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.
Sperm-specific phospholipase C zeta (PLC zeta) is known to induce intracellular Ca(2+) oscillations and egg activation when expressed in mouse eggs by injection of RNA encoding PLC zeta. We investigated the expression level and spatial distribution of PLC zeta in the egg in real time and in relation to the initiation and termination of Ca(2+) oscillations by monitoring fluorescence of a yellow fluorescent protein 'Venus' fused with PLC zeta. Ca(2+) oscillations similar to those at fertilization were induced at 40-50 min after RNA injection, when expressed PLC zeta reached 10-40 x 10(-15) g in the egg. PLC zeta-Venus increased up to 3 h and attained a steady level at 4-5 h. Interestingly, PLC zeta-Venus is accumulated to the pronucleus (PN) formed at 5-6 h and continuously increased there. Ca(2+) oscillations stopped in most eggs before initiation of the accumulation. A variant of PLC zeta that lacks three EF hand domains was much less effective in induction of Ca(2+) oscillations and little accumulated in the pronucleus, indicating a critical role of those domains. The ability of the accumulation to the pronucleus qualifies PLC zeta for a strong candidate of the Ca(2+) oscillation-inducing sperm factor, which is introduced into the ooplasm upon sperm-egg fusion and concentrated to the pronucleus after inducing egg activation.
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.
Sperm-specific phospholipase C-zeta (PLC) 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 PLC, which consists of four EF-hand domains (EF1-EF4) in the N terminus, X and Y catalytic domains, and C-terminal C2 domain. Those in the N terminus of EF1. Ca 2؉ 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, PLC 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 PLC␦1, seems to be required not only for enzymatic activity but also for nuclear translocation ability. PLC2 is a novel isozyme of PLC (the enzyme that hydrolyzes membrane PIP 2 into IP 3 and diacylglycerol) and a strong candidate of the mammalian sperm-derived egg-activating factor (1). PLC is specifically expressed in the sperm (2) and induces repetitive increase in [Ca 2ϩ ] i called Ca 2ϩ oscillations and subsequent early embryonic development when expressed in mouse eggs by injection of RNA encoding PLC (2, 3). In mammalian fertilization, accumulated evidence indicates that a cytosolic sperm factor is driven into the ooplasm upon sperm egg fusion and induces Ca 2ϩ oscillations (4, 5), which are caused by Ca 2ϩ release from the endoplasmic reticulum mainly through type 1 IP 3 receptor (6) and are a pivotal signal for egg activation characterized by resumption of the second meiosis and formation of PN (5). PLC is a strong candidate of the sperm factor, because 1) fertilization-like Ca 2ϩ oscillations are produced by PLC expressed in a mouse egg at an estimated level comparable to the content in single mouse sperm (2, 3). 2) Injection of recombinant PLC into mouse eggs induces Ca 2ϩ oscillations as well (7). 3) Ca 2ϩ oscillation-inducing ability of sperm extract injected into eggs (4, 8) is lost when pretreated with an antibody against PLC (2). 4) PLC content in the mouse sperm and the number of Ca 2ϩ spikes at fertilization are reduced by transgenic RNA interference of PLC (9). 5) PLC has such a high Ca 2ϩ sensitivity of PIP 2 -hydrolyzing activity that the enzyme can be active in the resting cells at ϳ100 nM Ca 2ϩ (7, 10), suitable for the sperm factor as the first stimulus in the egg cytoplasm at fertilization.Another important property of PLC is nuclear translocation ability. PLC expressed by RNA injection is accumulated into the formed PN (3,11,12). This is consistent with earlier observation that sperm-derived Ca 2ϩ oscillation-inducing activity is concentrated into PN formed several hours after fertilization, as examined by transfer of the oopla...
SUMMARYSelective inactivation of ␣ 1B -adrenoceptor (AR) by the sitedirected alkylating agent chlorethylclonidine (CEC) has been used as one of major pharmacological criteria to subclassify ␣ 1 -AR; however, the mechanism for the differential CEC sensitivity of the two subtypes is uncertain, and the extent of CEC inactivation varies depending on the treatment employed. In this study, we examined the correlation between the subcellular localization of ␣ 1 -AR subtypes (␣ 1A and ␣ 1B ) and CEC sensitivity. Constructing ␣ 1 -AR tagged with the FLAG epitope at the amino terminus and/or green fluorescent protein (GFP) at the carboxyl terminus, we examined the subcellular distribution of ␣ 1 -ARs expressed in COS-7 cells. Flow cytometry analysis showed that most populations of GFP-expressing ␣ 1B -AR cells, but very few GFP-expressing ␣ 1A -AR cells, were detected by the anti-amino terminus antibodies. The immunocytochemical and GFP-fluorescence confocal micrographs showed that ␣ 1A -ARs predominantly localize intracellularly, whereas ␣ 1B -ARs localize on the cell surface. Furthermore, CEC (10 M) treatment of intact cells resulted in an inactivation of approximately 42% of ␣ 1A -ARs and 93% of ␣ 1B -ARs, whereas treatment of the membrane preparations resulted in an inactivation of approximately 83% of ␣ 1A -ARs and 88% of ␣ 1B -ARs, respectively. Together, the results showed that a hydrophilic alkylating agent CEC preferentially inactivates ␣ 1 -AR on the cell surface irrespective of its subtype, and that the subtype-specific subcellular localization rather than the receptor structure is a major determinant for CEC inactivation of ␣ 1 -AR. Subtype-specific subcellular localization suggests an additional class of functional properties that provide new insight into drug action.
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 ␥ 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 (R 2 ϭ 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.
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 alphaSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant alphaSNAP, 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.
The low efficiency of transgenic animal production by microinjection has been a serious problem especially for the production of transgenic livestock. We developed a method to selectively produce transgenic mice using green fluorescent protein (GFP) as a marker. Using this method, we obtained eight fetuses and four live-born mice derived from 55 GFP-positive blastocysts. PCR analysis showed 11 out of 12 mice (fetuses and newborn mice) were transgenic. Southern blot analysis showed that 8 out of 12 were transgenic. GFP expression was also observed in bovine blastocysts, suggesting that this method should contribute to the efficient production of transgenic livestock.
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