Hideki Nikami

Expression and localization of insulin-regulatable glucose transporter (GLUT4) in rat brain

The localization of glucose transporters (GLUTs) was examined in various regions of the rat brain. The mRNA of GLUT1 and GLUT3 were found ubiquitously in every brain region (cortex, hippocampus, midbrain, striatum, hypothalamus, medulla oblongata and cerebellum). The mRNA and protein of GLUT4, an insulin-regulatable glucose transporter in peripheral tissues, were also identified, particularly abundantly in the cerebellum. In situ hybridization analysis revealed that GLUT4 mRNA was present in some discrete cells, such as Purkinje cells in the cerebellum, the vestibular nucleus in the medulla oblongata and also in ependymal cells along the cerebral ventricles. The GLUT4 mRNA level in the cerebellum changed little in fasted or experimentally induced diabetic rats while those in adipose tissues decreased much. The results suggest that insulin-sensitive glucose uptake may occur in some specific cells of the brain but is regulated in a different manner from those in peripheral cells.

Macrophage-derived cytokine and nitric oxide profiles in type I and type II diabetes mellitus: effect of thymoquinone.

Comparing macrophage-derived cytokine and nitric oxide (NO) profiles in type I and type II diabetes mellitus (DM); and determining whether thymoquinone (TQ) has any modulatory effect were the main objectives of the present study. Peritoneal macrophages have been collected from Otsuka Long-Evans Tokushima Fatty (OLETF) as a model for type II DM and its control Long-Evans Tokushima Otsuka (LETO) rats, as well as from streptozotocin (STZ)-injected LETO ones as a model for type I DM. The cells were cultured and incubated with or without TQ (10 µM) in the absence or presence of lipopolysaccharide (LPS; 1 µg/ml). The same parameters have been also assessed in sera of the used animals with or without TQ treatment (3 mg/kg) under both LPS-stimulated (10 mg/kg) and unstimulated conditions. Nitrite, IL-1β and TNF-α were significantly higher in macrophage supernatants and sera of the acutely affected STZ-LETO rats either with or without LPS stimulation compared to corresponding controls. On the other hand, chronically diabetic OLETF rats’ macrophage supernatants showed significant decreases of IL-1β and TNF-α levels upon LPS stimulation or even without stimulation (IL-1β); and insignificant increase in nitrite concentration, which turned significant upon LPS stimulation. Sera of these animals, however, showed significant increase in TNF-α level. TQ normalised the elevated nitrite and cytokine profiles both in vitro and in vivo, yet had no significant effect on the already decreased parameters in chronically affected OLETF rats. These data suggest that there is a tendency for macrophage inflammatory products to increase in acute type I and to decrease in chronic type II DM; and that TQ has the potential to normalise the elevated levels of these macrophage-derived inflammatory mediators.

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.

Pseudorabies virus (PRV) early protein 0 activates PRV gene transcription in combination with the immediate-early protein IE180 and enhances the infectivity of PRV genomic DNA.

Pseudorabies virus (PRV) early protein 0 (EP0) functions as a transactivator of the viral gene promoters. In transient expression assays employing chloramphenicol acetyl transferase (CAT) reporter constructs, EP0 and the immediate-early protein IE180 act in an additive manner to activate transcription from the thymidine kinase (TK) and glycoprotein G (gG) gene promoters. EP0 enhanced the synthesis of infectious virus in cotransfection experiments with the EP0-expression plasmid and PRV genomic DNA. EP0 was detected by Western blot analysis in the purified virions. These results may indicate that EP0 in the virions acts as an important transactivator to express the immediate-early gene efficiently in the first stage of infection, and IE180 and EP0 expressed after the infection cooperatively activate the early and late gene expression in the later stage of infection.

Induction of peroxisomal lipid metabolism in mice fed a high-fat diet.

Peroxisomes catalyze a range of essential metabolic functions, mainly related to lipid metabolism. However, their roles in obesity have yet to be clarified. The aim of this study was to investigate the correlation between obesity and peroxisomal lipid metabolism, particularly very long-chain fatty acid (VLCFA) metabolism, gene expression of peroxisomal β-oxidation enzymes, peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy (ABCD1) gene and its related gene, ABCD2, the elongation of the VLCFA (ELOVL) gene family and the transcriptional factors involved in the regulation of these genes, including peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein. These factors were analyzed in livers from mice fed a high-fat diet (HFD) or a regular diet (RD) for 20 weeks. Furthermore, the amounts of plasma saturated and unsaturated fatty acids, including VLCFAs, were measured. A HFD induced hepatic gene expression of not only hydroxysteroid 17-β dehydrogenase 4 (HSD17b4) and sterol carrier protein 2 (SCP2) in peroxisomal β-oxidation enzymes but also of ELOVL1, 2, 5 and 6, which are involved in the elongation of saturated and unsaturated VLCFAs. Furthermore, ABCD2 mRNA prominently increased in the HFD mice. The transcriptional regulator of these genes, PPARα, was also up-regulated in the HFD mice. VLCFA ratios including C24:0/C22:0, C25:0/C22:0 and C26:0/C22:0 are the most significant diagnostic markers of inherited peroxisomal diseases. These ratios were found to be low in the plasma of the HFD mice compared with the RD mice. The results suggest that HFD activates hepatic peroxisomal VLCFA metabolism, and may provide useful fundamental information to explain the role of peroxisomal function in obesity and lifestyle-related diseases.

An electrophysiological study of excitatory purinergic neuromuscular transmission in longitudinal smooth muscle of chicken anterior mesenteric artery

1 The object of the present study was to clarify the neurotransmitters controlling membrane responses to electrical field stimulation (EFS) in the longitudinal smooth muscle cells of the chicken anterior mesenteric artery. 2 EFS (5 pulses at 20 Hz) evoked a depolarization of amplitude 19.7±2.1 mV, total duration 29.6±3.1 s and latency 413.0±67.8 ms. This depolarization was tetrodotoxin (TTX)‐sensitive and its amplitude was partially decreased by atropine (0.5 μM); however, its duration was shortened by further addition of prazosin (10 μM). 3 Atropine/prazosin‐resistant component was blocked by the nonspecific purinergic antagonist, suramin, in a dose‐dependent manner, indicating that this component is mediated by the neurotransmitter adenosine 5′‐triphosphate (ATP). 4 Neither desensitization nor blocking of P2X receptor with its putative receptor agonist α,β‐methylene ATP (α,β‐MeATP, 1 μM) and its antagonist pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic (PPADS, up to 50 μM), had significant effect on the purinergic depolarization. In contrast, either desensitization or blocking of P2Y receptor with its putative agonist 2‐methylthioATP (2‐MeSATP, 1 μM) and its antagonist Cibacron blue F3GA (CBF3GA, 10 μM) abolished the purinergic depolarization, indicating that this response is mediated through P2Y but not P2X receptor. 5 The purinergic depolarization was inhibited by pertussis toxin (PTX, 600 ng ml−1). Furthermore, it was significantly inhibited by a phospholipase C (PLC) inhibitor, U‐73122 (10 μM), indicating that the receptors involved in mediating the purinergic depolarization are linked to a PTX‐sensitive G‐protein, which is involved in a PLC‐mediated signaling pathway. 6 Data of the present study suggest that the EFS‐induced excitatory membrane response occurring in the longitudinal smooth muscle of the chicken anterior mesenteric artery is mainly purinergic in nature and is mediated via P2Y purinoceptors.

Capsaicin pretreatment attenuates LPS-induced hypothermia through TRPV1-independent mechanisms in chicken

It has been demonstrated that chicken TRPV1 (transient receptor potential vanilloid of subtype-1) is insensitive to capsaicin (CAP), and therefore, a chicken model is suitable to analyze the CAP-sensitive TRPV1-independent pathway. We elucidated here the possible involvement of the pathway in hypothermia induced by bacterial endotoxin (lipopolysaccharide, LPS) in chickens. Chicks were pretreated with CAP (10 mg/kg, iv) at 1, 2 and 3 days of age to desensitize them towards the CAP-sensitive pathway. An intravenous injection of LPS in 4-day-old chicks caused progressive hypothermia, ending with collapse and 78% mortality within 12 h after injection. The CAP pretreatment rescued the LPS-induced endotoxin shock and hypothermia in chicks. LPS-induced iNOS expression as well as NO production in liver and lung was suppressed by CAP pretreatment. CAP pretreatment also attenuated hypothermia due to exposure of chicks to cold ambient temperature. These findings suggest that a CAP-sensitive TRPV1-independent pathway may be involved in pathophysiological hypothermic reactions through the mediation of NO in chickens.

Combined effects of organochlorine pesticides heptachlor and hexachlorobenzene on the promotion stage of hepatocarcinogenesis in rats

We aimed to investigate the combined effect of organochlorine pesticides heptachlor (HEP) and hexachlorobenzene (HCB) by using a medium-term rat liver bioassay. Male F344 rats were initially administered diethylnitrosamine (DEN, 200mg/kgi.p.); after a 2-week non-dosing period, they were given diets containing HEP (5 or 25ppm), HCB (70 or 350ppm), or their mixtures (5 and 70ppm or 25 and 350ppm) for 6weeks. All rats were subjected to partial hepatectomy at week 3 and killed at week 8. We observed additive or synergistic effects of HEP and HCB in groups treated with mixtures of these pesticides. Number and area of preneoplastic foci positive for glutathione S-transferase placental form (GST-P) were consistently higher in these groups than the sum of individual values in the groups treated with HEP or HCB alone. Consistent with these findings, HEP and HCB had additive or synergistic effects on cell proliferation induction within the preneoplastic foci and cytochrome P450 (CYP) 2B1 and 3A1 induction, which may lead to more efficient metabolic activation of HEP and HCB. On the basis of these findings, we conclude that HEP and HCB have additive and synergistic effects on the development of GST-P-positive foci and that higher risks are associated with a combination of residual organochlorine pesticides in foods than with individual residual organochlorine pesticides.

Suppression of pseudorabies virus replication by a mutant form of immediate-early protein IE180 repressing the viral gene transcription.

A mutant form of the immediate-early (IE) protein IE180 of pseudorabies virus (PRV), dIN454-C1081 is a strong repressor of the PRV IE gene promoter. In order to assess the antiviral potential of the IE180 mutant, HeLa cells were transformed with the mutant gene and then infected with PRV and herpes simplex virus type 1 (HSV-1). The transformed cell lines showed marked resistance to PRV infection, but were susceptible to infection with HSV-1, indicating that the IE180 mutant expressed in the stable cell line specifically inhibited PRV growth. In those cells infected with PRV, transcription of the PRV IE gene was repressed. In addition, the IE180 mutant exhibited a dominant-negative property in transient expression assay. The present results indicate that the resistance of the cells to PRV infection was due to repression of the IE gene transcription by the IE 180 mutant.

A neurophysiological evidence of capsaicin-sensitive nerve components innervating interscapular brown adipose tissue

Neurophysiological basis for the heterogeneity of the nerve components in the brown adipose tissue (BAT) was examined in this experiment. Efferent nerve signals were recorded from the central cut end of the small nerve filament dissected from the nerve fibers innervating the interscapular BAT (IBAT). By focusing on qualitative aspects of observed compound action potentials (spikes), we found two distinctive types of spikes exhibited by the intercostal nerves innervating IBAT. The spikes mainly appeared upon sympathetic stimulations (cold stimulation and glucose administration) were characterized by low amplitude with relatively short duration (small spike) and their sensitivity to the ganglion blocker, hexamethonium (C6). On the other hand, the spikes seen throughout the experiments were characterized by high amplitude with long duration (large spike) and their insensitivity to C6. Since BAT is activated by cold and feeding via sympathetic nervous system, the small spikes seemed to be exhibited by sympathetic fibers. On the other hand, appearance of the large C6-insensitive spikes was strongly attenuated in capsaicin-desensitized rats. Even though the functional link between IBAT and C6 insensitive fibers remains unanswered, our results suggest that IBAT is under control of various nerve types including capsaicin-sensitive fibers in addition to the control of sympathetic nervous system.