Antipsychotics induce distinctive IEG expression changes in nucleus accumbens and striatum, mainly due to their prominent D2R antagonistic activity.

<p>(a) Time courses of seven IEG expressions induced by 0.3 mg/kg (p.o.) of haloperidol. Data are shown as mean± SEM (n = 5). Filled circles, p<0.05 in unpaired t-test with Welch’s correction, compound (n = 5) versus vehicle (n = 5) at the same time point; open circles, not significant. The changes of expression levels are shown as fold change over vehicle. (b) TFP of seven IEGs over time induced by antipsychotic agents in mouse brain. Antipsychotics; haloperidol (0.3 mg/kg, p.o.), aripiprazole (3 mg/kg, p.o.), olanzapine (10 mg/kg, p.o.) and clozapine (30 mg/kg, p.o.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

Temporal and Spatial Transcriptional Fingerprints by Antipsychotic or Propsychotic Drugs in Mouse Brain

<div><p>Various types of antipsychotics have been developed for the treatment of schizophrenia since the accidental discovery of the antipsychotic activity of chlorpromazine. Although all clinically effective antipsychotic agents have common properties to interact with the dopamine D2 receptor (D2R) activation, their precise mechanisms of action remain elusive. Antipsychotics are well known to induce transcriptional changes of immediate early genes (IEGs), raising the possibility that gene expressions play an essential role to improve psychiatric symptoms. Here, we report that while different classes of antipsychotics have complex pharmacological profiles against D2R, they share common transcriptome fingerprint (TFP) profile of IEGs in the murine brain <i>in vivo</i> by quantitative real-time PCR (qPCR). Our data showed that various types of antipsychotics with a profound interaction of D2R including haloperidol (antagonist), olanzapine (antagonist), and aripiprazole (partial agonist) all share common spatial TFPs closely homologous to those of D2R antagonist sulpiride, and elicited greater transcriptional responses in the striatum than in the nucleus accumbens. Meanwhile, D2R agonist quinpirole and propsychotic NMDA antagonists such as MK-801 and phencyclidine (PCP) exhibited the contrasting TFP profiles. Clozapine and propsychotic drug methamphetamine (MAP) displayed peculiar TFPs that reflect their unique pharmacological property. Our results suggest that transcriptional responses are conserved across various types of antipsychotics clinically effective in positive symptoms of schizophrenia and also show that temporal and spatial TFPs may reflect the pharmacological features of the drugs. Thus, we propose that a TFP approach is beneficial to evaluate novel drug candidates for antipsychotic development.</p></div

D2R antagonist shows spatial TFPs homologous to those by antipsychotics, while D2 agonist and propsychotic agents exhibit their peculiar TFPs.

<p>TFPs of seven IEGs over time induced by D2R antagonist/agonist and propsychotic drugs in mouse brain; (a) Selective D2R antagonist sulpiride (100 mg/kg, i.p.) and agonist quinpirole (10 mg/kg, i.p.), and (b) Propsychotic agents MAP (3 mg/kg, s.c.), PCP (10 mg/kg, s.c.) and MK-801 (1 mg/kg, s.c.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

TFPs of seven IEGs by treatment of propsychotic agents showing dose-response pattern in mouse brain.

<p>Significant reduction of <i>Egr2</i> in nucleus accumbens, striatum and prefrontal cortex, and increases of <i>c-fos</i> and <i>Ccn1</i> in all tested regions are characteristic of NMDA antagonists, whilst MAP serves robust IEGs induction in all four regions studied here. The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression. Propsychotic agents; MAP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), PCP (1 hr treatment, 1 mg/kg to 10 mg/kg, s.c.), MK-801 (1hr treatment, 0.1 mg/kg to 3 mg/kg, s.c.).</p

Antipsychotics treatment except clozapine commonly induce three IEGs, <i>c-fos</i>, <i>Arc</i> and <i>Egr2</i> in a dose-dependent manner in nucleus accumbens and striatum.

<p>(a) Dose-dependent effects of haloperidol (1 hr treatment, 0.03 mg/kg to 1 mg/kg, p.o.). Note that four IEGs (<i>c-fos</i>, <i>Arc</i>, <i>Egr1</i> and <i>Egr2</i>) are dose-dependently elevated in nucleus accumbens and striatum. The dashed horizontal line indicates baseline (the value of 1).Data are shown as mean± SEM (n = 4~5). *p<0.025, significantly different from vehicle at a dose of 0 mg/kg in Williams’ test. (b) TFPs of seven IEGs showing dose-response pattern by treatment of four antipsychotics in mouse brain. Significant induction for three IEGs (<i>c-fos</i>, <i>Arc</i> and <i>Egr2</i>) were commonly observed in nucleus accumbens and striatum among three antipsychotics, haloperidol, aripiprazole and olanzapine. Antipsychotics; haloperidol (1 hr treatment, 0.03 mg/kg to 1 mg/kg, p.o.), aripiprazole (2 hrs treatment, 0.1 mg/kg to 3 mg/kg, p.o.), olanzapine (1 hr treatment, 0.3 mg/kg to 10 mg/kg, p.o.) and clozapine (1 hr treatment, 1 mg/kg to 30 mg/kg, p.o.). The bar depicted in the bottom right corner represents the expression level of the gene searched in a TFP. Red represents high expression, green represents vehicle-control level expression, and blue represents low expression.</p

Fasiglifam does not exacerbate FFA-induced apoptotic signaling in MIN6 cells.

<p>Caspase 3/7 activity in the mouse pancreatic β cell line MIN6 after 72-h exposure to 0.25–1 mM palmitic acid (<i>A</i>) or γ-linolenic acid (γ-LA) (<i>B</i>) in combination with fasiglifam (Fas, 0.1–10 µM). “PA+Fas” and “γ-LA+Fas” indicate “1 mM palmitic acid +10 µM fasiglifam” and “1 mM γ-LA +10 µM fasiglifam”, respectively. Data shown are mean ± s.e.m. (n = 3).</p

A Novel Antidiabetic Drug, Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

<div><p>Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs <i>in vivo.</i> Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.</p></div

Partial agonist activity of fasiglifam is affected by FFAR1/GPR40 expression levels.

<p>(<i>A</i>) The chemical structure of fasiglifam. (<i>B</i> and <i>C</i>) FFAR1 agonist activities of fasiglifam and free fatty acids (FFAs) in the intracellular Ca²⁺ mobilization assay using CHO cell lines expressing hFFAR1 (clone #104) (<i>B</i>) or mFFAR1 (<i>C</i>). Data are representative of three experiments. (<i>D</i>) hFFAR1 mRNA levels of hFFAR1-expressing CHO clones were evaluated by qRT-PCR. (<i>E</i><b>-</b><i>H</i>) Relative Ca²⁺ influx activities of γ-LA and fasiglifam in CHO clones #104 (<i>E</i>), #19 (<i>F</i>), #2 (<i>G</i>), and #4 (<i>H</i>) with various hFFAR1 expression levels. Error bars indicate s.e.m. (n = 3).</p

Point mutations of FFAR1/GPR40 differentially affect Ca²⁺ influx activities of fasiglifam and γ-LA.

<p>(<i>A</i>) Relative cell surface expression levels of FLAG-tagged FFAR1 wild<b>-</b>type and mutant receptors in transfected HEK293T cells were determined using flow cytometric analysis (FACS). (<i>B</i><b>-</b><i>J</i>) Effects of FFAR1 point mutations on the Ca²⁺ influx activities of FFAR1 agonists. HEK293T cells were transiently transfected with mock vector (<i>B</i>), wild-type (<i>C</i>), S8A (<i>D</i>), Y91A (<i>E</i>), H137A (<i>F</i>), R183A (<i>G</i>), L186F (<i>H</i>), N244A (<i>I</i>), and R258A (<i>J</i>) constructs. Data are representative of three independent experiments. Error bars indicate s.e.m. (n = 3); γ-LA, γ-linolenic acid.</p

Insulinotropic effects of fasiglifam are attenuated by pharmacological reduction of plasma FFA levels <i>in vivo</i>.

<p>(<i>A</i>) Effects of the lipolysis inhibitors acipimox (30 mg/kg) and fasiglifam (10 mg/kg) on plasma free fatty acids (FFAs) during the oral glucose tolerance test (OGTT) in N-STZ-1.5 rats. (<i>B</i>) Area under the curve (AUC) of plasma FFA during 0–120 min. Fas, fasiglifam. (<i>C</i>) Plasma glucose levels after coadministration of acipimox (30 mg/kg) and fasiglifam (10 mg/kg). (<i>D</i>) AUC of plasma glucose levels during 0–120 min. *<i>P</i><0.05, **<i>P</i><0.01 versus vehicle by Student’s t-test, ^$$<i>P</i><0.01 versus vehicle by Aspin–Welch test. (<i>E</i>) Plasma insulin concentrations after coadministration of acipimox and fasiglifam during OGTT. (<i>F</i>) Insulinotropic effects of fasiglifam (Fas) just before glucose load (time 0) shown in (<i>E</i>) in the absence and presence of acipimox. **<i>P</i><0.01 versus vehicle, ^$<i>P</i><0.05 versus acipimox alone by Student’s t-test, followed by Bonferroni’s correction for four time point comparisons. Data represent mean ± s.e.m. (n = 6).</p