Cavβ3 regulates Ca2+-signalling and insulin expression in pancreatic β-cells in a cell-autonomous manner

<p>Voltage-gated Ca²⁺ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic [Ca²⁺] in pancreatic β-cells. Here, we deleted the Cavβ3-encoding gene <i>Cacnb3</i> in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca²⁺-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO cells. In the absence of Cavβ3, Cav-currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca²⁺ release and the frequency of Ca²⁺-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca²⁺-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.</p>

Cavβ3 regulates Ca2+-signalling and insulin expression in pancreatic β-cells in a cell-autonomous manner

<p>Voltage-gated Ca²⁺ (Cav) channels consist of a pore-forming Cavα1 subunit and auxiliary Cavα2-δ and Cavβ subunits. In fibroblasts, Cavβ3, independent of its role as a Cav subunit, reduces the sensitivity to low concentrations of inositol-1,4,5-trisphosphate (IP3). Similarly, Cavβ3 could affect cytosolic [Ca²⁺] in pancreatic β-cells. Here, we deleted the Cavβ3-encoding gene <i>Cacnb3</i> in insulin-secreting rat β-(Ins-1) cells using CRISPR/Cas9. These cells were used as controls to investigate the role of Cavβ3 on Ca²⁺-signalling, glucose-induced insulin secretion (GIIS), Cav-channel activity and gene expression in wild-type cells in which Cavβ3 and the IP3-receptor were co-immunoprecipitated. Transcript and protein profiling revealed significantly increased levels of insulin transcription factor Mafa, CaMKIV, neuroendocrine convertase1 (Pcsk1) and nitric oxide synthase-1 (NOS-1) in Cavβ3-KO cells. In the absence of Cavβ3, Cav-currents were not altered. In contrast, CREB activity, the amount of MAFA protein and GIIS, the extent of IP3-dependent Ca²⁺ release and the frequency of Ca²⁺-oscillations were increased. These processes were decreased by the Cavβ3 protein in a concentration-dependent manner. Our study shows that Cavβ3 interacts with the IP3-receptor in isolated β-cells, controls IP3-dependent Ca²⁺-signalling independently of Cav channel functions, and thereby regulates insulin expression and its glucose-dependent release in a cell-autonomous manner.</p>

Association Analysis of Neuronal Nitric Oxide Synthase 1 Gene Polymorphism With Psychopathological Symptoms in Chronic Ketamine Users

Objective: We previously found that chronic ketamine usages were associated with various psychotic and cognitive symptoms mimicking schizophrenia. The blockade of the NMDA receptor and subsequent nitric oxide synthase 1 (NOS1) dysfunction were found to be closely correlated with schizophrenia including NOS1 gene polymorphisms. We examined the allelic variants of the gene coding neuronal nitric oxide synthase 1 (NOS1) in chronic ketamine users in the Chinese population and analyzed the association between NOS1 gene polymorphism and psychopathological symptoms in chronic ketamine users. The association between the NOS1 polymorphism and ketamine use characteristics was also examined.Methods: One hundred ninety seven male chronic ketamine users and 82 controls were recruited. Four common SNPs of the NOS1 gene, rs6490121, rs41279104, rs3782206, and rs3782219, were examined by real-time PCR with the TaqMan® assay system. Psychopathological symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS), Beck Depression Inventory (BDI), and the Beck Anxiety Inventory (BAI).Results: The genotype distribution of rs6490121 and rs41279104 in chronic ketamine users was significantly different from that in the control (p = 0.0001 and p = 0.002). The G allele frequency of rs6490121 in ketamine users was higher than that in the control (p = 2.23 * 10−6, OR = 3.07, 95% CI = 1.93–4.90). The T allele frequency of rs41279104 in chronic ketamine users was higher than that in the control (p = 0.01, OR = 1.76, 95% CI = 1.14–2.72). The BAI score was significantly different among the three genotypic groups of rs6490121 (F = 6.21, p = 0.002) in ketamine users; subjects of genotype AG and GG had a lower score than subjects of genotype AA. The score of the negative symptom subscale of PANSS was significantly different among the three genotypic groups of rs41279104 (F = 5.39, p = 0.005); in ketamine users, subjects of genotype CT and TT had a higher score than subjects of genotype CC. There was no difference in drug use characteristics in different genotypes of the four NOS1 gene polymorphisms tested in ketamine users (p &gt; 0.05).

Zinc Finger Transcription Factor Zbtb16 Coordinates the Response to Energy Deficit in the Mouse Hypothalamus

The central nervous system controls feeding behavior and energy expenditure in response to various internal and external stimuli to maintain energy balance. Here we report that the newly identified transcription factor zinc finger and BTB domain containing 16 (Zbtb16) is induced by energy deficit in the paraventricular (PVH) and arcuate (ARC) nuclei of the hypothalamus via glucocorticoid (GC) signaling. In the PVH, Zbtb16 is expressed in the anterior half of the PVH and co-expressed with many neuronal markers such as corticotropin-releasing hormone (Crh), thyrotropin-releasing hormone (Trh), oxytocin (Oxt), arginine vasopressin (Avp), and nitric oxide synthase 1 (Nos1). Knockdown (KD) of Zbtb16 in the PVH results in attenuated cold-induced thermogenesis and improved glucose tolerance without affecting food intake. In the meantime, Zbtb16 is predominantly expressed in agouti-related neuropeptide/neuropeptide Y (Agrp/Npy) neurons in the ARC and its KD in the ARC leads to reduced food intake. We further reveal that chemogenetic stimulation of PVH Zbtb16 neurons increases energy expenditure while that of ARC Zbtb16 neurons increases food intake. Taken together, we conclude that Zbtb16 is an important mediator that coordinates responses to energy deficit downstream of GCs by contributing to glycemic control through the PVH and feeding behavior regulation through the ARC, and additionally reveal its function in controlling energy expenditure during cold-evoked thermogenesis via the PVH. As a result, we hypothesize that Zbtb16 may be involved in promoting weight regain after weight loss.

Evaluating Common Variants in NOS1AP in Patients with Implantable Cardioverter Defibrillator for Secondary Prevention

Background Contemporary researchers found single nucleotide polymorphisms (SNPs) in nitric oxide synthase 1 adaptor protein (NOS1AP) gene are associated with altered QT intervals and SCD. However, the clinical utility and implications of these SNP have not been described. This study aims to explore the clinical utility and implications of SNPs in nitric oxide synthase 1 adaptor protein (NOS1AP) in patients with implantable cardioverter defibrillator (ICD) for secondary prevention. Methods We firstly conducted a case-control study to evaluate the associations between hot-spot SNPs in NOS1AP (rs12143842, rs10494366, rs12567209 and rs16847548) and patients with ICD for secondary prevention. Then the clinical values of the positive SNPs were further evaluated in these patients. All patients were divided into three groups according to different genotypes of the positive SNPs. ICD interrogation data at 3, 12 months, and 3 years after implantation, which include rapid ventricular arrhythmia episodes and appropriate therapies, were analyzed in three genotypes. Results Case-control study revealed significant allelic association between rs10494366 and ICD recipients who experienced appropriate therapies. After a mean follow-up time of 31.70 ± 9.15 months, we detected not only significant difference among three genotypes on the distribution of ICD shocks and appropriate therapies, but apparently also the correlation of rs10494366 and ICD shocks. Furthermore, under kaplan-meier and cox regression analysis, TT genotype both showed higher risk for sudden cardiac death (SCD) compared with GG genotype. Conclusions The present study revealed that SNP rs10494366 was associated with appropriate therapies and SCD in patients with ICD for secondary prevention for the first time.