) approximately F75A (15-fold) > T64A (4-fold) = T62A (4-fold) approximately E54A (3-fold). The decreases in binding affinity observed for the latter three mutants are mainly due to decreased association rate constants while alterations in rate constants both, for association and dissociation, result in the drastically reduced affinities for the former two mutants. These results allow us to conclude that sBMPR-IA recognizes the ligand using the concave face of the molecule. The major ligand-binding site of the BMP type IA receptor consists of Phe75 in loop 2 and Ile52, Glu54, Thr62 and Thr64 on the three-stranded beta-sheet. These findings should provide a general basis for the ligand/type I receptor recognition in the TGF-beta superfamily.     

Induction of apoptosis in human cancer cells by TZT-1027, an antimicrotubule agent

TZT-1027, a newly synthesized dolastatin 10 derivative, is a potent antitumor agent which inhibits microtubule polymerization and perturbs microtubule dynamics. In this report, we investigated whether TZT-1027 inhibited the growth of various human cancer cells, and the cell death caused by TZT-1027 was due to apoptosis. In addition, we elucidated the apoptosis machinery induced by treatment with TZT-1027. The 50% growth-inhibitory concentrations (IC₅₀ values) of TZT-1027 on cancer cells derived from various sources were not more than 5.9 ng/ml. TZT-1027 showed superior cytotoxicity than any other antitumor agents. Next, we evaluated morphological nuclear change, namely, chromatin condensation and DNA fragmentation. We used three cancer cell lines derived from different types in view of having apoptosis related protein, human leukemia HL-60 (in the presence of both Caspase-3 and Bcl-2), human breast cancer MCF-7 (in the absence of Caspase-3), and human prostate cancer DU145 (in the absence of Bcl-2). TZT-1027 induced DNA fragmentation in the presence but not absence of Caspase-3. Nevertheless, apoptic chromatin condensation was observed in all cancer cells even if there was no Caspase-3. Furthermore, we examined whether TZT-1027, microtubule-disrupting agent, influenced cell cycle progression. Flow cytometric analysis revealed the cells treated with TZT-1027, and with the other antimicrotubule agents, to be arrested at the G₂/M phase and subsequently to show fragmented DNA smaller than that of G₁ phase cells. Moreover, we tested TZT-1027 for its ability to induce Bcl-2 phosphorylation in human cancer cell lines. TZT-1027 and other agents which interacted with microtubules induced Bcl-2 phosphorylation, whereas DNA-damaging agents did not. The present results suggested an association of the growth-inhibitory effect of TZT-1027 with the induction of apoptosis and indicated that the apoptosis induced by TZT-1027 was followed by G₂/M arrest even if there was no Caspase-3 or Bcl-2.