Impact of Intensive Low-Density Lipoprotein Cholesterol-Lowering Therapy on Coronary Artery Plaques in Acute Coronary Syndrome.

Acute coronary syndrome (ACS) is associated with a high incidence of unstable plaques beyond the culprit lesion, leading to early recurrence of cardiovascular events. Coronary computed tomography angiography (CCTA) can be used to noninvasively observe plaques throughout the coronary arteries. To evaluate the impact of intensive low-density lipoprotein cholesterol (LDL-C)-lowering therapy on quantitative changes in coronary plaque, assessed using CCTA in a study population with ACS. In total, 81 consecutive patients with ACS who underwent CCTA at discharge and at 1-year follow-up from April 2018 to March 2020 were analyzed. The patients were divided into 2 groups: those who achieved LDL-C <70 mg/100 ml and those who did not. Changes in plaque morphology within and between the 2 groups were compared using CCTA. A total of 198 vessels were analyzed. The calcified plaque volume was significantly increased in the LDL-C <70 group (65.8 ± 80.1 mm3 to 73.6 ± 83.7 mm3, p = 0.007), whereas no significant change was observed in the LDL-C ≥70 group (106.9 ± 161.7 mm3 to 105.7 ± 137.5 mm3, p = 0.552). Percent change in low-attenuation plaque volume in the LDL <70 group was significantly lower than in the LDL-C ≥70 group (17.2 ± 90.9% vs 84.4 ± 162.6%, p = 0.020). Receiver operating characteristic curve analysis demonstrated that the target LDL-C level for low-attenuation plaque volume regression was 64 mg/100 ml. In conclusion, noninvasive CCTA demonstrated that intensive LDL-C lowering in high-risk patients with ACS could potentially lead to plaque stabilization.

Phosphorylation of α-synuclein at T64 results in distinct oligomers and exerts toxicity in models of Parkinson's disease.

α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.

Efficacy of catheter ablation from the noncoronary aortic cusp of verapamil-sensitive atrial tachycardia arising near the atrioventricular node.

BACKGROUND: The efficacy of catheter ablation from the noncoronary aortic cusp (NCC) of verapamil-sensitive atrial tachycardia arising near the atrioventricular node (AVN-AT) has yet to be fully clarified. OBJECTIVE: We elucidated the determinant of an effective AVN-AT ablation from the NCC. METHODS: After identifying the earliest atrial activation site (EAAS) during tachycardia, the direction of the slow conduction zone (SCZ) of the reentry circuit was identified by demonstrating manifest entrainment in 26 patients with AVN-AT. Catheter ablation was initially performed from the NCC irrespective of the local activation time. If NCC ablation was ineffective, catheter ablation was performed targeting the SCZ entrance. Then the anatomical relationship between the SCZ and the successful ablation site was elucidated. RESULTS: NCC catheter ablation terminated AVN-AT in 14 patients (NCC group) but not in 12 (non-NCC group). Catheter ablation targeting the SCZ entrance terminated all non-NCC group ATs. The local activation time at the NCC relative to the EAAS did not differ between the NCC and non-NCC groups (10.1 ± 6.5 ms vs 11.2 ± 4.8 ms; P = .6333). The direction of the SCZ was posterior to the EAAS in all NCC group patients; however, it was posterolateral (n = 5) and lateral (n = 7) to the EAAS in the non-NCC group, suggesting that the SCZ existed in the direction of the NCC in the NCC group but was away from the NCC in the non-NCC group. CONCLUSION: A close proximity between the NCC and the SCZ of the reentry circuit, but not the local activation time at the NCC, determined the efficacy of NCC catheter ablation in AVN-ATs.

Verification of Coronary Computed Tomography-Derived Fractional Flow Reserve Measurement Site for Detection of Significant Coronary Artery Disease.

Background: The optimal site for measuring computed tomography (CT)-derived fractional flow reserve (FFRCT) to detect significant coronary artery disease (CAD) remains unknown. We investigated how diagnostic performance changes with FFRCT measurement site. Methods and Results: The diagnostic performance of FFRCT, measured 1-2 cm distal to the stenosis vs. a far-distal site, in detecting significant CAD with invasive fractional flow reserve ≤0.8 was evaluated in 254 diseased vessels from 146 patients with stable or suspected CAD diagnosed by coronary CT angiography. Receiver operating characteristic curve analysis revealed a significantly larger area under the curve for FFRCT measured 1-2 cm distal to the stenosis than at a far-distal site (0.829 vs. 0.791, respectively; P=0.0305). The rate of reclassification of positive FFRCT was 19% for measurements made 1-2 cm distal to the stenosis, and diagnostic accuracy for FFRCT 0.71-0.80 improved from 36% to 58% (P=0.0052). Vessel-based diagnostic accuracy of FFRCT 1-2 cm distal to the stenosis and at a far-distal site was 75% and 65%, respectively (P<0.0001), with corresponding sensitivity of 87% and 94% (P=0.0039), specificity of 60% and 29% (P<0.0001), a positive predictive value of 73% and 62% (P=0.028), and a negative predictive value of 78% and 79% (P=0.958). Conclusions: Our data suggest measuring FFRCT 1-2 cm distal to the stenosis has better diagnostic performance for detecting physiologically significant CAD.

Comparison of Diagnostic Performance of Fractional Flow Reserve Derived from Coronary Computed Tomographic Angiography Versus Single-Photon Emission Computed Tomographic Myocardial Perfusion Imaging.

Fraction flow reserve (FFR) derived from computed tomography (FFRCT) has been proposed to be an effective gatekeeper for invasive angiographic referral. The purpose of the present study is to examine the real-world diagnostic performance of FFRCT and myocardial perfusion imaging as well as to assess the utility of FFRCT as a gatekeeper for invasive coronary angiography in patients suspected of having obstructive coronary artery disease. Total of 146 consecutive patients underwent both single-photon emission computed tomography (SPECT) and invasive FFR were evaluated. An FFRCT value 1 to 2 cm distal to a stenosis ≤0.80 was defined as positive for ischemia and a summed stress score ≥2 or transient ischemic dilatation ≥1.2 were positive for ischemia with the invasive FFR value of <0.80 serving as the gold standard. The patient-based sensitivity of FFRCT was significantly higher than SPECT (91 vs 52%, p <0.001) and exhibited similar positive predictive value (82 vs 82%, p = 0.91). These trends were observed even in patients with multivessel and left main trunk disease and those with severe coronary calcification. In conclusion, our data suggest that FFRCT has higher diagnostic performance characteristics than SPECT and details the superior FFRCT analysis in detecting patients with hemodynamically significant coronary artery disease. Our results support the clinical utility of FFRCT analysis as a gatekeeper for invasive coronary angiography in clinical practice.

Discovery of a Potent and Orally Bioavailable Melatonin Receptor Agonist.

To develop potent and orally bioavailable melatonin receptor (MT1 and MT2) agonists, a novel series of 5-6-5 tricyclic derivatives was designed, synthesized, and evaluated. The synthesized indeno[5,4-d][1,3]oxazole, cyclopenta[c]pyrazolo[1,5-a]pyridine, indeno[5,4-d][1,3]thiazole, and cyclopenta[e]indazole derivatives showed potent binding affinities for MT1/MT2 receptors. Further optimization of these derivatives based on their metabolic stability in human hepatic microsomes revealed that (S)-3b ((S)-N-[2-(2-methyl-7,8-dihydro-6H-indeno[5,4-d][1,3]oxazol-8-yl)ethyl]acetamide) was a potent MT1 and MT2 ligand (MT1, Ki = 0.031 nM; MT2, Ki = 0.070 nM) with good metabolic stability in human hepatic microsomes. Moreover, compound (S)-3b showed good BBB permeability in rats, and its in vivo pharmacological effects were confirmed by its sleep-promotion ability in cats.

The fission yeast gmn2+ gene encodes an ERD1 homologue of Saccharomyces cerevisiae required for protein glycosylation and retention of luminal endoplasmic reticulum proteins.

The gmn2 mutant of Schizosaccharomyces pombe has previously been shown to exhibit defects in protein glycosylation of N-linked oligosaccharides (Ballou, L. and Ballou, CE., Proc. Natl. Acad. Sci. USA, 92, 2790-2794 (1995)). Like most glycosylation-defective mutants, the S. pombe gmn2 mutant was found to be sensitive to hygromycin B, an aminoglycoside antibiotic. As a result of complementation analysis, the gmn2+ gene was found to be a single open reading frame that encodes a polypeptide of 373 amino acids consisting of multiple membrane-spanning regions. The Gmn2 protein shares sequence similarity with Kluyveromyces lactis and Saccharomyces cerevisiae Erd1 proteins, which are required for retention of luminal endoplasmic reticulum (ER) proteins. Although disruption of the gmn2+ gene is not lethal, the secreted glycoprotein showed a significant glycosylation defect with destabilization of the glycosyltransferase responsible for N-glycan elongation. It was also shown that a significant amount of BiP was missorted to the cell surface according to ADEL receptor destabilization. Fluorescent microscopy revealed that the functional Gmn2-EGFP fusion protein is mainly localized in the Golgi membrane. These results indicate that the Gmn2 protein is required for protein glycosylation and for retention of ER-resident proteins in S. pombe cells.

Comparison of the catheter ablation outcome in patients between targeting the entrance and exit of the reentry circuit in verapamil-sensitive atrial tachycardia originating from the atrioventricular-node vicinity.

Verapamil-sensitive atrial tachycardia originating from the atrioventricular node vicinity (AVN-AT) can be eliminated with radiofrequency energy (RF) deliveries targeting either the entrance or exit of its reentry circuit. However, the outcome of these different approaches has not been clarified well. Thus, we compared the catheter ablation outcome targeting the entrance of reentry circuit, identified by the entrainment method (Ent-Group; 21 patients) with that targeting the earliest atrial activation site (EAAS) during AT (Exit-Group; 16 patients). There was no significant difference in the tachycardia cycle length (441.4 ± 87.4 vs. 392.8 ± 64.8 ms, p = 0.0704) or distance from the His bundle (HB) site to the EAAS (6.5 ± 2.0 vs. 7.6 ± 1.8 mm, p = 0.0822) between the Ent- and Exit-Groups. However, distance from the successful ablation site to the HB site in the Ent-Group was significantly longer than that in the Exit-Group (13.4 ± 3.1 vs. 7.6 ± 1.8 mm, p < 0.0001), resulting in more frequent transient atrioventricular block episodes in the Exit-Group than Ent-Group (31.3 vs. 0%, p < 0.01). Initial ATs (AT1s) were terminated in all patients in both Groups. However, ATs accompanied by shifting in the EAAS (AT2) were induced more frequently in the Exit-Group than Ent-Group (50.0 vs. 14.3%, p < 0.02) after eliminating AT1. RF deliveries to the EAAS eliminated all AT2s. The number of RF deliveries was greater in the Exit-Group than Ent-Group (6.9 ± 3.3 vs. 3.9 ± 1.6, p < 0.001). In conclusion, RF ablation targeting the entrance sites can avoid AVN injury and is superior in reducing the number of RF deliveries and occurrence of different ATs than targeting the exit sites in the AVN-AT.

Stent Fracture Induced Infected Pseudo Coronary Artery Aneurysm with Massive Abscess Successfully Treated by Coronary Stent Graft.

Infected pseudo coronary artery aneurysm (CAA) is extremely rare, and currently, there is no established treatment. We experienced a rare case of an infected pseudo CAA brought on due to a stent fracture. Following prolonged successful antimicrobial administration, which proved effective in successfully treating the patient, we performed coronary stent graft placement. Although a surgical procedure should fundamentally be the first course of action considered in such cases, when there are concerns as to the degree of invasiveness, our strategy represents a viable option.

Efficient production of 1,3-propanediol by psychrophile-based simple biocatalysts in Shewanella livingstonensis Ac10 and Shewanella frigidimarina DSM 12253.

1,3-Propanediol (1,3-PDO) is a valuable compound with a large potential market in many industries. This study aimed to evaluate the abilities of the Psychrophile-based Simple bioCatalyst (PSCat) reaction system to biosynthesize 1,3-PDO. This biocatalyst has a potential platform that replaces the chemical-based production counterparts. The two genes involved in the metabolic pathway were expressed both individually and together in the psychrophilic host bacterium. The intracellular metabolic flux was deactivated using heat treatment, at 45 °C for 15 min. After individual gene expression (25.0 mM), 1,3-PDO productivity of the cells increased by approximately 2.5 times, in comparison to when genes were expressed together (10.2 mM). Productivity was boosted (31.1 mM) when the cofactor regeneration system was activated in the biocatalyst. Hence, both the ability of individual gene expression and the cofactor regeneration system were verified in the PSCat approach. Nonetheless, further research is necessary to develop and optimize this process for industrial production.

Determinants of insufficient improvement in fractional flow reserve following percutaneous coronary intervention.

Fractional flow reserve (FFR) has become an increasingly important index for decision making concerning coronary revascularization. It is commonly accepted that significant improvement in FFR following percutaneous coronary intervention (PCI) is associated with better symptomatic relief and a lower event rate. However, in lesions with insufficient FFR improvement, PCI may not improve prognosis. Leading to the observation that the clinical and angiographic characteristics associated with insufficient FFR improvement have not been fully explored. The purpose of this study was to investigate the factors associated with insufficient improvement in FFR. Using our own PCI database, established between January 2014 and December 2018, we identified 220 stable coronary artery lesions, which had been evaluated for both pre- and post-PCI FFR values. All 220 of these lesions were included in this study. The improvement in FFR (ΔFFR) was calculated in each lesion with the lowest quartile of ΔFFR being defined as the lowest ΔFFR group, and the other quartiles being defined as the intermediate-high ΔFFR group. The mean ΔFFR in the lowest and intermediate-high ΔFFR groups was 0.07 ± 0.02 and 0.21 ± 0.11, respectively. In multivariate logistic regression analysis, a short total stent length (10 mm increase: OR 0.67, 95% CI 0.47-0.96, P = 0.030), higher pre-PCI FFR (0.1 increase: OR 4.07, 95% CI 1.83-9.06, P = 0.001), in-stent restenosis (ISR) (OR 8.02, 95% CI 1.26-51.09, P = 0.028), myocardial infarction (MI) in the target vessel (OR 6.87, 95% CI 1.19-39.69, P = 0.031) and non-use of intravascular imaging (OR 0.35, 95% CI 0.12-0.99, P = 0.048) were significantly associated with the lowest ΔFFR group. The use of short stents, higher pre-PCI FFR values, ISR, MI in the target vessel, and non-use of intravascular imaging were significantly associated with insufficient FFR improvement. It was conversely suggested that full coverage and adequate dilatation of the lesions under an intravascular imaging guidance might contribute to an improvement in FFR.

Nucleus accumbens pathways control cell-specific gene expression in the medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is a critical component of a cortico-basal ganglia-thalamo-cortical loop regulating limbic and cognitive functions. Within this circuit, two distinct nucleus accumbens (NAc) output neuron types, dopamine D1 or D2 receptor-expressing neurons, dynamically control the flow of information through basal ganglia nuclei that eventually project back to the mPFC to complete the loop. Thus, chronic dysfunction of the NAc may result in mPFC transcriptomal changes, which in turn contribute to disease conditions associated with the mPFC and basal ganglia. Here, we used RNA sequencing to analyse differentially expressed genes (DEGs) in the mPFC following a reversible neurotransmission blocking technique in D1 or D2 receptor-expressing NAc neurons, respectively (D1-RNB, or D2-RNB). Gene Set Enrichment Analysis revealed that gene sets of layer 5b and 6 pyramidal neurons were enriched in DEGs of the mPFC downregulated in both NAc D1- and D2-RNB mice. In contrast, gene sets of layer 5a pyramidal neurons were enriched in upregulated DEGs of the mPFC in D1-RNB mice, and downregulated DEGs of the mPFC in D2-RNB mice. These findings reveal for the first time that NAc output pathways play an important role in controlling mPFC gene expression.

Clinical impact of successful recanalization for chronic total occlusion: insights from stress myocardial perfusion imaging.

The clinical benefits of percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) is still controversial. The purpose of this study is to assess the quantitative therapeutic benefits of successful PCI for CTO from the clinical data acquired by myocardial perfusion imaging (MPI). Consecutive 42 patients, who were successfully revascularized of CTO between August 2013 and March 2018, were examined. A stress MPI was performed before CTO PCI and at follow-up, and the changes in quantitative gated and perfusion single photon emission computed tomography parameters were examined. The follow-up interval was 18 ± 9 (median 14) months, during which 36 patients were maintained patency (patent CTO), while 6 were re-occluded (R/O CTO). The reduction in the % myocardial ischemia and the improvement in the ejection fraction were significantly higher in the patent CTO group than those in the R/O CTO group (67.5 ± 37.0% vs. - 56.4 ± 84.9%, p < 0.0001, 20.7 ± 49.8% vs. - 9.2 ± 20.6%, p = 0.0247, respectively). Interestingly, the improvements we observed were predominantly in the patients with LAD CTO rather than those with RCA or LCx CTO. Successful CTO PCI was able to reduce myocardial ischemia and improve the cardiac function when the patency after CTO PCI was maintained, with the most notable significance in the patients with LAD CTO.

Identification of evolutionarily conserved gene networks mediating neurodegenerative dementia.

Identifying the mechanisms through which genetic risk causes dementia is an imperative for new therapeutic development. Here, we apply a multistage, systems biology approach to elucidate the disease mechanisms in frontotemporal dementia. We identify two gene coexpression modules that are preserved in mice harboring mutations in MAPT, GRN and other dementia mutations on diverse genetic backgrounds. We bridge the species divide via integration with proteomic and transcriptomic data from the human brain to identify evolutionarily conserved, disease-relevant networks. We find that overexpression of miR-203, a hub of a putative regulatory microRNA (miRNA) module, recapitulates mRNA coexpression patterns associated with disease state and induces neuronal cell death, establishing this miRNA as a regulator of neurodegeneration. Using a database of drug-mediated gene expression changes, we identify small molecules that can normalize the disease-associated modules and validate this experimentally. Our results highlight the utility of an integrative, cross-species network approach to drug discovery.

Determinants of high device cost in current percutaneous coronary interventions.

BACKGROUND: Percutaneous coronary interventions (PCI), especially medical devices, consume large amounts of medical resources. It is important to know which type of lesions requires high device costs among current PCI. The purpose of this study was to investigate the association between lesion characteristics and medical device costs in current PCI. METHODS: We identified 593 coronary artery lesions in our PCI database between January 1, 2015 and December 31, 2015. The total PCI cost was calculated for each lesion. The highest quartile (Q1) of total PCI costs was defined as the highest cost group, whereas the other quartiles (Q2, Q3, Q4) were defined as the low-intermediate cost group. RESULTS: The mean PCI cost in the highest cost and low-intermediate cost groups was ¥1,032,943 ±â€¯211,912 and ¥532,547 ±â€¯112,127, respectively. In a multivariate logistic regression analysis, lesion length (10 mm increase: OR 2.93, 95% CI 2.25-3.82, P < 0.001), left main lesion (OR 2.96, 95% CI 1.02-8.60, P = 0.046), moderate to severe calcification (OR 16.43, 95% CI 7.97-33.88, P < 0.001), chronic total occlusion (CTO) (OR 5.83, 95% CI 2.07-16.39, P = 0.001), and bifurcation (OR 2.01, 95% CI 1.08-3.75, P = 0.027) were significantly associated with the highest cost group. CONCLUSIONS: Lesion characteristics including CTO, diffuse long lesion, calcification, and bifurcation were significantly associated with the highest device cost. Non-CTO complex lesions including bifurcation and calcification as well as CTO lesions require higher PCI device costs than non-complex lesions.

Discovery of a Slow Tight Binding LPA1 Antagonist (ONO-0300302) for the Treatment of Benign Prostatic Hyperplasia.

Scaffold hopping from the amide group of lead compound ONO-7300243 (1) to a secondary alcohol successfully gave a novel chemotype lysophosphatidic acid receptor 1 (LPA1) antagonist 4. Wash-out experiments using rat isolated urethra showed that compound 4 possesses a tight binding feature to the LPA1 receptor. Further modification of two phenyl groups of 1 to pyrrole and an indane moiety afforded an optimized compound ONO-0300302 (19). Despite its high i.v. clearance, 19 inhibited significantly an LPA-induced increase of intraurethral pressure (IUP) in rat (3 mg/kg, p.o.) and dog (1 mg/kg, p.o.) over 12 h. Binding experiments with [3H]-ONO-0300302 suggest that the observed long duration action is because of the slow tight binding character of 19.

Characterization of CRF1 receptor antagonists with differential peripheral vs central actions in CRF challenge in rats.

The aim of this study was to investigate peripheral and central roles of corticotropin-releasing factor (CRF) in endocrinological and behavioral changes. Plasma adrenocorticotropin (ACTH) concentration was measured as an activity of hypothalamic-pituitary-adrenal (HPA) axis. As behavioral changes, locomotion and anxiety behavior were measured after CRF challenge intravenously (i.v.) for the peripheral administration or intracerebroventricularly (i.c.v.) for the central administration. Plasma ACTH concentration was significantly increased by both administration routes of CRF; however, hyperlocomotion and anxiety behavior were induced only by the i.c.v. administration. In the drug discovery of CRF1 receptor antagonists, we identified two types of compounds, Compound A and Compound B, which antagonized peripheral CRF-induced HPA axis activation to the same extent, but showed different effects on the central CRF signal. These had similar in vitro CRF1 receptor binding affinities (15 and 10nM) and functional activities in reporter gene assay (15 and 9.5nM). In the ex vivo binding assays using tissues of the pituitary, oral treatment with Compound A and Compound B at 10mg/kg inhibited [125I]-CRF binding, whereas in the assay using tissues of the frontal cortex, treatment of Compound A but not Compound B inhibited [125I]-CRF binding, indicating that only Compound A inhibited central [125I]-CRF binding. In the peripheral CRF challenge, increase in plasma ACTH concentration was significantly suppressed by both Compound A and Compound B. In contrast, Compound A inhibited the increase in locomotion induced by the central CRF challenge while Compound B did not. Compound A also reduced central CRF challenge-induced anxiety behavior and c-fos immunoreactivity in the cortex and the hypothalamic paraventricular nucleus. These results indicate that the central CRF signal, rather than the peripheral CRF signal would be related to anxiety and other behavioral changes, and CRF1 receptor antagonism in the central nervous system may be critical for identifying drug candidates for anxiety and mood disorders.

In vitro comparison of duration of action of melatonin agonists on melatonin MT1 receptor: possible link between duration of action and dissociation rate from receptor.

Melatonin MT1 and MT2 receptors are Gi protein-coupled receptors and promising therapeutic targets for a number of diseases. A proportion of G protein-coupled receptor agonists and antagonists have been classified according to their duration of action, which influences their pharmacological efficacy. However, the duration of action of melatonin agonists remains unclear. In this study, we investigated the duration of action of melatonin agonists (melatonin, 2-iodomelatonin, ramelteon, and the ramelteon metabolite M-II) at the melatonin MT1 receptor, which is more resistant to agonist-induced desensitization than the melatonin MT2 receptor. In Chinese hamster ovary cells stably expressing the human melatonin MT1 receptor, significant differences in the duration of action were observed after 2-h pretreatment with agonists followed by washout. In contrast to melatonin and M-II, the agonist activities of ramelteon and 2-iodomelatonin were persistent (i.e. inhibition of forskolin-stimulated cAMP formation and increase in ERK 1/2 phosphorylation) even after repeated washouts. Similar activities were observed for INS-1 cells endogenously expressing the rat MT1 receptor. Further, we examined potential factors linked to the duration of action. Residual activities of melatonin agonists after washout strongly correlated with their dissociation rates from the human melatonin MT1 receptor, but not their lipophilicity or extent of desensitization. These data suggest that the in vitro duration of action significantly differs between melatonin agonists and might dictate dissociation kinetics. Characterization of these in vitro properties may facilitate further in vivo study of the duration of action.

The melatonin agonist ramelteon induces duration-dependent clock gene expression through cAMP signaling in pancreatic INS-1 ß-cells.

Prolonged exposure to melatonin improves glycemic control in animals. Although glucose metabolism is controlled by circadian clock genes, little is known about the role of melatonin signaling and its duration in the regulation of clock gene expression in pancreatic ß-cells. Activation of MT1 and MT2 melatonin receptors inhibits cAMP signaling, which mediates clock gene expression. Therefore, this study investigated exposure duration-dependent alterations in cAMP element-binding protein (CREB) phosphorylation and clock gene expression that occur during and after exposure to ramelteon, a selective melatonin agonist used to treat insomnia. In rat INS-1 cells, a pancreatic ß-cell line endogenously expressing melatonin receptors, ramelteon persistently decreased CREB phosphorylation during the treatment period (2-14 h), whereas the subsequent washout induced an enhancement of forskolin-stimulated CREB phosphorylation in a duration- and concentration-dependent manner. This augmentation was blocked by forskolin or the melatonin receptor antagonist luzindole. Similarly, gene expression analyses of 7 clock genes revealed the duration dependency of the effects of ramelteon on Rev-erbα and Bmal1 expression through melatonin receptor-mediated cAMP signaling; longer exposure times (14 h) resulted in greater increases in the expression and signaling of Rev-erbα, which is related to ß-cell functions. Interestingly, this led to amplified oscillatory Rev-erbα and Bmal1 expression after agonist washout and forskolin stimulation. These results provide new insights into the duration-dependent effects of ramelteon on clock gene expression in INS-1 cells and may improve the understanding of its effect in vivo. The applicability of these results to pancreatic islets awaits further investigation.

Pharmacological characterization of M-II, the major human metabolite of ramelteon.

The duration of action of melatonin may be important for improvements in sleep efficiency in insomniacs. Ramelteon, a selective melatonin agonist, is primarily metabolized to the active metabolite M-II, which has a longer half-life and greater systemic exposure than ramelteon. Hence, M-II may contribute significantly to the hypnotic benefits of ramelteon. We assessed the ramelteon-like activity of M-II in vitro and in vivo using cats. Binding and functional studies in Chinese hamster ovary cells expressing human melatonin receptors (MT1 or MT2) revealed that M-II binds melatonin receptors with lower affinity (Ki: 114 and 566 pmol/l for MT1 and MT2, respectively) and has lower potency (IC50: 208 and 1,470 pmol/l for MT1 and MT2, respectively) compared with ramelteon. However, higher M-II doses significantly improved sleep in cats. Thus, M-II may contribute to the clinical efficacy of ramelteon.