Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Ischemic Postconditioning Reduces Reperfusion Arrhythmias by Adenosine Receptors and Protein Kinase C Activation but Is Independent of KATP Channels or Connexin 43.

Ischemic postconditioning (IPoC) reduces reperfusion arrhythmias but the antiarrhythmic mechanisms remain unknown. The aim of this study was to analyze IPoC electrophysiological effects and the role played by adenosine A1, A2A and A3 receptors, protein kinase C, ATP-dependent potassium (KATP) channels, and connexin 43. IPoC reduced reperfusion arrhythmias (mainly sustained ventricular fibrillation) in isolated rat hearts, an effect associated with a transient delay in epicardial electrical activation, and with action potential shortening. Electrical impedance measurements and Lucifer-Yellow diffusion assays agreed with such activation delay. However, this delay persisted during IPoC in isolated mouse hearts in which connexin 43 was replaced by connexin 32 and in mice with conditional deletion of connexin 43. Adenosine A1, A2A and A3 receptor blockade antagonized the antiarrhythmic effect of IPoC and the associated action potential shortening, whereas exogenous adenosine reduced reperfusion arrhythmias and shortened action potential duration. Protein kinase C inhibition by chelerythrine abolished the protective effect of IPoC but did not modify the effects on action potential duration. On the other hand, glibenclamide, a KATP inhibitor, antagonized the action potential shortening but did not interfere with the antiarrhythmic effect. The antiarrhythmic mechanisms of IPoC involve adenosine receptor activation and are associated with action potential shortening. However, this action potential shortening is not essential for protection, as it persisted during protein kinase C inhibition, a maneuver that abolished IPoC protection. Furthermore, glibenclamide induced the opposite effects. In addition, IPoC delays electrical activation and electrical impedance recovery during reperfusion, but these effects are independent of connexin 43.

Involvement of Connexin Hemichannels in the Inflammatory Response of Chronic Diseases.

Over the last decade it has become evident that under normal conditions connexin hemichannels are either not expressed (e.g., skeletal muscle) or are expressed in very low numbers with low open probability in various mammalian tissues (e.g., liver and central nervous system (CNS)).[...].

Insulin stimulus-secretion coupling is triggered by a novel thiazolidinedione/sulfonylurea hybrid in rat pancreatic islets.

New compounds with promising antidiabetic activity were synthesized. For the first time, a portion of the glibenclamide molecule was bound to a part of the core structure of thiazolidinedione to evaluate insulin secretagogue activity. Following studies in our laboratory, 4-{2-[2-(3,4-dichlorophenyl)-4-oxo-1,3-thiazolidin-3-yl]ethyl}benzene-1-sulfonamide (DTEBS) was selected to evaluate glycemia using the glucose tolerance test and insulin secretagogue activity by E.L.I.S.A. The mechanism of action of this compound was studied by 45 Ca2+ influx and whole-cell patch-clamp in rat pancreatic isolated islets. Furthermore, AGE formation in vitro was investigated. We herein show that this novel hybrid compound (DTEBS) exhibits an insulinogenic index and a profile of serum insulin secretion able to maintain glucose homeostasis. Its mechanism of action is mediated by ATP-sensitive potassium channels (KATP) and L-type voltage-dependent calcium channels (VDCC) and by activating protein kinase C and A (PKC and PKA). In addition, the stimulatory action of the compound on calcium influx and insulin secretion indicates that the potentiation of voltage-sensitive K+ currents (Kv) is due to the repolarization phase of the action potential after secretagogue excitation-secretion in pancreatic islets. Furthermore, under these experimental conditions, the compound did not induce toxicity and the in vitro late response of the compound to protein glycation reinforces its use to prevent complications of diabetes. DTEBS exerts an insulin secretagogue effect by triggering KATP, VDCC, and Kv ionic currents, possibly via PKC and PKA pathway signal transduction, in beta-cells. Furthermore, DTEBS may hold potential for delaying the late complications of diabetes.

Polymorphism E23K (rs5219) in the KCNJ11 gene in Euro-Brazilian subjects with type 1 and 2 diabetes.

Insulin secretion is regulated by ATP-sensitive potassium channels (KATP). The potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11) gene, located on chromosome 11p15.1, encodes the subunit Kir6.2 that forms the pore region of KATP channels in pancreatic ß-cells. Among the single nucleotide polymorphisms (SNPs) associated with KCNJ11, the E23K polymorphism (rs5219) promotes a substitution (G > A) of a glutamic acid residue for lysine at position 23. The E23K SNP has been associated with diabetes in several populations, although with controversial results. The aim of this study was to evaluate the association of the E23K SNP with type 1 and 2 diabetes in a case-control study approved by the Ethics Committee. We genotyped 458 Euro-Brazilian individuals, classified as healthy (control group, CTRL, N = 217), patients with type 1 diabetes mellitus (T1D, N = 102), and patients with type 2 diabetes mellitus (T2D, N = 139). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using BanII restriction digestion. The restriction fragments were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining. The genotype (EE/EK/KK) frequencies (%) for the CTRL group (38.2/50.2/11.6), T1D (34.3/52.0/13.7), and T2D (38.2/48.9/12.9) were in Hardy-Weinberg equilibrium and there were no significant differences (CRTL vs T1D, P = 0.771; CRTL vs T2D, P = 0.937; T1D vs T2D, P = 0.831). The minor allele frequencies (MAF; K) for CTRL (37.0%), T1D (39.7%), and T2D (37.4%) were not different among the groups (P > 0.05). The MAF value for healthy subjects was similar to other Caucasian populations (34.5-37.5%). In summary, the E23K polymorphism (rs5219) was not associated with type 1 or 2 diabetes mellitus in the studied population.

Acute effect of 3ß-hidroxihop-22(29)ene on insulin secretion is mediated by GLP-1, potassium and calcium channels for the glucose homeostasis.

The effect of 3ß-hidroxihop-22(29)ene (3-BHO) on insulin and glucagon-like peptide 1 (GLP-1) secretion as well as the mechanism of action of the compound in pancreatic islet on glucose homeostasis was investigated. The data from in vivo treatment show that 3-BHO significantly reduces the hyperglycemia by increasing the insulin and GLP-1 secretion, as well as by accumulating hepatic glycogen in hyperglycemic rats. In rat pancreatic ß-cell, 3-BHO stimulates the glucose uptake, insulin vesicles translocation to the plasma membrane and thus the insulin secretion through the involvement of potassium channels (ATP- and Ca(2+)-dependent K(+) channels) and calcium channels (L-type voltage-dependent calcium channels (L-VDCC)). Furthermore, this study also provides evidence for a crosstalk between intracellular high calcium concentration, PKA and PKC in the signal transduction of 3-BHO to stimulate insulin secretion. In conclusion, 3-BHO diminishes glycaemia, stimulates GLP-1 secretion and potentiates insulin secretion and increase hepatic glycogen content. Moreover, this triterpene modulates calcium influx characterizing ATP-K(+), Ca(2+)-K(+) and L-VDCC channels-dependent pathways as well as PKA and PKC activity in pancreatic islets underlying the signaling of 3-BHO for the secretory activity and contribution on glucose homeostasis.

Increased plasma incretin concentrations identifies a subset of patients with persistent congenital hyperinsulinism without KATP channel gene defects.

Congenital hyperinsulinism causes profound hypoglycemia, which may persist or resolve spontaneously. Among 13 children with congenital hyperinsulinism, elevated incretin hormone concentrations were detected in 2 with atypical, persistent disease. We suggest that incretin biomarkers may identify these patients, and that elevated hormone levels may contribute to their pathophysiology.

Decreasing cx36 gap junction coupling compensates for overactive KATP channels to restore insulin secretion and prevent hyperglycemia in a mouse model of neonatal diabetes.

Mutations to the ATP-sensitive K(+) channel (KATP channel) that reduce the sensitivity of ATP inhibition cause neonatal diabetes mellitus via suppression of ß-cell glucose-stimulated free calcium activity ([Ca(2+)]i) and insulin secretion. Connexin-36 (Cx36) gap junctions also regulate islet electrical activity; upon knockout of Cx36, ß-cells show [Ca(2+)]i elevations at basal glucose. We hypothesized that in the presence of overactive ATP-insensitive KATP channels, a reduction in Cx36 would allow elevations in glucose-stimulated [Ca(2+)]i and insulin secretion to improve glucose homeostasis. To test this, we introduced a genetic knockout of Cx36 into mice that express ATP-insensitive KATP channels and measured glucose homeostasis and islet metabolic, electrical, and insulin secretion responses. In the normal presence of Cx36, after expression of ATP-insensitive KATP channels, blood glucose levels rapidly rose to >500 mg/dL. Islets from these mice showed reduced glucose-stimulated [Ca(2+)]i and no insulin secretion. In mice lacking Cx36 after expression of ATP-insensitive KATP channels, normal glucose levels were maintained. Islets from these mice had near-normal glucose-stimulated [Ca(2+)]i and insulin secretion. We therefore demonstrate a novel mechanism by which islet function can be recovered in a monogenic model of diabetes. A reduction of gap junction coupling allows sufficient glucose-stimulated [Ca(2+)]i and insulin secretion to prevent the emergence of diabetes.

Glibenclamide inhibits cell growth by inducing G0/G1 arrest in the human breast cancer cell line MDA-MB-231.

BACKGROUND: Glibenclamide (Gli) binds to the sulphonylurea receptor (SUR) that is a regulatory subunit of ATP-sensitive potassium channels (KATP channels). Binding of Gli to SUR produces the closure of KATP channels and the inhibition of their activity. This drug is widely used for treatment of type 2-diabetes and it has been signaled as antiproliferative in several tumor cell lines. In previous experiments we demonstrated the antitumoral effect of Gli in mammary tumors induced in rats. The aim of the present work was to investigate the effect of Gli on MDA-MB-231 breast cancer cell proliferation and to examine the possible pathways involved in this action. RESULTS: The mRNA expression of the different subunits that compose the KATP channels was evaluated in MDA-MB-231 cells by reverse transcriptase-polymerase chain reaction. Results showed the expression of mRNA for both pore-forming isoforms Kir6.1 and Kir6.2 and for the regulatory isoform SUR2B in this cell line. Gli inhibited cell proliferation assessed by a clonogenic method in a dose dependent manner, with an increment in the population doubling time. The KATP channel opener minoxidil increased clonogenic proliferation, effect that was counteracted by Gli. When cell cycle analysis was performed by flow cytometry, Gli induced a significant cell-cycle arrest in G0/G1 phase, together with an up-regulation of p27 levels and a diminution in cyclin E expression, both evaluated by immunoblot. However, neither differentiation evaluated by neutral lipid accumulation nor apoptosis assessed by different methodologies were detected. The cytostatic, non toxic effect on cell proliferation was confirmed by removal of the drug.Combination treatment of Gli with tamoxifen or doxorubicin showed an increment in the antiproliferative effect only for doxorubicin. CONCLUSIONS: Our data clearly demonstrated a cytostatic effect of Gli in MDA-MB-231 cells that may be mediated through KATP channels, associated to the inhibition of the G1-S phase progression. In addition, an interesting observation about the effect of the combination of Gli with doxorubicin leads to future research for a potential novel role for Gli as an adjuvant in breast cancer treatment.

INGAP-PP up-regulates the expression of genes and proteins related to K+ ATP channels and ameliorates Ca2+ handling in cultured adult rat islets.

Islet Neogenesis Associated Protein (INGAP) increases pancreatic beta-cell mass and potentiates glucose-induced insulin secretion. Here, we investigated the effects of the pentadecapeptide INGAP-PP in adult cultured rat islets upon the expression of proteins constitutive of the K(+)(ATP) channel, Ca(2+) handling, and insulin secretion. The islets were cultured in RPMI medium with or without INGAP-PP for four days. Thereafter, gene (RT-PCR) and protein expression (Western blotting) of Foxa2, SUR1 and Kir6.2, cytoplasmic Ca(2+) ([Ca(2+)](i)), static and dynamic insulin secretion, and (86)Rb efflux were measured. INGAP-PP increased the expression levels of Kir6.2, SUR1 and Foxa2 genes, and SUR1 and Foxa2 proteins. INGAP-PP cultured islets released significantly more insulin in response to 40 mM KCl and 100 muM tolbutamide. INGAP-PP shifted to the left the dose-response curve of insulin secretion to increasing concentrations of glucose (EC(50) of 10.0+/-0.4 vs. 13.7+/-1.5 mM glucose of the controls). It also increased the first phase of insulin secretion elicited by either 22.2 mM glucose or 100 microM tolbutamide and accelerated the velocity of glucose-induced reduction of (86)Rb efflux in perifused islets. These effects were accompanied by a significant increase in [Ca(2+)](i) and the maintenance of a considerable degree of [Ca(2+)](i) oscillations. These results confirm that the enhancing effect of INGAP-PP upon insulin release, elicited by different secretagogues, is due to an improvement of the secretory function in cultured islets. Such improvement is due, at least partly, to an increased K(+)(ATP) channel protein expression and/or changing in the kinetic properties of these channels and augmented [Ca(2+)](i) response. Accordingly, INGAP-PP could potentially be used to maintain the functional integrity of cultured islets and eventually, for the prevention and treatment of diabetes.

[Neonatal diabetes mellitus]. / Diabetes melito neonatal.

Neonatal diabetes is a rare condition characterized by hyperglycemia, requiring insulin treatment, diagnosed within the first months of life. The disorder may be either transient, resolving in infancy or early childhood with possible relapse later, or permanent in which case lifelong treatment is necessary. Both conditions are genetically heterogeneous; however, the majority of the cases of transient neonatal diabetes are due to abnormalities of an imprinted region of chromosome 6q24. For permanent neonatal diabetes, the most common causes are heterozygous activating mutations of KCNJ11, the gene encoding the Kir6.2 sub-unit of the ATP-sensitive potassium channel. In this article we discuss the clinical features of neonatal diabetes, the underlying genetic defects and the therapeutic implications.

Diabetes melito neonatal: [revisão] / Neonatal diabetes mellitus: [review]

O diabetes neonatal (DN) é uma condição rara caracterizada por hiperglicemia, que necessita de tratamento com insulina, diagnosticado nos primeiros meses de vida. Clinicamente pode ser classificado em DN transitório quando ocorre remissão da doença em poucos meses, podendo haver recorrência posterior; ou permanente quando, como o nome indica, não ocorre remissão. Ambas as condições são geneticamente heterogêneas; entretanto a maioria dos casos de DN transitório é decorrente de anormalidades da região de imprinted no cromossomo 6q24. Mutações ativadoras em heterozigose no gene KCNJ11, que codifica a subunidade Kir6.2 do canal de potássio ATP-sensível, são a causa mais comum de DN permanente. No presente artigo, discutimos as características clínicas do DN, os mecanismos moleculares envolvidos e suas implicações terapêuticas.

Neonatal diabetes is a rare condition characterized by hyperglycemia, requiring insulin treatment, diagnosed within the first months of life. The disorder may be either transient, resolving in infancy or early childhood with possible relapse later, or permanent in which case lifelong treatment is necessary. Both conditions are genetically heterogeneous; however, the majority of the cases of transient neonatal diabetes are due to abnormalities of an imprinted region of chromosome 6q24. For permanent neonatal diabetes, the most common causes are heterozygous activating mutations of KCNJ11, the gene encoding the Kir6.2 sub-unit of the ATP-sensitive potassium channel. In this article we discuss the clinical features of neonatal diabetes, the underlying genetic defects and the therapeutic implications.