A High-Throughput Assay for the Pancreatic Islet Beta-Cell Potassium Channel: Use in the Pharmacological Characterization of Insulin Secretagogues Identified from Phenotypic Screening.

Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.

Diazoxide Preconditioning of Nonhuman Primate Pancreas Improves Islet Isolation Outcomes by Mitochondrial Protection.

OBJECTIVES: Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model. METHODS: Cynomolgus monkey pancreata were flushed with UW or UW + 150 µM DZ during procurement and preserved for 8 hours before islet isolation. RESULTS: First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model. CONCLUSIONS: This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

Diazoxide affects mitochondrial bioenergetics by the opening of mKATP channel on submicromolar scale.

BACKGROUND: Cytoprotection afforded by mitochondrial ATP-sensitive K+-channel (mKATP-channel) opener diazoxide (DZ) largely depends on the activation of potassium cycle with eventual modulation of mitochondrial functions and ROS production. However, generally these effects were studied in the presence of Mg∙ATP known to block K+ transport. Thus, the purpose of our work was the estimation of DZ effects on K+ transport, K+ cycle and ROS production in rat liver mitochondria in the absence of Mg∙ATP. RESULTS: Without Mg·ATP, full activation of native mKATP-channel, accompanied by the increase in ATP-insensitive K+ uptake, activation of K+-cycle and respiratory uncoupling, was reached at ≤0.5 µM of DZ,. Higher diazoxide concentrations augmented ATP-insensitive K+ uptake, but not mKATP-channel activity. mKATP-channel was blocked by Mg·ATP, reactivated by DZ, and repeatedly blocked by mKATP-channel blockers glibenclamide and 5-hydroxydecanoate, whereas ATP-insensitive potassium transport was blocked by Mg2+ and was not restored by DZ. High sensitivity of potassium transport to DZ in native mitochondria resulted in suppression of mitochondrial ROS production caused by the activation of K+-cycle on sub-micromolar scale. Based on the oxygen consumption study, the share of mKATP-channel in respiratory uncoupling by DZ was found. CONCLUSIONS: The study of mKATP-channel activation by diazoxide in the absence of MgATP discloses novel, not described earlier, aspects of mKATP-channel interaction with this drug. High sensitivity of mKATP-channel to DZ results in the modulation of mitochondrial functions and ROS production by DZ on sub-micromolar concentration scale. Our experiments led us to the hypothesis that under the conditions marked by ATP deficiency affinity of mKATP-channel to DZ can increase, which might contribute to the high effectiveness of this drug in cardio- and neuroprotection.

Diazoxide Modulates Cardiac Hypertrophy by Targeting H2O2 Generation and Mitochondrial Superoxide Dismutase Activity.

BACKGROUND: Cardiac hypertrophy involves marked wall thickening or chamber enlargement. If sustained, this condition will lead to dysfunctional mitochondria and oxidative stress. Mitochondria have ATP-sensitive K+ channels (mitoKATP) in the inner membrane that modulate the redox status of the cell. OBJECTIVE: We investigated the in vivo effects of mitoKATP opening on oxidative stress in isoproterenol- induced cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced in Swiss mice treated intraperitoneally with isoproterenol (ISO - 30 mg/kg/day) for 8 days. From day 4, diazoxide (DZX - 5 mg/kg/day) was used in order to open mitoKATP (a clinically relevant therapy scheme) and 5-hydroxydecanoate (5HD - 5 mg/kg/day) or glibenclamide (GLI - 3 mg/kg/day) were used as mitoKATP blockers. RESULTS: Isoproterenol-treated mice had elevated heart weight/tibia length ratios (HW/TL). Additionally, hypertrophic hearts had elevated levels of carbonylated proteins and Thiobarbituric Acid Reactive Substances (TBARS), markers of protein and lipid oxidation. In contrast, mitoKATP opening with DZX avoided ISO effects on gross hypertrophic markers (HW/TL), carbonylated proteins and TBARS, in a manner reversed by 5HD and GLI. Moreover, DZX improved mitochondrial superoxide dismutase activity. This effect was also blocked by 5HD and GLI. Additionally, ex vivo treatment of isoproterenol- induced hypertrophic cardiac tissue with DZX decreased H2O2 production in a manner sensitive to 5HD, indicating that this drug also acutely avoids oxidative stress. CONCLUSION: Our results suggest that diazoxide blocks oxidative stress and reverses cardiac hypertrophy. This pharmacological intervention could be a potential therapeutic strategy to prevent oxidative stress associated with cardiac hypertrophy.

Diazoxide prevents H2O2-induced chondrocyte apoptosis and cartilage degeneration in a rat model of osteoarthritis by reducing endoplasmic reticulum stress.

Osteoarthritis (OA) is a common disease affecting elderly individuals. Its incidence rises sharply with age, and chondrocyte apoptosis plays a vital role in its pathogenesis. Diazoxide opens mitochondrial ATP-sensitive potassium (mitoKATP) channels and exerts multiple pharmacological effects, including reductions in blood pressure and blood sugar levels. It also exerts anti-apoptotic activities, but the cellular and molecular mechanisms by which diazoxide inhibits chondrocyte apoptosis are unknown, as is whether apoptosis is related to endoplasmic reticulum stress (ERS). In the present study, we explored the mechanism underlying the chondroprotective effect of diazoxide on hydrogen peroxide (H2O2)-stimulated chondrocyte apoptosis in rats with surgically induced OA. A cell counting kit-8 (CCK-8) assay showed that the viability of H2O2-stimulated chondrocytes was enhanced in a dose-dependent manner. However, at a concentration ≥400µM, diazoxide had other, negative effects. The protective effect of diazoxide in vitro included inhibition of the ERS response and of mitochondrial dysfunction induced by H2O2 stimulation. These responses were related to activation of the PERK1/2 and ERK1/2 signaling pathways; the prevention of chondrocyte apoptosis; the down-regulation of caspase-3, Bax, ATF-6 and C/EBP-homologous protein (CHOP) expression; and the up-regulation of Bcl-2 and Col II. In vivo, histological and immunohistochemical analyses of caspase-3 and CHOP expression revealed that diazoxide ameliorated cartilage degeneration in a rat model of OA, as revealed by histological and immunohistochemical analyses of caspase-3 and CHOP expression. Diazoxide suppressed H2O2-triggered chondrocyte apoptosis, and ameliorated cartilage degeneration, by inhibiting the development of ERS.

Bioenergetic and volume regulatory effects of mitoKATP channel modulators protect against hypoxia-reoxygenation-induced mitochondrial dysfunction.

The mitochondrial ATP-sensitive K(+) (mitoKATP) channel plays a significant role in mitochondrial physiology and protects against ischemic reperfusion injury in mammals. Although fish frequently face oxygen fluctuations in their environment, the role of the mitoKATP channel in regulating the responses to oxygen stress is rarely investigated in this class of animals. To elucidate whether and how the mitoKATP channel protects against hypoxia-reoxygenation (H-R)-induced mitochondrial dysfunction in fish, we first determined the mitochondrial bioenergetic effects of two key modulators of the channel, diazoxide and 5-hydroxydecanoate (5-HD), using a wide range of doses. Subsequently, the effects of low and high doses of the modulators on mitochondrial bioenergetics and volume under normoxia and after H-R using buffers with and without magnesium and ATP (Mg-ATP) were tested. In the absence of Mg-ATP (mitoKATP channel open), both low and high doses of diazoxide improved mitochondrial coupling, but only the high dose of 5-HD reversed the post-H-R coupling-enhancing effect of diazoxide. In the presence of Mg-ATP (mitoKATP channel closed), diazoxide at the low dose improved coupling post-H-R, and this effect was abolished by 5-HD at the low dose. Interestingly, both low and high doses of diazoxide reversed H-R-induced swelling under mitoKATP channel open conditions, but this effect was not sensitive to 5-HD. Under mitoKATP channel closed conditions, diazoxide at the low dose protected the mitochondria from H-R-induced swelling and 5-HD at the low dose reversed this effect. In contrast, diazoxide at the high dose failed to reduce the swelling caused by H-R, and the addition of the high dose of 5-HD enhanced mitochondrial swelling. Overall, our study showed that in the presence of Mg-ATP, both opening of mitoKATP channels and bioenergetic effects of diazoxide were protective against H-R in fish mitochondria, while in the absence of Mg-ATP only the bioenergetic effect of diazoxide was protective.

Cucurbita ficifolia Bouché increases insulin secretion in RINm5F cells through an influx of Ca(2+) from the endoplasmic reticulum.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cucurbita ficifolia Bouché(C. ficifolia) is a plant used in Mexican traditional medicine to control type 2 diabetes (T2D). The hypoglycemic effect of the fruit of C. ficifolia has been demonstrated in different experimental models and in T2D patients. It has been proposed that D-chiro-inositol (DCI) is the active compound of the fruit. Additionally, it has been reported that C. ficifolia increases the mRNA expression of insulin and Kir 6.2 (a component of the ATP-sensitive potassium (K(+)ATP) channel, which is activated by sulphonylurea) in RINm5F cells. However, it remains unclear whether C. ficifolia and DCI causes the secretion of insulin by increasing the concentration of intracellular calcium ([Ca(2+)]i) through K(+)ATP channel blockage or from the reservoir in the endoplasmic reticulum (ER). MATERIAL AND METHODS: The aqueous extract of C. ficifolia was obtained and standardized with regard to its DCI content. RINm5F pancreatic ß-cells were incubated with different concentrations (50, 100, 200 and 400µM) of DCI alone or C. ficifolia (9, 18, 36 and 72µg of extract/mL), and the [Ca(2+)]i of the cells was quantified. The cells were preloaded with the Ca(2+) fluorescent dye fluo4-acetoxymethyl ester (AM) and visualized by confocal microscopy. Insulin secretion was measured by an ELISA method. Subsequently, the effect of C. ficifolia on the K(+)ATP channel was evaluated. In this case, the blocker activator diazoxide was used to inhibit the C. ficifolia-induced calcium influx. In addition, the inositol 1,4,5-trisphosphate (IP3)-receptor-selective inhibitor 2-amino-thoxydiphenylborate (2-APB) was used to inhibit the influx of calcium from the ER that was induced by C. ficifolia. RESULTS: It was found that DCI alone did not increase [Ca(2+)]i or insulin secretion. In contrast, treatment with C. ficifolia increased [Ca(2+)]i 10-fold compared with the control group. Insulin secretion increased by 46.9%. In the presence of diazoxide, C. ficifolia decreased [Ca(2+)]i by 50%, while insulin secretion increased by 36.4%. In contrast, in the presence of 2-APB, C. ficifolia increased [Ca(2+)]i 18-fold, while insulin secretion remained constant, indicating an additive effect. Therefore, C. ficifolia was not found to block the K(+)ATP channel. However, it did exert an effect by increasing [Ca(2+)]i from the ER, which may partly explain the insulin secretion observed following treatment with C. ficifolia. CONCLUSIONS: The hypoglycemic properties of C. ficifolia can be explained in part by its effect as a secretagogue for insulin through an increase in [Ca(2+)]i from the calcium reservoir in the ER. Therefore, the mechanism of action of C. ficifolia is different to those of the currently used hypoglycemic drugs, such as sulfonylureas. These results support that C. ficifolia may be a potential natural resource for new agents to control T2D.

Central Regulation of Glucose Production May Be Impaired in Type 2 Diabetes.

The challenges of achieving optimal glycemic control in type 2 diabetes highlight the need for new therapies. Inappropriately elevated endogenous glucose production (EGP) is the main source of hyperglycemia in type 2 diabetes. Because activation of central ATP-sensitive potassium (KATP) channels suppresses EGP in nondiabetic rodents and humans, this study examined whether type 2 diabetic humans and rodents retain central regulation of EGP. The KATP channel activator diazoxide was administered in a randomized, placebo-controlled crossover design to eight type 2 diabetic subjects and seven age- and BMI-matched healthy control subjects. Comprehensive measures of glucose turnover and insulin sensitivity were performed during euglycemic pancreatic clamp studies following diazoxide and placebo administration. Complementary rodent clamp studies were performed in Zucker Diabetic Fatty rats. In type 2 diabetic subjects, extrapancreatic KATP channel activation with diazoxide under fixed hormonal conditions failed to suppress EGP, whereas matched control subjects demonstrated a 27% reduction in EGP (P = 0.002) with diazoxide. Diazoxide also failed to suppress EGP in diabetic rats. These results suggest that suppression of EGP by central KATP channel activation may be lost in type 2 diabetes. Restoration of central regulation of glucose metabolism could be a promising therapeutic target to reduce hyperglycemia in type 2 diabetes.

Diazoxide, a K(ATP) channel opener, prevents ischemia-reperfusion injury in rodent pancreatic islets.

Diazoxide (DZ) is a pharmacological opener of ATP-sensitive K(+) channels that has been used for mimicking ischemic preconditioning and shows protection against ischemic damage. Here we investigated whether diazoxide supplementation to University of Wisconsin (UW) solution has cellular protection during islet isolation and improves in vivo islet transplant outcomes in a rodent ischemia model. C57/B6 mice pancreata were flushed with UW or UW + DZ solution and cold preserved for 6 or 10 h prior to islet isolation. Islet yield, in vitro and in vivo function, mitochondrial morphology, and apoptosis were evaluated. Significantly higher islet yields were observed in the UW + DZ group than in the UW group (237.5 ± 25.6 vs. 108.7 ± 49.3, p < 0.01). The islets from the UW + DZ group displayed a significantly higher glucose-induced insulin secretion (0.97 ng/ml ± 0.15 vs. 0.758 ng/ml ± 0.21, p = 0.009) and insulin content (60.96 ng/islet ± 13.94 vs. 42.09 ng/islet ± 8.15, p = 0.002). The DZ-treated islets had well-preserved mitochondrial morphology with superior responses of mitochondrial potentials, and calcium influx responded to glucose. A higher number of living cells and less late apoptotic cells were observed in the UW + DZ group (p < 0.05). Additionally, the islets from the UW + DZ group had a significantly higher cure rate and improved glucose tolerance. This study is the first to report mitoprotective effects of DZ for pancreas preservation and islet isolation. In the future, it will be necessary to further understand the underlying mechanism for the mitoprotection and to test this promising approach for pancreas preservation and the islet isolation process in nonhuman primates and ultimately humans.

Nox2 as a potential target of mitochondrial superoxide and its role in endothelial oxidative stress.

Superoxide (O2(·-)) production by the NADPH oxidases is implicated in the pathogenesis of many cardiovascular diseases, including hypertension. We have previously shown that activation of NADPH oxidases increases mitochondrial O2(·-) which is inhibited by the ATP-sensitive K(+) channel (mitoKATP) inhibitor 5-hydroxydecanoic acid and that scavenging of mitochondrial or cytoplasmic O2(·-) inhibits hypertension. We hypothesized that mitoKATP-mediated mitochondrial O2(·-) potentiates cytoplasmic O2(·-) by stimulation of NADPH oxidases. In this work we studied Nox isoforms as a potential target of mitochondrial O2(·-). We tested contribution of reverse electron transfer (RET) from complex II to complex I in mitochondrial O2(·-) production and NADPH oxidase activation in human aortic endothelial cells. Activation of mitoKATP with low dose of diazoxide (100 nM) decreased mitochondrial membrane potential (tetramethylrhodamine methyl ester probe) and increased production of mitochondrial and cytoplasmic O2(·-) measured by site-specific probes and mitoSOX. Inhibition of RET with complex II inhibitor (malonate) or complex I inhibitor (rotenone) attenuated the production of mitochondrial and cytoplasmic O2(·-). Supplementation with a mitochondria-targeted SOD mimetic (mitoTEMPO) or a mitochondria-targeted glutathione peroxidase mimetic (mitoEbselen) inhibited production of mitochondrial and cytoplasmic O2(·-). Inhibition of Nox2 (gp91ds) or Nox2 depletion with small interfering RNA but not Nox1, Nox4, or Nox5 abolished diazoxide-induced O2(·-) production in the cytoplasm. Treatment of angiotensin II-infused mice with RET inhibitor dihydroethidium (malate) significantly reduced blood pressure. Our study suggests that mitoKATP-mediated mitochondrial O2(·-) stimulates cytoplasmic Nox2, contributing to the development of endothelial oxidative stress and hypertension.

Nuciferine stimulates insulin secretion from beta cells-an in vitro comparison with glibenclamide.

ETHNOPHARMACOLOGICAL RELEVANCE: Several Asian plants are known for their anti-diabetic properties and produce alkaloids and flavonoids that may stimulate insulin secretion. MATERIALS AND METHODS: Using Vietnamese plants (Nelumbo nucifera, Gynostemma pentaphyllum, Smilax glabra, and Stemona tuberosa), we extracted two alkaloids (neotuberostemonine, nuciferine) and four flavonoids (astilbin, engeletin, smitilbin, and 3,5,3'-trihydroxy-7,4'-dimethoxyflavone), and studied their insulin stimulatory effects. RESULTS: Nuciferine, extracted from Nelumbo nucifera, stimulated both phases of insulin secretion in isolated islets, whereas the other compounds had no effect. The effect of nuciferine was totally abolished by diazoxide and nimodipine, and diminished by protein kinase A and protein kinase C inhibition. Nuciferine and potassium had additive effects on insulin secretion. Nuciferine also stimulated insulin secretion in INS-1E cells at both 3.3 and 16.7 mM glucose concentrations. Compared with glibenclamide, nuciferine had a stronger effect on insulin secretion and less beta-cell toxicity. However, nuciferine did not compete with glibenclamide for binding to the sulfonylurea receptor. CONCLUSIONS: Among several compounds extracted from anti-diabetic plants, nuciferine was found to stimulate insulin secretion by closing potassium-adenosine triphosphate channels, explaining anti-diabetic effects of Nelumbo nucifera.

High-dose fasudil preserves postconditioning against myocardial infarction under hyperglycemia in rats: role of mitochondrial KATP channels.

BACKGROUND: The current study was carried out to determine whether fasudil hydrochloride (fasudil), a Rho-kinase inhibitor, has myocardial postconditioning (PostC) activity under hyperglycemia as well as normoglycemia, and if so, whether the effects could be mediated by mitochondrial ATP-sensitive potassium (m-KATP) channels. METHODS: Male Sprague-Dawley rats were anesthetized with sodium pentobarbital. After opening the chest, all rats underwent 30-min coronary artery occlusion followed by 2-h reperfusion. The rats received low-dose (0.15 mg/kg) or high-dose (0.5 mg/kg) fasudil or diazoxide, an m-KATP channel opener, at 10 mg/kg, just before reperfusion under normoglycemic or hyperglycemic conditions. In another group, rats received 5-hydroxydecanoic acid (5HD), an m-KATP channel blocker, at 10 mg/kg, before high-dose fasudil. Myocardial infarct size was expressed as a percentage of area at risk (AAR). RESULTS: Under normoglycemia, low-dose and high-dose fasudil and diazoxide reduced myocardial infarct size (23 ± 8%, 21 ± 9% and 21 ± 10% of AAR, respectively) compared with that in the control (42 ± 7%). Under hyperglycemia, low-dose fasudil (40 ± 11%) and diazoxide (44 ± 14%) could not exert this beneficial effect, but high-dose fasudil reduced myocardial infarct size in the same manner as under normoglycemia (21 ± 13%). 5HD prevented fasudil-induced reduction of myocardial infarct size (42 ± 13%). CONCLUSION: Fasudil induces PostC against myocardial infarction via activation of m-KATP channels in the rat. Although hyperglycemia attenuates the PostC, high-dose fasudil can restore cardioprotection.

Activation of K(ATP) channels suppresses glucose production in humans.

Increased endogenous glucose production (EGP) is a hallmark of type 2 diabetes mellitus. While there is evidence for central regulation of EGP by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels in rodents, whether these central pathways contribute to regulation of EGP in humans remains to be determined. Here we present evidence for central nervous system regulation of EGP in humans that is consistent with complementary rodent studies. Oral administration of the K(ATP) channel activator diazoxide under fixed hormonal conditions substantially decreased EGP in nondiabetic humans and Sprague Dawley rats. In rats, comparable doses of oral diazoxide attained appreciable concentrations in the cerebrospinal fluid, and the effects of oral diazoxide were abolished by i.c.v. administration of the K(ATP) channel blocker glibenclamide. These results suggest that activation of hypothalamic K(ATP) channels may be an important regulator of EGP in humans and that this pathway could be a target for treatment of hyperglycemia in type 2 diabetes mellitus.

Bioenergetic defect associated with mKATP channel opening in a mouse model carrying a mitofusin 2 mutation.

Charcot-Marie-Tooth disease type 2A (CMT2A) is an autosomal dominant axonal form of peripheral neuropathy caused by mutations in the mitofusin 2 gene (MFN2), which encodes a mitochondrial outer membrane protein that promotes mitochondrial fusion. Emerging evidence also points to a role of MFN2 in the regulation of mitochondrial metabolism. To examine whether mitochondrial dysfunction is a feature of CMT2A, we used a transgenic mouse model expressing in neurons a mutated R94Q form of human MFN2 shown to induce a CMT2A phenotype. Oxygraphic and enzymatic measurements both revealed a combined defect of mitochondrial complexes II and V (40 and 30% decrease, respectively) in the brain of Tg-R94 mice, leading to a drastic decrease of ATP synthesis. These deficiencies were reversed by the mitochondrial ATP-sensitive potassium channel (mK(ATP)) inhibitor 5-hydroxydecanoate. Conversely, in controls and wild-type human MFN2 mice, the mK(ATP) activator diazoxide mimicked the deficiency observed with the R94Q mutation. The physical links between complexes II and V, previously proposed as part of mK(ATP), were reinforced in Tg-R94Q mice. Our results show that the R94Q MFN2 mutation induces a combined defect of complexes II and V linked to the opening of mK(ATP), which could participate in the pathophysiology of the disease.

Diazoxide reduces status epilepticus neuron damage in diabetes.

Diabetic hyperglycemia is associated with seizure severity and may aggravate brain damage after status epilepticus. Our earlier studies suggest the involvement of ATP-sensitive potassium channels (K(ATP)) in glucose-related neuroexcitability. We aimed to determine whether K(ATP) agonist protects against status epilepticus-induced brain damage. Adult male Sprague-Dawley rats were divided into two groups: the streptozotocin (STZ)-induced diabetes (STZ) group and the normal saline (NS) group. Both groups were treated with either diazoxide (15 mg/kg, i.v.) (STZ + DZX, NS + DZX) or vehicle (STZ + V, NS + V) before lithium-pilocarpine-induced status epilepticus. We evaluated seizure susceptibility, severity, and mortality. The rats underwent Morris water-maze tests and hippocampal histopathology analyses 24 h post-status epilepticus. A multi-electrode recording system was used to study field excitatory postsynaptic synaptic potentials (fEPSP). RNA interference (RNAi) to knockdown Kir 6.2 in a hippocampal cell line was used to evaluate the effect of diazoxide in the presence of high concentration of ATP. Seizures were less severe (P < 0.01), post-status epilepticus learning and memory were better (P < 0.05), and neuron loss in the hippocampal CA3 area was lower (P < 0.05) in the STZ + DZX than the STZ + V group. In contrast, seizure severity, post-status epilepticus learning and memory, and hippocampal CA3 neuron loss were comparable in the NS + DZX and NS + V groups. fEPSP was lower in the STZ + DZX but not in the NS + DZX group. The RNAi study confirmed that diazoxide, with its K(ATP)-opening effects, could counteract the K(ATP)-closing effect by high dose ATP. We conclude that, by opening K(ATP), diazoxide protects against status epilepticus-induced neuron damage during diabetic hyperglycemia.

Chloro-substituted 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides as ATP-sensitive potassium channel activators: impact of the position of the chlorine atom on the aromatic ring on activity and tissue selectivity.

The synthesis of 5-chloro-, 6-chloro-, and 8-chloro-substituted 3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides is described. Their inhibitory effect on the insulin releasing process and their vasorelaxant activity was compared to that of previously reported 7-chloro-3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides. "5-Chloro" compounds were found to be essentially inactive on both the insulin-secreting and the smooth muscle cells. By contrast, "8-chloro" and "6-chloro" compounds were found to be active on insulin-secreting cells, with the "6-chloro" derivatives emerging as the most potent drugs. Moreover, the "6-chloro" analogues exhibited less myorelaxant activity than their "7-chloro" counterparts. 8-Chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (25b) and 6-chloro-3-cyclobutylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (19e) were further identified as K(ATP) channel openers by radioisotopic measurements conducted on insulin-secreting cells. Likewise, current recordings on HEK293 cells expressing human SUR1/Kir6.2 channels confirmed the highly potent activity of 19e (EC(50) = 80 nM) on such types of K(ATP) channels. The present work indicates that 6-chloro-3-alkylamino/cycloalkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides appear to be more attractive than their previously described 7-chloro-substituted analogues as original drugs activating the SUR1/Kir6.2 K(ATP) channels.

The effect of "Teucrium polium L." extracts on insulin release from in situ isolated perfused rat pancreas in a newly modified isolation method: the role of Ca2+ and K+ channels.

BACKGROUND: "Teucrium polium L." (TP) has been long recommended in Iranian folk medicine for its anti-diabetic activities. We attempt here to evaluate the effect of TP extract on insulin secretion in rat pancreas. METHODS: Rat pancreas was isolated in situ and perfused with Krebs solution containing low glucose (LG, 2.8 mM) or high glucose (HG, 16.7 mM) as perfusate. The aqueous extract (Aq. E) and methanolic extract (Met. E) of TP aerial parts and two partition fractions of Met. E were added to perfusate to evaluate insulin release. Diazoxide (DZX) and verapamil (VPM) were also used for assessing the probable mechanism of the effects. In each experimental group, the peak and baseline of insulin levels in effluent samples were compared. The GC/MS analysis was carried out to detect active ingredients in the extracts. RESULTS: Adding Met. E to the LG caused a significant increase (P<0.05) in insulin release from the basal level of 0.17 ± 0.05 µg/l to a peak value of 3.94 ± 1.29 µg/l. when Met. E was introduced to the HG, there was a further protracted stimulation of insulin release from 2.15 ± 1.35 µg/l to 6.16 ± 0.52 µg/l. Both DZX and VPM when added separately to the LG, led to inhibition of Met. E induced insulin secretion. The Aq. E and fractions had no significant effect on insulin secretion. Only in the Met. E, the component 5-hydroxy-4',7-dimethoxyflavone (apigenin-4',7-dimethylether) was detected. CONCLUSION: It can be concluded that the insulinotropic properties of TP extracts can be attributed to the presence of apigenin existing only in Met. E, but not in Aq. E and fractions. Moreover, certain types of K+ and Ca2+ channels take part in this effect.

Terminalia bellirica stimulates the secretion and action of insulin and inhibits starch digestion and protein glycation in vitro.

Traditional plant treatments have been used throughout the world for the therapy of diabetes mellitus. The aim of the present study was to investigate the efficacy and mode of action of Terminalia bellirica used traditionally for the treatment of diabetes in India. T. bellirica aqueous extract stimulated basal insulin output and potentiated glucose-stimulated insulin secretion concentration-dependently in the clonal pancreatic beta-cell line, BRIN-BD11 (P < 0.001). The insulin-secretory activity of the plant extract was abolished in the absence of extracellular Ca2+ and by inhibitors of cellular Ca2+ uptake, diazoxide and verapamil (P < 0.001; n 8). Furthermore, the extract did not increase insulin secretion in depolarised cells and did not further augment insulin secretion triggered by tolbutamide or glibenclamide. T. bellirica extract also displayed insulin-mimetic activity and enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes by 300 %. At higher concentrations, the extract also produced a 10-50 % (P < 0.001) decrease in starch digestion in vitro and inhibited protein glycation (P < 0.001). The present study has revealed that components in T. bellirica extract stimulate insulin secretion, enhance insulin action and inhibit both protein glycation and starch digestion. The former actions are dependent on the active principle(s) in the plant being absorbed intact. Future work assessing the use of T. bellirica as a dietary adjunct or as a source of active anti-diabetic agents may provide new opportunities for the treatment of diabetes.

The role of ATP-sensitive potassium channel blockers in ischemia-reperfusion-induced renal injury versus their effects on cardiac ischemia reperfusion in rats.

In renal ischemia reperfusion (V/R), opening of adenosine-triphosphate (ATP)-sensitive potassium (K(ATP)) channels results in massive influx of neutrophils in both renal and lung tissues. Our study was focused on the role of ATP-dependent potassium channel modulators, glimepiride and glibenclamide on I/R induced renal injury in rats. Additionally we evaluated their effects on normal heart and on ischemic reperfused heart subjected to ischemic preconditioning protection afforded by diazoxide. To test this hypothesis, we used renal I/R and cardiac I/R experiment. Renal ischemia reperfusion induced marked renal dysfunction associated with significant increase in arterial pressure, TNF-alpha levels, superoxide anion production, and myeloperoxidase activity. Treatment with glibenclamide or glimepiride, demonstrated a significant improvement in the reperfusion-induced injury in both kidney and lung. Glimepiride has no effect on superoxide anion production. However glibenclamide induced a significant improvement in these measurements as compared to glimepiride group. Before coronary artery ligation, neither diazoxide nor glimepiride pretreatment influenced significantly the electrocardiographic parameters in comparison with control group. Conversely, glibenclamide supplementation induced a significant elevation in these parameters. After left coronary artery ligation, reperfusion of the ischemic hearts caused a significant elevation in the measured electrocardiographic parameters. These elevations were significantly ameliorated by the pretreatment with diazoxide. In conclusion, the administration of glibenclamide significantly abolished the protective effects of diazoxide, while the pretreatment with glimepiride didn't abolish it. So, glimepiride offers some promise for therapy of renal I/R with minimizing the undesirable cardiac side effects.

Sesquiterpenoids from antidiabetic Psacalium decompositum block ATP sensitive potassium channels.

ETHNOPHARMACOLOGICAL RELEVANCE: The hypoglycemic effect of root and rhizome aqueous decoction of Psacalium decompositum (Asteraceae), a medicinal herb from Mexico, has been experimentally demonstrated, leading to the identification of several hypoglycemic sesquiterpenoids, such as cacalol, and the mixture of 3-hydroxycacalolide, and epi-3-hydroxycacalolide; however, the mechanism of action of these compounds is unknown. AIM OF THE STUDY: To establish whether cacalol, cacalone epimer mixture and cacalol acetate may block adenosine triphosphate-sensitive potassium channels (K(ATP) channels) in a similar way to the antidiabetic drug glibenclamide. MATERIALS AND METHODS: Cacalol, cacalone epimer mixture, and cacalol acetate were tested on the diazoxide-induced relaxation of male rat aortic rings precontracted with phenylephrine (3.2x10(-6)M). RESULTS: Cacalol (10(-5)M), cacalol acetate and the cacalone epimer mixture (10(-4)M) inhibited the diazoxide effect, in a similar manner and concentration as glibenclamide (10(-5)M). Cacalone epimer mixture exerted this effect in a concentration-dependent manner (P<0.01). Cacalol (10(-4)M), irreversibly inhibited the diazoxide-induced relaxation, and displayed activity at a lower concentration (10(-5)M) than cacalone epimer mixture and cacalol acetate. CONCLUSIONS: These results suggest that the studied compounds block K(ATP) channels in a similar way to glibenclamide in rat aorta. However, controversial data indicate that Psacalium decompositum sesquiterpenoids are less effective than glibenclamide in lowering plasma glucose levels, suggesting that cacalol and cacalone epimer mixture, as well as cacalol acetate, may display a higher affinity to SUR2 subunit of K(ATP) channels in aortic smooth muscle than to SUR1 subunit in pancreatic beta-cells.