Methanol (80%) leaf extract of Otostegia integrifolia Benth (Lamiaceae) lowers blood pressure in rats through interference with calcium conductance.

BACKGROUND: Otostegia integrifolia Benth. (Lamiaceae) leaves are used to treat hypertension in Ethiopian folk medicine. However, the claim has so far not been investigated scientifically. Thus, the objective of this study was to evaluate the antihypertensive activity of 80% methanol leaf extract of O. integrifolia in animal model of hypertension and possible underlying mechanisms in isolated rat aorta. METHODS: Antihypertensive effect of various oral doses of the extract (250, 500 and 1000 mg/kg) was determined in fructose-induced hypertensive rats using the non-invasive tail-cuff method. Thoracic aortic strips of rats were isolated and suspended in organ bath, and the vasodepressor effect as well as the possible mechanism (s) of action were studied by means of isometric tension recording experiments ex vivo. Phytochemical analysis was also performed to suggest possible constituents related to the activity. RESULTS: Blood pressure was significantly lowered in a dose-dependent manner following extract administration, suggesting that the extract possesses antihypertensive activity. The extract also caused a dose-dependent relaxation of aortic strip precontracted with KCl at a concentration of 6.25-125 µg/L, with a maximum relaxation (100%) achieved at a cumulative concentration of 318.75 µg/ml. The relaxation mechanism was found to be independent of muscarinic receptors, prostanoids, histamine receptors, ATP dependent K+ channels, sarcoplasmic reticulum stored Ca2+ and the endothelium system. The extract shifted the Ca2+ concentration-response curve to the right similar to that caused by nifedipine, suggesting that vasorelaxation could possibly be mediated via calcium channel blockade. The extract was found to contain phenolic compounds (164.3 mg/g, expressed as gallic acid equivalents) and flavonoids (125.7 mg/g, expressed as quercetin equivalents). CONCLUSION: The findings revealed that the plant is endowed with antihypertensive activity, providing evidence for its traditional use. The effect maybe, at least in part, due to dilation of blood vessels through blockade of Ca+ 2 channels mediated by phenolic and flavonoid constituents.

ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia-Reperfusion Injury and Inflammatory Reaction.

Inflammatory response during myocardial ischemia reperfusion injury (MIRI) is essential for cardiac healing, while excessive inflammation extends the infarction and promotes adverse cardiac remodeling. Understanding the mechanism of these uncontrolled inflammatory processes has a significant impact during the MIRI therapy. Here, we found a critical role of ATP-sensitive potassium channels (KATP) in the inflammatory response of MIRI and its potential mechanism and explored the effects of Panax Notoginseng Saponins (PNS) during this possess. Rats underwent 40 min ischemia by occlusion of the left anterior descending (LAD) coronary artery and 60 min of reperfusion. PNS was treated at the corresponding time point before operation; 5-hydroxydecanoate (5-HD) and glybenclamide (Gly) (or Nicorandil (Nic)) were used as pharmacological blocker (or nonselective opener) of KATP. Cardiac function and pathomorphology were evaluated and a set of molecular signaling experiments was tested. KATP current density was measured by patch-clamp. Results revealed that in MIRI, PNS pretreatment restored cardiac function, reduced infarct size, and ameliorated inflammation through KATP. However, inhibiting KATP by 5-HD and Gly significantly reversed the effects, including NLRP3 inflammasome and inflammatory mediators IL-6, MPO, TNF-α, and MCP-1. Moreover, PNS inhibited the phosphorylation and nuclear translocation of NF-κB in I/R myocardium when the KATP was activated. Importantly, PNS promoted the expression of subunits and activation of KATP. The study uncovered KATP served as a new potential mechanism during PNS modulating MIRI-induced inflammation and promoting injured heart recovery. The manipulation of KATP could be a potential therapeutic approach for MIRI and other inflammatory diseases.

Functional characterization of ABCC9 variants identified in sudden unexpected natural death.

BACKGROUND: Genetic variation in ion channel genes ('channelopathies') are often associated with inherited arrhythmias and sudden death. Genetic testing ('molecular autopsies') of channelopathy genes can be used to assist in determining the likely causes of sudden unexpected death. However, different in silico approaches can yield conflicting pathogenicity predictions and assessing their impact on ion channel function can assist in this regard. METHODS AND RESULTS: We performed genetic testing of cases of sudden expected death in the New York City metropolitan area and found four rare or novel variants in ABCC9, which codes for the regulatory SUR2 subunit of KATP channels. All were missense variants, causing amino acid changes in the protein. Three of the variants (A355S, M941V, and K1379Q) were in cases of infants less than six-months old and one (H1305Y) was in an adult. The predicted pathogenicities of the variants were conflicting. We have introduced these variants into a human SUR2A cDNA, which we coexpressed with the Kir6.2 pore-forming subunit in HEK-293 cells and subjected to patch clamp and biochemical assays. Each of the four variants led to gain-of-function phenotypes. The A355S and M941V variants increased in the overall patch current. The sensitivity of the KATP channels to inhibitory 'cytosolic' ATP was repressed for the M941V, H1305Y and K1379Q variants. None of the variants had any effect on the unitary KATP channel current or the surface expression of KATP channels, as determined with biotinylation assays, suggesting that all of the variants led to an enhanced open state. CONCLUSIONS: All four variants caused a gain-of-function phenotype. Given the expression of SUR2-containing KATP channels in the heart and specialized cardiac conduction, vascular smooth muscle and respiratory neurons, it is conceivable that electrical silencing of these cells may contribute to the vulnerability element, which is a component of the triple risk model of sudden explained death in infants. The gain-of-function phenotype of these ABCC9 variants should be considered when assessing their potential pathogenicity.

KATP Channel Expression and Genetic Polymorphisms Associated with Progression and Survival in Amyotrophic Lateral Sclerosis.

The ATP-sensitive potassium (KATP) channel directly regulates the microglia-mediated inflammatory response following CNS injury. To determine the putative role of the KATP channel in amyotrophic lateral sclerosis (ALS) pathology, we investigated whether ALS induces changes in KATP channel expression in the spinal cord and motor cortex. We also characterized new functional variants of human ABCC8, ABCC9, KCNJ8, and KCNJ11 genes encoding for the KATP channel and analyzed their association with ALS risk, rate of progression, and survival in a Spanish ALS cohort. The expression of ABCC8 and KCNJ8 genes was enhanced in the spinal cord of ALS samples, and KCNJ11 increased in motor cortex of ALS samples, as determined by real-time polymerase chain reaction. We then sequenced the exons and regulatory regions of KATP channel genes from a subset of 28 ALS patients and identified 50 new genetic variants. For the case-control association analysis, we genotyped five selected polymorphisms with predicted functional relevance in 185 Spanish ALS (134 spinal ALS and 51 bulbar ALS) patients and 493 controls. We found that bulbar ALS patients presenting the G/G genotype of the rs4148646 variant of ABCC8 and the T/T genotype of the rs5219 variant of KCNJ11 survived longer than other ALS patients presenting other genotypes. Also, the C/C genotype of the rs4148642 variant of ABCC8 and the T/C genotype of the rs148416760 variant of ABCC9 modified the progression rate in spinal ALS patients. Our results suggest that the KATP channel plays a role in the pathophysiological mechanisms of ALS.

ATP sensitive K+ channel subunits (Kir6.1, Kir6.2) are the candidate mediators regulating ameliorating effects of pulsed magnetic field on aortic contractility in diabetic rats.

Diabetes mellitus is a metabolic disease that causes increased morbidity and mortality in developed and developing countries. With recent advancements in technology, alternative treatment methods have begun to be investigated in the world. This study aims to evaluate the effect of pulsed magnetic field (PMF) on vascular complications and contractile activities of aortic rings along with Kir6.1 and Kir6.2 subunit expressions of ATP-sensitive potassium channels (KATP ) in aortas of controlled-diabetic and non-controlled diabetic rats. Controlled-diabetic and non-controlled diabetic adult male Wistar rats were exposed to PMF for a period of 6 weeks according to the PMF application protocol (1 h/day; intensity: 1.5 mT; consecutive frequency: 1, 10, 20, and 40 Hz). After PMF exposure, body weight and blood glucose levels were measured. Then, thoracic aorta tissue was extracted for relaxation-contraction and Kir6.1 and Kir6.2 expression experiments. Blood plasma glucose levels, body weight, and aortic ring contraction percentage decreased in controlled-diabetic rats but increased in non-controlled diabetic rats. PMF therapy repressed Kir6.1 mRNA expression in non-controlled diabetic rats but not in controlled diabetic rats. Conversely, Kir6.2 mRNA expressions were repressed both in controlled diabetic and non-controlled diabetic rats by PMF. Our findings suggest that the positive therapeutic effects of PMF may act through (KATP ) subunits and may frequently occur in insulin-free conditions. Bioelectromagnetics. 39:299-311, 2018. © 2018 Wiley Periodicals, Inc.

Overexpression of WNK1 in POMC-expressing neurons reduces weigh gain via WNK4-mediated degradation of Kir6.2.

"With no lysine" (WNK) kinases have been shown to regulate various ion transporters in various tissues, but studies on the function of WNK kinases in the brain have been limited. In this study, we discovered that WNK1 and WNK4 in POMC-expressing neuronal cells in WNK1 overexpressed transgenic mice (WNK1 TG) decrease appetite via degradation of Kir6.2. Weight gain after 20 weeks of age was delayed in WNK1 TG mice as a result of reduced food intake. Expression of WNK1 and proopiomelanocortin (POMC) was higher in POMC-expressing neurons in the hypothalamus of WNK1 TG mice than in WT mice. Immunostaining of serial sections of the hypothalamus revealed that POMC-expressing neurons were smaller in WNK1 TG mice than in WT mice. In addition, expression of Kir6.2 was significantly reduced in WNK1 TG mice. Overexpression and knockdown of WNK4 demonstrated that WNK4 regulates protein expression of Kir6.2 via protein-protein interaction. Accordingly, reduced age-dependent weight gain of WNK1 TG mice seems to be related with the decreased Kir6.2 expression via WNK1- and WNK4-regulated protein stability of Kir6.2.

Gastrodin causes vasodilation by activating KATP channels in vascular smooth muscles via PKA-dependent signaling pathway.

Gastrodin, one of the major components extracted from the Chinese herb Gastrodia elata Bl., has been widely used as an anticonvulsant, sedative, analgesic and hypotensive. In our study, we aimed to investigate the effects and possible mechanisms of gastrodin on vascular KATP channels. Tension experiments were used on rat mesenteric artery rings without an endothelium. Patch clamp experiments were executed to investigate the influences of gastrodin on the membrane current in mesenteric artery smooth muscle cells. Gastrodin induced vasorelaxation in a concentration dependent manner when rat mesenteric artery rings were pre-contracted with Phenylephrine. The vasorelaxation effect was partially diminished by pre-treating with a KATP channel inhibitor, or a PKA inhibitor. With whole-cell patch-clamp recording techniques, we found that gastrodin is a activator of KATP in rat mesenteric artery smooth muscle cells, and this effect was eliminate by pre-treating with H89or PKI, PKA inhibitor. In addition, when rat vascular smooth muscle cells were treated with 100 µM gastrodin for 24 h, maximum KATP current density increased by 28.1%. The results indicate that gastrodin exerts vasorelaxation effect through activation of PKA and subsequent opening of smooth muscle KATP channels.

Treating young adults with type 2 diabetes or monogenic diabetes.

It is increasingly recognised that diabetes in young adults has a wide differential diagnosis. There are many monogenic causes, including monogenic beta-cell dysfunction, mitochondrial diabetes and severe insulin resistance. Type 2 diabetes in the young is becoming more prevalent, particularly after adolescence. It's important to understand the clinical features and diagnostic tools available to classify the different forms of young adult diabetes. Classic type 1 diabetes is characterised by positive ß-cell antibodies and absence of endogenous insulin secretion. Young type 2 diabetes is accompanied by metabolic syndrome with obesity, hypertension and dyslipidaemia. Monogenic ß-cell dysfunction is characterised by non-autoimmune, C-peptide positive diabetes with a strong family history, while mitochondrial diabetes features deafness and other neurological involvement. Severe insulin resistance involves a young-onset metabolic syndrome often with a disproportionately low BMI. A suspected diagnosis of monogenic diabetes is confirmed with genetic testing, which is widely available in specialist centres across the world. Treatment of young adult diabetes is similarly diverse. Mutations in the transcription factors HNF1A and HNF4A and in the ß-cell potassium ATP channel components cause diabetes which responds to low dose and high dose sulfonylurea agents, respectively, while glucokinase mutations require no treatment. Monogenic insulin resistance and young-onset type 2 diabetes are both challenging to treat, but first line management involves insulin sensitisers and aggressive management of cardiovascular risk. Outcomes are poor in young-onset type 2 diabetes compared to both older onset type 2 and type 1 diabetes diagnosed at a similar age. The evidence base for treatments in monogenic and young-onset type 2 diabetes relies on studies of moderate quality at best and largely on extrapolation from work conducted in older type 2 diabetes subjects. Better quality, larger studies, particularly of newer agents would improve treatment prospects for young adults with diabetes.

Effect of electronic stimulation at Neiguan (PC 6) acupoint on gene expression of adenosine triphosphate-sensitive potassium channel and protein kinases in rats with myocardial ischemia.

OBJECTIVE: To investigate the effects of electronic stimulation at acupoints Neiguan (PC 6) and Lieque (LU 7) on the gene expression of the adenosine triphosphate (ATP)-Sensitive potassium channel (KATP: Kir6.1, Kir6.2, SUR2A, and SUR2B) and protein kinases (PKA, PKG, and PKCß2) in myocardial cells of rats with myocardial ischemia (MI) induced by isoproterenol (ISO). METHODS: Rats were randomly divided into a control, model, Neiguan (PC 6), Lieque (LU 7), and non-acupoint groups. The MI model was established by injecting rats with ISO. Electro-acupuncture treatment was given to the acupuncture groups, once a day for 7 days. Gene expression was analyzed with real-time PCR. RESULTS: The gene expression of KATP and protein kinases in the model group was higher than those in the control group (P < 0.05). After acupuncture treatment, the KATP and protein kinase expression levels were significantly lower in the Neiguan (PC 6) and Lieque (LU 7) groups compared with the mod- el group (P < 0.05). The Neiguan (PC 6) group lowered these levels significantly more than that of the Lieque (LU 7) group (P < 0.05). No significant differences were observed between the model and non-acupoint groups (P> 0.05). CONCLUSION: Our findings suggest that electronic needling of Neiguan (PC 6) can both reduce the gene expression of KATP and protein kinases in rats with ISO-induced MI.

Total flavonoids from Ganshanbian (Herba Hyperici Attenuati) effect the expression of CaL-alpha1C and K(ATP)-Kir6.1 mRNA of the myocardial cell membrane in myocardial ischemia-reperfusion arrhythmia rats.

OBJECTIVE: To observe the impact of total flavonoids from Ganshanbian (Herba Hyperici Attenuati) on the expression of vascular smooth muscle membrane L-type calcium channel alpha1 C subunit (CaL-alpha1C) and ATP-sensitive K+ channel (K(ATP))-Kir6.1 mRNA, and explore the mechanisms of the antiarrhythmic effect of Ganshanbian (Herba Hyperici Attenuati) total flavonoids. METHODS: The treatment group was fed total flavonoids from Ganshanbian (Herba Hyperici Attenuati) for 7 days by gavage with 100 mg x kg(-1) x d(-1). The blank control group and model control group were given the same amount of normal saline for 7 d. Arrhythmias were induced by performing a myocardial ischemia-reperfusion and electrocardiogram was observed. Reverse transcription-polymerase chain reaction was used to detect the expression of CaL-alpha 1C and K(ATP)-Kir6.1 mRNA in the myocardial cell membrane of all groups of rats. RESULTS: Total flavonoids from Ganshanbian (Herba Hyperici Attenuati) can delay the appearance of myocardial ischemia reperfusion arrhythmias, shorten the duration of myocardial ischemia reperfusion arrhythmias, reduce heart rate, reduce cell membrane expression of CaL-a1C mRNA and enhance the expression of K(ATP)-Kir6.1 mRNA in myocardial ischemia-reperfusion arrhythmic rats. CONCLUSION: Total flavonoids from Ganshanbian (Herba Hyperici Attenuati) can alleviate arrhythmias by affecting the expression of L-type calcium channels and ATP-sensitive K+ channels.

Label-free cell phenotypic profiling decodes the composition and signaling of an endogenous ATP-sensitive potassium channel.

Current technologies for studying ion channels are fundamentally limited because of their inability to functionally link ion channel activity to cellular pathways. Herein, we report the use of label-free cell phenotypic profiling to decode the composition and signaling of an endogenous ATP-sensitive potassium ion channel (KATP) in HepG2C3A, a hepatocellular carcinoma cell line. Label-free cell phenotypic agonist profiling showed that pinacidil triggered characteristically similar dynamic mass redistribution (DMR) signals in A431, A549, HT29 and HepG2C3A, but not in HepG2 cells. Reverse transcriptase PCR, RNAi knockdown, and KATP blocker profiling showed that the pinacidil DMR is due to the activation of SUR2/Kir6.2 KATP channels in HepG2C3A cells. Kinase inhibition and RNAi knockdown showed that the pinacidil activated KATP channels trigger signaling through Rho kinase and Janus kinase-3, and cause actin remodeling. The results are the first demonstration of a label-free methodology to characterize the composition and signaling of an endogenous ATP-sensitive potassium ion channel.

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.

Nicorandil ameliorates hypertension-related bladder dysfunction in the rat.

AIMS: There is increasing evidence that ischemia is one of the main etiology in overactive bladder (OAB), and that nicorandil prevents OAB. We investigated the effect of nicorandil on hypertension-related bladder dysfunction in spontaneously hypertensive rats (SHRs). METHODS: Twelve-week-old SHRs received six-weeks treatment with nicorandil (0, 3, or 10 mg/kg, i.p. every day). Wistar rats were used for normotensive controls. Six weeks after nicorandil treatment, the bladder blood flow was estimated by hydrogen clearance method, and the bladder functions were estimated by voiding behavior studies and functional studies. Tissue levels of nerve growth factor (NGF) were measured by ELISA method. Furthermore, the participation levels of K(ATP) channel pores were investigated by real-time PCR. RESULTS: SHRs showed significant increases in blood pressure, micturition frequency, tissue levels of NGF and expressions of both K(IR) 6.1 and K(IR) 6.2 mRNAs, and a significant decrease in the bladder blood flow. The carbachol-induced contractile responses were similar in all groups. Although both doses of nicorandil failed to decrease the blood pressure, nicorandil significantly decreased the micturition frequency, tissue levels of NGF and increased the bladder blood flow in a dose dependent manner. The expressions of K(IR) 6.1 and K(IR) 6.2 mRNAs were slightly up-regulated by the low dose of nicorandil, whereas the high dose of nicorandil significantly up-regulated those expressions compared to non-treated SHRs. CONCLUSIONS: These data indicate that nicorandil prevents hypertension-related bladder dysfunction in the SHR, which may be related to its effect on the increased blood flow in the bladder.

Measuring and evaluating the role of ATP-sensitive K+ channels in cardiac muscle.

Since ion channels move electrical charge during their activity, they have traditionally been studied using electrophysiological approaches. This was sometimes combined with mathematical models, for example with the description of the ionic mechanisms underlying the initiation and propagation of action potentials in the squid giant axon by Hodgkin and Huxley. The methods for studying ion channels also have strong roots in protein chemistry (limited proteolysis, the use of antibodies, etc.). The advent of the molecular cloning and the identification of genes coding for specific ion channel subunits in the late 1980s introduced a multitude of new techniques with which to study ion channels and the field has been rapidly expanding ever since (e.g. antibody development against specific peptide sequences, mutagenesis, the use of gene targeting in animal models, determination of their protein structures) and new methods are still in development. This review focuses on techniques commonly employed to examine ion channel function in an electrophysiological laboratory. The focus is on the K(ATP) channel, but many of the techniques described are also used to study other ion channels.

An open sarcolemmal adenosine triphosphate-sensitive potassium channel is necessary for detrimental myocyte swelling secondary to stress.

BACKGROUND: Stress (exposure to hyperkalemic cardioplegia, metabolic inhibition, or osmotic) results in significant myocyte swelling and reduced contractility. In contrast to wild-type mice, these detrimental consequences are not observed in mice lacking the Kir6.2 subunit of the sarcolemmal ATP-sensitive potassium (sK(ATP)) channel after exposure to hyperkalemic cardioplegia. The hypothesis for this study was that an open sK(ATP) channel (Kir6.2 and SUR2A subunits) is necessary for detrimental myocyte swelling to occur in response to stress. METHODS AND RESULTS: To investigate the role of the sK(ATP) channel in stress-induced myocyte swelling, high-dose pharmacological sK(ATP) channel blockade and genetic deletion (knockout of Kir6.2 subunit) were used. Myocytes were exposed sequentially to Tyrode control (20 minutes), test (stress) solution (20 minutes), and Tyrode control (20 minutes). To evaluate pharmacological channel blockade, myocytes were exposed to hyperkalemic cardioplegia (stress) with and without a K(ATP) channel blocker. To evaluate the effects of genetic deletion, wild-type and sK(ATP) knockout [Kir6.2(-/-)] myocytes were exposed to metabolic inhibition (stress). Myocyte volume was recorded using image-grabbing software. Detrimental myocyte swelling was prevented by high-dose sK(ATP) channel blockade (glibenclamide or HMR 1098) but not mitochondrial K(ATP) channel blockade (5-hydroxydecanoate) during exposure to hyperkalemic cardioplegia. Genetic deletion of the sK(ATP) channel prevented significant myocyte swelling in response to metabolic inhibition. CONCLUSIONS: K(ATP) channel openers prevent detrimental myocyte swelling and reduce contractility in response to stress through an unknown mechanism. Paradoxically, the present data support a role for sK(ATP) channel activation in myocyte volume derangement in response to stress.

Acute sulfonylurea therapy at disease onset can cause permanent remission of KATP-induced diabetes.

OBJECTIVE: Neonatal diabetes mellitus (NDM) can be caused by gain-of-function ATP-sensitive K(+) (K(ATP)) channel mutations. This realization has led to sulfonylurea therapy replacing insulin injections in many patients. In a murine model of K(ATP)-dependent NDM, hyperglycemia and consequent loss of ß-cells are both avoided by chronic sulfonylurea treatment. Interestingly, K(ATP) mutations may underlie remitting-relapsing, transient, or permanent forms of the disease in different patients, but the reason for the different outcomes is unknown. RESEARCH DESIGN AND METHODS: To gain further insight into disease progression and outcome, we examined the effects of very early intervention by injecting NDM mice with high-dose glibenclamide for only 6 days, at the beginning of disease onset, then after the subsequent progression with measurements of blood glucose, islet function, and insulin sensitivity. RESULTS: Although ∼70% of mice developed severe diabetes after treatment cessation, ∼30% were essentially cured, maintaining near-normal blood glucose until killed. Another group of NDM mice was initiated on oral glibenclamide (in the drinking water), and the dose was titrated daily, to maintain blood glucose <200 mg/dL. In this case, ∼30% were also essentially cured; they were weaned from the drug after ∼4 weeks and again subsequently maintained near-normal blood glucose. These cured mice maintain normal insulin content and were more sensitive to insulin than control mice, a compensatory mechanism that together with basal insulin secretion may be sufficient to maintain near-normal glucose levels. CONCLUSIONS: At least in a subset of animals, early sulfonylurea treatment leads to permanent remission of NDM. These cured animals exhibit insulin-hypersensitivity. Although untreated NDM mice rapidly lose insulin content and progress to permanently extremely elevated blood glucose levels, early tight control of blood glucose may permit this insulin-hypersensitivity, in combination with maintained basal insulin secretion, to provide long-term remission.

Astragaloside IV regulates expression of ATP-sensitive potassium channel subunits after ischemia-reperfusion in rat ventricular cardiomyocytes.

OBJECTIVE: Astragaloside IV (AsIV) is the major effective component extracted from the Chinese herb Astragalus membranaceus, which has been widely used to treat cardiovascular disease. Recent studies have shown that AsIV can potentially protect the heart from myocardial ischemic injury, but the mechanisms of action are unknown. ATP-sensitive potassium (KATP) channels are activated during ischemia and exert a compensatory protective effect on cardiomyocytes. We therefore examined the effects of AsIV on KATP channel currents and channel expression in isolated rat ventricular cardiomyocytes after ischemia-reperfusion injury. METHODS: Forty Wistar rats were divided into five groups: control group, ischemia-reperfusion (IP) group, IP + glibenclamide group, IP + pinacidil group and IP + AsIV group. The ischemia-reperfusion injury model was established in enzymatically isolated ventricular cardiomyocytes by perfusion with calcium-free Tyrode solution for 10 min, arrest for 30 min, and reperfusion for 45 min. The different drugs were applied for 10-15 min, and the KATP channel current (I(KATP)) was recorded with voltage-clamp mode by whole-cell patch-clamp technique. Protein and mRNA expression of the KATP channel subunits Kir6.1, Kir6.2, SUR2A and SUR2B was quantified using western blotting and real-time PCR. RESULTS: The KATP current in IP group was significantly greater than that in control group (211.45 +/- 33.67 vs 83.51 +/- 23.67 pA; P < 0.01). Glibenclamide (10 micromol/L) blocked KATP currents, whereas both AsIV (1 mg/L) and the known channel opener pinacidil (50 micromol/L) significantly increased I(KATP) (P < 0.05). Consistent with this, AsIV significantly up-regulated protein and mRNA expression of Kir6.1, Kir6.2, SUR2A, SUR2B (P < 0.01 vs IP group). CONCLUSION: The protective effects of AsIV in ischemia-reperfusion injury may be related to the up-regulation of several KATP channel subunits and facilitation of KATP currents.

Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.

Blood glucose levels are tightly controlled, a process thought to be orchestrated primarily by peripheral mechanisms (insulin secretion by ß cells, and insulin action on muscle, fat, and liver). The brain also plays an important, albeit less well-defined role. Subsets of neurons in the brain are excited by glucose; in many cases this involves ATP-mediated closure of K(ATP) channels. To understand the relevance of this, we are manipulating glucose sensing within glucose-excited neurons. In the present study, we demonstrate that glucose excitation of MCH-expressing neurons in the lateral hypothalamus is mediated by K(ATP) channels and is negatively regulated by UCP2 (a mitochondrial protein that reduces ATP production), and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis. Combined, the glucose-excited neurons are likely to play key, previously unexpected roles in regulating blood glucose.

Altered dietary carbohydrates significantly affect gene expression of the major glucosensing components in Brockmann bodies and hypothalamus of rainbow trout.

Carnivorous fish have a limited capacity to utilize dietary carbohydrates even though glucosensing components exist in the hypothalamus and Brockmann bodies. Therefore, we fed trout for 10 days with two experimental diets containing a high level of carbohydrates (20%) or a carbohydrate-free level (<0.3%) to test the capacity of dietary carbohydrates to regulate gene expression of glucosensing components. Fish were fed and killed 1, 6, and 24 h after the meal to analyze plasma glucose levels, glucosensing-related biochemical parameters, and gene expression of the major components of the glucosensing system in the hypothalamus and Brockmann bodies. Glucose facilitative transporter type 2 and glucokinase gene expression were confirmed by real-time PCR data and two new components of the glucosensing mechanism, Kir6.-like and sulfonylurea receptor-like, were detected for the first time in fish in both tissues. In addition, a clear adaptation to dietary carbohydrates was found in trout Brockmann bodies, based on increased gene expression of major components of the system as well as enhanced glucokinase activities and glycogen levels. In contrast, in the hypothalamus, only glucokinase gene expression and activity showed a response to dietary carbohydrates, supporting the key role of that enzyme in glucosensing mechanism. Finally, a differential postprandial profile was found between tissues regarding the glucosensing potential, since the hypothalamus seems to respond to hyperglycemia earlier than the Brockmann bodies, whose response took place later. Altogether, these data describe for the first time in fish a complete response of major glucosensing components to dietary carbohydrates in trout hypothalamus and Brockmann bodies, supporting an efficient adaptation of both tissues to those dietary components.