Identification of protein kinase CK2 inhibitors using solvent dipole ordering virtual screening.

Novel protein kinase CK2 inhibitors were identified using the solvent dipole ordering virtual screening method. A total of 26 compounds categorized in 15 distinct scaffold classes inhibited greater than 50% of enzyme activity at 50 µM, and eight exhibited IC50 values less than 10 µM. Most of the identified compounds are lead-like and dissimilar to known inhibitors. The crystal structures of two of the CK2 complexes revealed the high accuracy of the predicted binding modes.

Administration of perilla oil coated with Calshell increases glucagon-like peptide secretion.

Recently, we found that unsaturated long-chain fatty acids (such as alpha-linolenic acid) promote the secretion of glucagon-like peptide-1 (GLP-1) via G protein-coupled receptor GPR120, which is expressed predominantly in the colon. In order to ensure that the triglycerides or free fatty acids, such as alpha-linolenic acid, reach the distal intestinal tract effectively, we developed a Calshell technique. Following single treatment of Calshell perilla oil powder, the GLP-1 secretion level was significantly higher than following vehicle treatment, 120 min after treatment. Next, we examined the effects of long-term Calshell perilla oil powder treatment on GLP-1 secretion. Plasma GLP-1 level of Calshell perilla oil powder treatment was significantly higher than of vehicle treatment for 1, 14, 28 and 56 d. We thereby demonstrated for the first time the utility of Calshell oil powder treatment for effective and sustainable GLP-1 secretion. The Calshell technique is apparently useful as a drug delivery system, since Calshell unsaturated oil powder is protected from gastric acid, reaches enteroendocrine cells in the gastrointestinal tract, and then induces effective incretin secretion.

Cloning and characterization of the rat free fatty acid receptor GPR120: in vivo effect of the natural ligand on GLP-1 secretion and proliferation of pancreatic beta cells.

We have recently found that GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain free fatty acids (FFAs) and that GPR120 stimulation promotes the secretion of glucagons-like peptide-1 (GLP-1) in the mouse (Hirasawa et al., Nat Med 11:90-94, 2005). In this study, we cloned and characterized rat GPR120 (rGPR120), and then we examined the in vivo effects of acute and long-term administration of the natural ligand alpha-linolenic acid (alpha-LA). The cloned rat GPR120 complimentary DNA had a seven transmembrane structure, and a homology comparison of human, mouse, and rat GPR120 revealed that the rat GPR120 (rGPR120) shares 85 and 98% sequence identity with the human and mouse GPR120 proteins, respectively. The tissue distribution and ligand properties of rGPR120 were similar to those of mouse GPR120. In addition, alpha-LA provoked a transient increase in [Ca2+]i levels in HEK293 cells expressing rGPR120. Furthermore, administration of alpha-LA to the rat increased plasma GLP-1 levels, and long-term administration of alpha-LA led to proliferation of pancreatic beta cells, probably because of the enhanced GLP-1 secretion. These results show that rat GPR120 is a G-protein-coupled receptor whose ligand is a free fatty acid, and it may play an important role in the FFA-associated physiological responses.

Effects of hyperbaric exposure with high oxygen concentration on glucose and insulin levels and skeletal muscle-fiber properties in diabetic rats.

The effects of hyperbaric exposure with high oxygen concentration on glucose and insulin levels and skeletal muscle-fiber properties were investigated in type 2 diabetic Goto-Kakizaki rats. Five-week-old rats were exposed to a hyperbaric environment (1.25 atmospheric pressure) with a high oxygen concentration (36%) for 6 h daily. Glucose and insulin levels and properties including fiber-type distribution, cross-sectional area, and oxidative enzyme activity in the soleus muscle were examined after hyperbaric exposure for 4 weeks. The growth-related increase in glucose level was inhibited by hyperbaric exposure, and insulin also showed lower levels compared with control rats. The percentage of low-oxidative type I fibers in the muscle decreased and high-oxidative type IIA and type IIC fibers, which were not detected in the muscle of control rats, were observed after hyperbaric exposure. The oxidative enzyme activity of type I fibers in the muscle increased after hyperbaric exposure. Hyperbaric exposure with high oxygen concentration might therefore provide a new approach to improve the glucose tolerance, insulin resistance, and altered skeletal muscle metabolism that are caused by diabetes mellitus. Muscle Nerve, 2006.

Light activates the adrenal gland: timing of gene expression and glucocorticoid release.

Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamo-adenohypophysial axis. The abolishment after SCN lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The magnitude of corticostereone response is dose dependently correlated with the light intensity. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.

Disruption of the prepulse inhibition of the startle reflex in vasopressin V1b receptor knockout mice: reversal by antipsychotic drugs.

In the present study, we investigated whether mice lacking the arginine vasopressin (AVP) V1b receptor (V1bR) exhibit deficits of prepulse inhibition (PPI) of the startle reflex, reminiscent of the sensorimotor gating deficits observed in a large majority of schizophrenic patients. V1bR knockout (KO) mice displayed significantly reduced levels of PPI of the startle reflex. In addition to PPI deficits, V1bR KO mice showed increased acoustic startle response. However, acoustic startle response was not significantly correlated to the PPI of the startle reflex in V1bR KO mice. V1bR KO mice also showed a decrease in basal levels of extracellular dopamine (DA) in the medial prefrontal cortex, which is thought to be an important brain region for PPI. Moreover, PPI deficits observed in the V1bR KO mice are significantly reversed by atypical antipsychotics such as risperidone and clozapine but not by a typical neuroleptic haloperidol, like in schizophrenic patients. By contrast, we did not observe any significant differences between V1bR KO mice and wild-type mice in the open-field, light/dark, elevated plus maze, and forced swimming tests. The results of the present study indicate that V1bR may be involved in the regulation of PPI of the startle reflex. The V1bR has been considered an important molecular target for the development of antipsychotic drugs.

Free fatty acids inhibit serum deprivation-induced apoptosis through GPR120 in a murine enteroendocrine cell line STC-1.

Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.

Comprehensive analysis of the effect of phytoestrogen, daidzein, on a testicular cell line, using mRNA and protein expression profile.

In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17beta-estradiol (E2), diethylstilbestrol (DES) and staurosporin on the TM4 testicular cell line, using comprehensive analysis, such as cDNA microarray and two-dimension polyacrylamide gel electropholesis (2D-PAGE) analysis, and we demonstrated if these toxicogenomic analyses could classify the chemical compounds. First, RNA was extracted from TM4 cells that had been treated with daidzein (80 microM), DES, E2 (40 microM) and stauroporin (100 nM) for 30 min. We performed cDNA microarray analysis, and the expression ratio data thus obtained were then analyzed using hierarchical clustering. This hierarchical clustering showed that daidzein exposure induced a different effect on gene expression change from that of E2, DES and staurosporin. Next, protein extracted from TM4 cells also underwent cDNA microarray analysis for 3 h. We performed 2D-PAGE analysis, and the spot intensity ratio data thus obtained were analyzed using hierarchical clustering. As with cDNA microarray, the hierarchical clustering of protein spot ratios showed that daidzein exposure induced a different effect on gene expression change from that of the other substances. In conclusion, we have demonstrated for the first time that classification of these chemicals can be performed by clustering analysis, using data from cDNA microarray and 2D-PAGE analyses, and that exposure to daidzein induces effects different from those of E2, DES and staurosporin.

Characteristics of behavioral abnormalities in alpha1d-adrenoceptors deficient mice.

To investigate the functional role of alpha1d-adrenergic receptor (alpha1d-AR) in the CNS, we have generated mutant mice lacking the alpha1d-AR using a gene targeting approach and examined in detail the effects of alpha1d-AR knockout mice on motor function, sensory function, and learning and memory. alpha1d-AR knockout mice showed better motor coordination at the highest rotating speed of the rotarod performance and stronger muscle tone using the traction meter, but their locomotor activity and swimming ability in the water maze were not affected. In the water maze requiring reference memory, alpha1d-AR knockout mice showed normal spatial learning. In the Y-maze task requiring working memory or attention, alpha1d-AR knockout mice displayed an impaired spontaneous alternation performance. The alpha1d-AR knockout mice tended to display lower levels of acoustic startle responses than the wild-type group at lower pulse intensities, although the acoustic prepulse inhibition was not impaired in the alpha1d-AR knockout mice. Furthermore, the NMDA receptor antagonist, MK-801-induced deficits of acoustic prepulse inhibition were not observed in the alpha1d-AR knockout mice. These results clearly demonstrate that the alpha1d-AR receptor plays an important role in the process of auditory sensory function, attention or working memory rather than reference memory, and the sensorimotor gating deficits induced by the NMDA receptor antagonist.