Takeo Awaji

Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120

Diabetes, a disease in which the body does not produce or use insulin properly, is a serious global health problem. Gut polypeptides secreted in response to food intake, such as glucagon-like peptide-1 (GLP-1), are potent incretin hormones that enhance the glucose-dependent secretion of insulin from pancreatic beta cells. Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules in various cellular processes, including the secretion of gut incretin peptides. Here we show that a G-protein-coupled receptor, GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain FFAs. Furthermore, we show that the stimulation of GPR120 by FFAs promotes the secretion of GLP-1 in vitro and in vivo, and increases circulating insulin. Because GLP-1 is the most potent insulinotropic incretin, our results indicate that GPR120-mediated GLP-1 secretion induced by dietary FFAs is important in the treatment of diabetes.

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP‐2, essential proteins for neurotransmitter release, accumulated in the synaptic‐releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co‐localized with synaptotagmin I and synaptobrevin/VAMP‐2 after 3–4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse‐enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and αSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant αSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

Structure-Activity Relationships of GPR120 Agonists Based on a Docking Simulation

GPR120 is a G protein-coupled receptor expressed preferentially in the intestinal tract and adipose tissue, that has been implicated in mediating free fatty acid-stimulated glucagon-like peptide-1 (GLP-1) secretion. To develop GPR120-specific agonists, a series of compounds (denoted as NCG compounds) derived from a peroxisome proliferator-activated receptor γ agonist were synthesized, and their structure-activity relationships as GPR120 agonists were explored. To examine the agonistic activities of these newly synthesized NCG compounds, and of compounds already shown to have GPR120 agonistic activity (grifolic acid and MEDICA16), we conducted docking simulation in a GPR120 homology model that was developed on the basis of a photoactivated model derived from the crystal structure of bovine rhodopsin. We calculated the hydrogen bonding energies between the compounds and the GPR120 model. These energies correlated well with the GPR120 agonistic activity of the compounds (R2 = 0.73). NCG21, the NCG compound with the lowest calculated hydrogen bonding energy, showed the most potent extracellular signal-regulated kinase (ERK) activation in a cloned GPR120 system. Furthermore, NCG21 potently activated ERK, intracellular calcium responses and GLP-1 secretion in murine enteroendocrine STC-1 cells that express GPR120 endogenously. Moreover, administration of NCG21 into the mouse colon caused an increase in plasma GLP-1 levels. Taken together, our present study showed that a docking simulation using a GPR120 homology model might be useful to predict the agonistic activity of compounds.

Difference in Ca2+ Oscillation-Inducing Activity and Nuclear Translocation Ability of PLCZ1, an Egg-Activating Sperm Factor Candidate, Between Mouse, Rat, Human, and Medaka Fish

Mouse phospholipase C, zeta 1 (PLCZ1), a strong candidate of egg-activating sperm factor, induces Ca(2+) oscillations and accumulates into formed pronucleus (PN) when expressed by cRNA injection. These activities were compared among mouse and human PLCZ1, newly cloned rat Plcz1, and medaka fish plcz1. The PLCZ1 proteins of the four species have an approximately homologous sequence of nuclear localization signal. However, the nuclear translocation ability was defective in rat, human, and medaka PLCZ1 expressed in mouse eggs. Rat PLCZ1 could not enter rat PN, whereas mouse PLCZ1 could. Mouse and human PLCZ1 translocated into the nucleus of COS-7 cells transfected with cDNA. There was little medaka PLCZ1 accumulated in the nucleus, and rat PLCZ1 was never located in the nucleus. All PLCZ1 proteins including fish could induce Ca(2+) oscillations in mouse eggs, but the activity was variable in the order of human >> mouse > medaka >> rat, estimated from minimal RNA concentration to induce Ca(2+) spikes. Ca(2+) oscillations by human PLCZ1 continued far beyond the time of PN formation (T(PN)), whereas those by mouse PLCZ1 ceased slightly before T(PN). High-frequency Ca(2+) spikes by overexpressed rat PLCZ1 stopped far before T(PN), possibly by feedback inhibition. Ca(2+) oscillations by fertilization of rat eggs stopped at T(PN), despite defective nuclear translocation of rat PLCZ1. Thus, PLCZ1 sequestration into PN participates in termination of Ca(2+) oscillations at the interphase of mouse embryos but does not always operate in other mammals, notably in rat embryos.

The Role of X/Y Linker Region and N-terminal EF-hand Domain in Nuclear Translocation and Ca2+ Oscillation-inducing Activities of Phospholipase Cζ, a Mammalian Egg-activating Factor

Sperm-specific phospholipase C-zeta (PLCζ) causes intracellular Ca2+ oscillations and thereby egg activation and is accumulated into the formed pronucleus (PN) when expressed in mouse eggs by injection of cRNA encoding PLCζ, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those activities were analyzed by expressing PLCζ mutants tagged with fluorescent protein Venus by injection of cRNA into unfertilized eggs or 1-cell embryos after fertilization. Nuclear localization signal (NLS) existed at 374–381 in the X/Y linker region. Nuclear translocation was lost by replacement of Arg376, Lys377, Arg378, Lys379, or Lys381 with glutamate, whereas Ca2+ oscillations were conserved. Nuclear targeting was also absent for point mutation of Lys299 and/or Lys301 in the C terminus of X domain, or Trp13, Phe14, or Val18 in the N terminus of EF1. Ca2+ oscillation-inducing activity was lost by the former mutation and was remarkably inhibited by the latter. A short sequence 374–383 fused with Venus showed active translocation into the nucleus of COS-7 cells, but 296–309 or 1–19 did not. Despite the presence of these special regions, both activities were deprived by deletion of not only EF1 but also EF2–4 or C2 domain. Thus, PLCζ is driven into the nucleus primarily by the aid of NLS and putative regulatory sites, but coordinated three-dimensional structure, possibly formed by a folding in the X/Y linker and close EF/C2 contact as in PLCδ1, seems to be required not only for enzymatic activity but also for nuclear translocation ability.

Flow Cytometry-Based Binding Assay for GPR40 (FFAR1; Free Fatty Acid Receptor 1)

GPR40 is a G protein-coupled receptor (GPCR) whose endogenous ligands have recently been identified as medium- and long-chain free fatty acids (FFAs), and it is thought to play an important role in insulin release. Despite recent research efforts, much still remains unclear in our understanding of its pharmacology, mainly because the receptor-ligand interaction has not been analyzed directly. To study the pharmacology of GPR40 in a more direct fashion, we developed a flow cytometry-based binding assay. FLAG-tagged GPR40 protein was expressed in Sf9 cells, solubilized, immobilized on immunomagnetic beads, and labeled with the fluorescent probe C1-BODIPY-C12. Flow cytometry analysis showed that C1-BODIPY-C12 specifically labels a single class of binding site in a saturable and reversible manner with an apparent dissociation constant of ∼3 μM. The FFAs that activate GPR40 competed with C1-BODIPY-C12 binding; thus, medium- to long-chain FFAs could compete, whereas short-chain FFAs and methyl linoleate had no inhibitory effect. Furthermore, ligands that are known to activate GPR40 competed for binding in a concentration-dependent manner. All the ligands that inhibited the binding promoted phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 in human embryonic kidney (HEK) 293 cells that expressed GPR40 and [Ca2+]i responses in mouse insulinoma (MIN6) cells that natively express GPR40; however, pioglitazone, a thiazolidinedione that failed to compete for the binding, did not activate ERK or [Ca2+]i response. This study showed that a flow cytometry-based binding assay can successfully identify direct interactions between GPR40 and its ligands. This approach would be of value in studying the pharmacology of GPCRs.

Differential mechanism for the cell surface sorting and agonist‐promoted internalization of the α1B‐adrenoceptor

α1B‐adrenoceptors are localized at a steady state in the plasma membrane in untreated cells, and internalize to intracellular vesicles when exposed to agonist. Flow cytometry analysis with an anti‐N‐terminus‐antibody (1B‐N1‐C, ( Hirasawa et al., 1996 )) facilitated the quantification of cell surface α1B‐adrenoceptor. Also, the cellular distribution of α1B‐adrenoceptors was visually monitored by immunocytochemical confocal microscopy. Utilizing this combined approach, we have examined the molecular mechanism for cellular trafficking of α1B‐adrenoceptors, including the process of sorting of the synthesized receptor protein to the cell surface, and the agonist‐induced internalization. The two processes were separately examined by using α1B‐adrenoceptor inducible DDT1MF‐2 cells for the sorting process and CHO cells stably expressing α1B‐adrenoceptors for the agonist‐promoted internalization. We examined the effects of cytochalasin D and mycalolide B (actin depolymerization agents), demecolcine (a microtubule disrupting agent), brefeldin A (an inhibitor of vesicular transport and Golgi function), bafilomycin A1 (a specific inhibitor of the vacuolar proton pump) or hyperosmotic sucrose treatment (that may inhibit clathrin‐mediated endocytosis) on these processes. We found that the agonist‐promoted internalization was blocked by cytochalasin D and mycalolide B, while the cell surface sorting process was specifically blocked by brefeldin A, indicating that the two processes involve different components of the cellular endocytic machinery. The experimental approach as exemplified in this study would provide a valuable system to study further the molecular mechanism(s) of cellular trafficking of G protein‐coupled receptors.

Effects of a New Forskolin Derivative, Nkh477, on Canine Ventricular Arrhythmia Models

Summary: Using two-stage coronary ligation-, digitalisand epinephrine-induced canine ventricular arrhythmia models, we examined whether a new positive inotropic agent, NKH477, 6-(3-dimethylaminopropionyl)forskolin hydrochloride, a water-soluble derivative of forskolin, had deleterious effects on arrhythmias. NKH477 increased heart rate (HR) and decreased blood pressure (BP) in dogs with all the arrhythmia models. Unexpectedly, NKH477 suppressed digitalis- and epinephrineinduced arrhythmias, but did not suppress two-stage coronary ligation arrhythmia or aggravate it. These results indicate that NKH477, unlike other new positive inotropic agents such as amrinone, milrinone, sulmazole and vesnarinone, did not worsen these arrhythmias; thus, NKH477 may be a useful positive inotropic agent with little arrhythmogenic effect

Acute antiarrhythmic effects of intravenously administered amiodarone on canine ventricular arrhythmia.

We investigated antiarrhythmic effects of intravenously (i.v.) administered amiodarone using four canine ventricular arrhythmia models. Bolus injections of amiodarone 3 mg/kg suppressed epinephrine (EPI)-induced arrhythmia and 5-mg/kg bolus injections of amiodarone suppressed digitalis- and two-stage coronary ligation-induced arrhythmia models, but the antiarrhythmic effects did not correlate with the amiodarone plasma concentrations. The infusion of amiodarone 6.67 mg/kg/h did not prolong the QTc interval or produce antiarrhythmic effects in coronary ligation and reperfusion experiments. Amiodarone significantly decreased the mean blood pressure (MAP), and this effect lasted throughout the observation period. The results indicate that the antiarrhythmic effects of intravenously administered amiodarone may not be due to its class III action, but to other actions, such as class I, II and IV actions.

Regulation of subcellular localization of α1-adrenoceptor subtypes

Abstract α1-Adrenergic receptors (AR) are members of the superfamily of G protein-coupled receptors (GPCRs) which mediate the effects of the sympathetic nervous system. α1-AR comprise a heterogeneous family of three distinct isoforms of α1A, α1B and α1D; however, very little is known about their difference in physiological role or regulation. We have recently observed a subtype-specific differences in subcellular localization of α1-ARs; thus, α1A-AR predominantly localize intracellularly, while α1B-AR on the cell surface. To examine the molecular mechanism for the subtype-specific differences in subcellular localization, we conducted a search for novel proteins that interact with the α1B-AR, specifically focusing on the carboxyl-terminal cytoplasmic domain. Using interaction cloning and biochemical techniques, we demonstrate that gC1q-R interacts with α1B-AR in vitro and in vivo through the specific site, and that in cells which co-express α1B-AR and gC1q-R, the subcellular localization of α1B-AR is markedly altered and its expression is down-regulated. These results suggest that gC1q-R plays a role in the regulation of the subcellular localization as well as the function of α1B-ARs.