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BPDZ 154 Activates Adenosine 5'-Triphosphate-Sensitive Potassium Channels: In Vitro Studies Using Rodent Insulin-Secreting Cells and Islets Isolated from Patients with Hyperinsulinism

Institute of Molecular Physiology and Department of Biomedical Science (K.E.C., A.T.L., P.D.B., M.J.D.), University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom; Laboratory of Pharmacology (M.-H.A., P.L.), Faculty of Medicine, Université Libre de Bruxelles, Bruxelles 1070, Belgium; Department of Medicinal Chemistry (P.d.T., B.P.), Université de Liège, Liège 4000, Belgium; Manchester Children’s Hospital (P.C.), Manchester M27 4MA, United Kingdom; Department of Surgery (R.M.), Department of Medicine, Manchester Royal Infirmary, Manchester M13 9WL, United Kingdom; Department of Pediatrics, Hôpital Necker-Enfants Malades (P.D.L., C.N.-F., J.-J.R., J.-M.S.), 75743 Paris, France; Université Catholique de Louvain (J.R.), Bruxelles, Belgium; and Institute of Child Health (K.J.L., K.H., A.A.-G.), Great Ormond Street Hospital, London WC1N 1EH, United Kingdom

Address all correspondence and requests for reprints to: Prof. M. J. Dunne, Institute of Molecular Physiology and Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield S10 2TN, United Kingdom. E-mail: m.j.dunne{at}sheffield.ac.uk.

Abstract

A novel ATP-sensitive potassium channel (K_ATP) channel agonist, BPDZ 154 (6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), was synthesized, and its effects on insulin-secreting cells were evaluated using electrophysiology, ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ efflux, and RIA determinations of insulin secretion. BPDZ 154, an analog of diazoxide, inhibited both glucose-induced insulin secretion from isolated perifused islets and the secretion of insulin induced by glucose and tolbutamide. These effects were mediated by the activation of ATP-sensitive potassium channels because BPDZ 154 induced a concentration-dependent increase in channel activity that was inhibited by the sulfonylurea tolbutamide and the imidazoline efaroxan. In ß-cells isolated from patients with either nontypical hyperinsulinism (preserved K_ATP channel function) or from the control areas of the pancreas of patients with focal hyperinsulinism, BPDZ 154 activated K_ATP channels and was found to be more effective and less readily reversible than diazoxide. By contrast, it was not possible to activate K_ATP channels by either diazoxide or BPDZ 154 in ß-cells from patients with hyperinsulinism as a consequence of defects in K_ATP channel function. In ß-cells isolated from a patient with pancreatic insulinoma, K_ATP channels were readily recorded and modulated by BPDZ 154. These data suggest that BPDZ 154 or BPDZ 154-like compounds may have therapeutic potential in the treatment of certain forms of hyperinsulinism.

HYPERINSULINISM IN INFANCY (HI) is the most common cause of persistent hypoglycemia in infancy and childhood. The most severe forms of this condition arise from gene defects in pancreatic ß-cell ATP-sensitive K⁺ (K_ATP) channel subunits SUR1 (sulfonylurea receptor 1 encoded by ABCC8) and inwardly rectifying K⁺ channel Kir6.2 (encoded by KCNJ11) at Ch.11p15 (1). Two major histopathologically diverse forms of HI predominate (2): diffuse disease (Di-HI), in which all ß-cells of the pancreas express the gene defect, and focal disease or Fo-HI (3, 4). In this condition loss of maternal heterozygosity leads to expression of K_ATP channel gene defects in cells derived from the focal regions (5, 6, 7). ABCC8 and KCNJ11 mutations have currently been identified in approximately 50% of patients with HI, and defects that influence the function of SUR1 are found throughout the ABCC8 gene (1, 8). Other identified causes of hyperinsulinism include mutations in glucokinase [GCK1, Ch.7p15–13 (9)], glutamate dehydrogenase [GLUD1, Ch.10q23.3 (10)], and short-chain L-3-hydroxyacyl-CoA dehydrogenase [Ch4q22-q26 (11)].

In ß-cells K_ATP channels are composed of four Kir6.2 subunits forming the ion channel pore and four SUR1 subunits that regulate the conductance of Kir6.2. Under normal conditions, K_ATP channels regulate the ß-cell resting membrane potential and couple glucose metabolism to insulin secretion through changes in the cell membrane potential. Defects in ABCC8 or KCNJ11 cause HI because they are responsible for loss of channel function, which leads to spontaneous depolarization of the membrane, unregulated Ca²⁺ influx, and uncontrolled insulin release (12, 13). If untreated, hyperinsulinemia-induced hypoglycemia is potentially lethal because of neurological complications associated with low blood glucose levels (14).

Diazoxide is used in the treatment of hyperinsulinism because it acts to inhibit insulin release through activation of K_ATP channels (14). However, the clinical effectiveness of diazoxide is variable (15) and as a consequence of adverse side effects, which include fluid retention leading to cardiac failure, hyperuricemia, thrombocytopenia, hypotension, facial changes, and generalized hypertrichosis, the compound can be poorly tolerated in vivo (14, 16). As a result, patients that do not respond to diazoxide treatment require surgery in the form of a pancreatectomy to alleviate hypoglycemia.

To generate more active and selective inhibitors of insulin release, we have recently developed a series of diazoxide-like molecules, which include 3-alkylamino-4H-1,2,4-benzothiadiazine 1,1-dioxides (i.e. BPDZ 73) (17, 18) and 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides (i.e. BPDZ 44) (19, 20). In this study, we examined the actions of a new synthetic diazoxide analog, 6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide (BPDZ 154), and evaluated the effects of this compound using rodent insulin-secreting cells and ß-cell lines and tissues isolated from patients following surgery for hyperinsulinism as a consequence of HI and pancreatic insulinoma (INS). We show that BPDZ 154 is an activator of K_ATP channels in control and disease tissues and that this is the principal mechanism by which BPDZ 154 leads to the inhibition of insulin release in vitro.

Patients and Methods

Patients

Studies were performed using tissues isolated from seven patients with HI and one patient with hyperinsulinism as a consequence of INS. Patients N80, N89, N92, and N93 were diagnosed as having HI as a consequence of diffuse disease, whereas patients N88, N90, and N91 suffered from focal hyperinsulinism (Table 1). All HI patients presented with typical symptoms of the disease, which were defined by hypoglycemia (blood glucose, <2.6 mmol/liter; normal range, 3.5–5.5 mmol/liter) in association with hyperinsulinism (insulin >480 ng/ml in association with a raised C-peptide at the time of hypoglycemia; normal insulin value, <160 ng/ml). These and other established criteria confirmed the diagnosis of HI (11). All patients failed to respond adequately to medical therapy involving diazoxide (7.5–20 mg/kg·d) (Table 1), and a subtotal or near total (95%) pancreatectomy was performed to prevent persistent hypoglycemia. Patient INS3 presented symptoms of hypoglycemia later in life and after failure to respond to diazoxide underwent a limited pancreatectomy to remove a benign pancreatic INS. Patient N89 was found to carry a gene mutation in ABCC8 (Table 1), but the genetic basis of hyperinsulinism in all other patients has not been determined.

Measurements of insulin release, ⁸⁶Rb⁺ efflux, and ⁴⁵Ca²⁺ outflow from perifused pancreatic islets. All experiments were performed with pancreatic islets isolated by the collagenase method from fed Wistar rats. The methods used to measure ⁸⁶Rb⁺ (⁴²K⁺ substitute) outflow, ⁴⁵Ca²⁺ outflow, and insulin release from perifused islets have been described previously (21, 22, 23). In brief, batches of 100 islets were incubated in a bicarbonate-buffered medium (115 mmol/liter NaCl, 5 mmol/liter KCl, 2.56 mmol/liter CaCl₂, 1 mmol/liter MgCl₂, and 24 mmol/liter NaHCO₃) containing 16.7 mmol/liter glucose and either ⁸⁶Rb⁺ (0.15–0.25 mmol/liter; 50 µCi/ml) or ⁴⁵Ca²⁺ (0.02–0.04 mmol/liter; 100 µCi/ml) for 60 min. After incubation, islets were submitted to four washes with a nonradioactive medium to remove extracellular radioactivity. Groups of 100 islets were then placed on a cellulose filter (SCWP01300, Millipore Corp., Saint Quentin, France) that was inserted in a perifusion chamber (Swinnex SX00 01300, Millipore Corp.) connected to two reservoirs through a three-way valve. The reservoirs contained media kept at 38 C and continuously mixed with O₂ (95%) and CO₂ (5%). The perifusate was delivered at a constant rate (1.0 ml/min) by means of a peristaltic pump (Minipulse 2, Gilson, Villiers le Bel, France). The temperature in the perifusion chamber averaged 37 C, and the inside pressure corresponded to the atmospheric pressure with no significant variation during the experiment. After a stabilization period of 30 min, from the 31st to the 90th minute of the perifusion, the effluent was continuously collected over successive periods of 1 min each. An aliquot of this effluent (0.5 ml) was then used for scintillation counting, and the remainder was stored at -20 C for determination of insulin release by RIA, as described previously (24). At the end of the perifusion, the radioactive content of islets was measured. To determine the effect of the experimental conditions on ⁸⁶Rb⁺ or ⁴⁵Ca²⁺ outflow, the fractional outflow rate for ⁸⁶Rb⁺ or ⁴⁵Ca²⁺ was determined as that percentage of the instantaneous islet content released per minute (21, 22, 23).

Electrophysiological measurements. Pancreatic islets and ß-cells were isolated from human cadaver organ donors and postoperatively from patients with HI, as described previously (12, 25). For studies related to Fo-HI, lesions of focal adenomatous hyperplasia of ß-cells were first identified by pancreatic venous sampling (4, 5) and then surgically isolated from the remainder of the pancreas. All preparations of islets and cells were maintained under standard tissue culture conditions in RPMI 1640 medium (Sigma, Poole, UK) supplemented with 10% (vol/vol) newborn calf serum, 100 IU/ml penicillin G, and 100 µg/ml streptomycin. The glucose-responsive rat-derived ß-cell line BRIN-BD11 was maintained under standard tissue culture conditions, as described (26).

K_ATP channel currents were recorded using the cell-attached and inside-out recording configurations of the patch-clamp technique (27). Data were obtained using a List L/M EPC-7 patch clamp amplifier (List Electronic, Darmstadt, Germany) and recorded for subsequent replay and analysis using a digital tape recorder (Biologic, Echirolles, France). All experiments were performed at room temperature. Glass micropipettes with a resistance of 5–10 M were filled with a Na⁺-rich solution composed of 140 mmol/liter NaCl, 4.7 mmol/liter KCl, 1.13 mmol/liter MgCl₂, 2.5 mmol/liter CaCl₂, 2.5 mmol/liter glucose, and 10 mmol/liter HEPES (pH 7.4). The cells were bathed and perifused in a K⁺-rich solution of composition 140 mmol/liter KCl, 10 mmol/liter NaCl, 1.13 mmol/liter MgCl₂, 1 mmol/liter EGTA, 2.5 mmol/liter glucose, and 10 mmol/liter HEPES (pH 7.2). K_ATP channel open-state probability (P_o) was estimated off-line by analysis of a minimum of 30-sec stretches of continuous data using a CED 1401 interface and designated software (Cambridge Electronic Design, Cambridge, UK) as described previously (28). Alternatively, where determination of Po was not possible because of large numbers of single K_ATP channel currents, data were expressed as the fractional peak K_ATP channel current value relative to the control value (12, 25). All current traces were displayed according to the convention with outward current events represented as upward deflections from the baseline.

Drugs. BPDZ 154 was synthesized at the Department of Medicinal Chemistry (Université de Liège, Liège, Belgium). The compound (6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide) results from the reaction of 6,7-dichloro-3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide, as described (29) with an excess of isopropylamine (Fig. 1). Typically, 6,7-dichloro-3-(1H-imidazol-1-yl)-4H-1,2,4-benzothiadiazine 1,1-dioxide (0.5 g, 1.6 mmol) and isopropylamine (10 ml) were heated together at 150 C in a sealed vessel for 4–5 h. The excess of amine was removed by distillation under reduced pressure, and the residue was dissolved in an aqueous 5% wt/vol solution of NaOH (20 ml) supplemented with methanol (10 ml). The alkaline solution was treated with charcoal and filtered, and the filtrate was adjusted to pH 6 with formic acid. The precipitate was collected by filtration, washed with distilled water, and dried. The compound recrystallized in methanol-water was isolated as a monohydrate; yield: 0.31 g (59%); melting point greater than 280 C [literature: 289–291 C, (30)]; infrared (KBr): 3529 (O-H), 3316 (N-H), 3089 (C-H aromatic), 2977 (C-H aliphatic), 1631, 1577, 1457 (C = C, N-H), 1289, 1160 (S = O) cm^-1; nuclear magnetic resonance [dimethylsulfoxide (DMSO)-d₆, hexamethyldisiloxane, ppm]: 1.10 [d, 6H, CH(CH₃)₂], 3.20 (s, H₂O), 3.80 [m, 1H, CH(CH₃)₂], 7.30 (bd, 1H, N-H exocyclic), 7.45 (s, 1H, 5-H), 7.80 (s, 1H, 8-H), 10.35 (bs, 1H, N-H intracyclic). BPDZ 154, diazoxide, glibenclamide, and tolbutamide (Sigma) were dissolved in DMSO and were added to test solutions to give a final concentration of DMSO less than 0.1%. There were no actions of DMSO on K_ATP channels or islet function at this concentration (22, 28).

View larger version (9K): [in this window] [in a new window]   Figure 1. Comparison of the structures of diazoxide and BPDZ 154.

All results are expressed as the mean ± SE of the mean. Comparisons were made using t tests or ANOVA, followed by Tukey’s tests, where data were normally distributed. Mann-Whitney rank sum tests were used for pair-wise comparisons where data were non-normally distributed.

Results

Modulation of insulin release, ⁸⁶Rb⁺ efflux, and ⁴⁵Ca²⁺ efflux by BPDZ 154

Figure 2 shows the effects of BPDZ 154 on glucose and sulfonylurea-induced insulin release from perifused rat pancreatic islets. Insulin secretion induced by 16.7 mmol/liter glucose was inhibited by approximately 85% by 10 µmol/liter BPDZ 154; insulin release averaged 90 ± 12 pg/islet·min before (40th to 44th minute) and 14.4 ± 1.6 pg/islet·min after (60th to 68th minute) the addition of BPDZ 154, with little recovery of secretion for 20 min following removal of the drug (n = 4, Fig. 2A). In addition, we also found that insulin secretion induced by a combination of 8.3 mmol/liter glucose and 100 µmol/liter tolbutamide was inhibited by approximately 80% by 10 µmol/liter BPDZ 154 (six experiments, Fig. 2B); insulin release averaged 68.4 ± 5.2 pg/islet·min before (40th to 44th minute) and 15.2 ± 2.8 pg/islet·min after (60th to 68th minute) the addition of BPDZ 154. Measurements of insulin output from islets incubated for 90 min revealed a concentration-dependent inhibitory effect of BPDZ 154 on 16.7 mmol/liter glucose-induced insulin release. After the addition of 10 nM, 100 nM, 1 µM, 10 µM, and 50 µM BPDZ 154 to the incubation medium, the residual insulin release represented 95.40 ± 5.09% (n = 22), 84.90 ± 4.50% (n = 21),13.19 ± 0.98% (n = 26), 6.29 ± 0.67% (n = 12), and 4.96 ± 0.41% (n = 12) of the control value (100%, no added drug), respectively. This indicates an IC₅₀ value of 0.28 µM. Because we have previously reported under identical experimental conditions that the corresponding value for diazoxide was approximately 20 µmol/liter [i.e. 24.8 ± 3.4 µmol/liter (17) and 18.4 ± 2.2 µmol/liter (23)], these data indicate that BPDZ 154 is a far more potent antagonist of ß-cell function than diazoxide. Our findings also reveal that BPDZ 154 was more potent than the parent molecule BPDZ 73 (17).

View larger version (22K): [in this window] [in a new window]   Figure 2. Inhibition of insulin secretion by BPDZ 154 in perifused rat pancreatic islets. Effect of 10 µmol/liter BPDZ 154 on insulin release from rat pancreatic islets perifused in the presence of 16.7 mmol/liter glucose (A) or 8.3 mmol/liter glucose and 100 µmol/liter tolbutamide (B). Under basal conditions insulin output was between 4 and 8 pg/islet per minute. All media contained 2.56 mmol/liter CaCl2. Mean values (±SE) refer to four to six individual experiments.

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of BPDZ 154 on 86Rb+ outflow from rat pancreatic islets perifused in the presence of 5.6 mmol/liter glucose and absence (•) or presence () of 10 µmol/liter glibenclamide. Basal media contained 2.56 mmol/liter CaCl2. Mean values (±SE) refer to three to four individual experiments.

View larger version (23K): [in this window] [in a new window]   Figure 4. Effect of 10 µmol/liter BPDZ 154 on 45Ca2+ outflow from rat pancreatic islets perifused in the presence of 16.7 mmol/liter glucose (A) or 8.3 mmol/liter glucose and 100 µmol/liter tolbutamide (B). Basal media either contained 2.56 mmol/liter CaCl2 (•) or CaCl2 was omitted and 0.5 mmol/liter EGTA was added (). The rate of 45Ca outflow before drug administration (min 31–44) was lower in islets exposed to calcium depleted media. Mean values (±SE) refer to four to six individual experiments.

Cell-attached and inside-out patch recordings from the rat pancreatic ß-cell line BRIN-BD11 were used to study the actions of BPDZ 154 on K_ATP channels. In six intact cells and excised patches in the presence of internally applied ATP (500 µmol/liter), BPDZ 154 consistently activated ATPsensitive K⁺ channels. Figure 5A shows typical data from seven separate inside-out patch recordings when K_ATP channels were exposed to a range of concentrations of BPDZ 154. In these experiments, both ATP and BPDZ 154 were applied to the inside face of the membrane. BPDZ 154-induced activation was concentration dependent with increasing levels of K_ATP channel activity being recorded as the concentration of BPDZ 154 was raised from 0.1 µmol/liter to 10 µmol/liter (n = 7). The approximate EC₅₀ for activation of K_ATP channels in the presence of 500 µmol/liter ATP was estimated to occur between 0.1 and 1 µmol/liter (Fig. 5B). Activation of K_ATP channels by 1 µmol/liter BPDZ 154 in the presence of 500 µmol/liter ATP was inhibited by the imidazoline efaroxan (100 µmol/liter, n = 6, P < 0.001), and the sulfonylureas tolbutamide (100 µmol/liter, n = 6, P < 0.01) and glibenclamide (1 µmol/liter, n = 8, P < 0.001, Fig. 6). Similar effects were also found in human ß-cells (patient N90) with 25 µmol/liter BPDZ 154 (n = 13).

View larger version (33K): [in this window] [in a new window]   Figure 5. Concentration-dependent activation of KATP channels in BRIN-BD11 ß-cells by BPDZ 154. A, Typical inside-out patch recording. BPDZ 154 activated KATP channels in the presence of 500 µmol/liter ATP in a concentration-dependent manner. B, Summary of data from seven similar recordings. Results are expressed as a percentage of the peak KATP channel current measured under basal conditions at the start of the sequence. In this and other figures, control values refer to the level of KATP channel activity recorded in the absence of test substances. Half maximal activation of KATP channels under these conditions was achieved between 0.1 µmol/liter and 1 µmol/liter BPDZ 154.

View larger version (34K): [in this window] [in a new window]   Figure 6. Inhibition of BPDZ 154-activated KATP channels by efaroxan, tolbutamide, and glibenclamide. A, Illustration of an inside-out patch recording from a BRIN-BD11 ß-cell, typical of five others. On each occasion the activation of KATP channels elicited by 1 µmol/liter BPDZ 154 in the presence of 500 µmol/liter ATP could be reversed by the addition of either 100 µmol/liter efaroxan or 100 µmol/liter tolbutamide. B(I), Summary of data from six experiments. Peak KATP channel current values under each condition were evaluated and are expressed as a percentage of the initial control current measured at the start of the sequence. Both efaroxan and tolbutamide significantly attenuated 1 µmol/liter BPDZ 154-induced channel activation (n = 6, Student’s paired t tests, P < 0.001, P < 0.01, respectively). B(II), Summary of the actions of 1 µmol/liter glibenclamide on 1 µmol/liter BPDZ 154-induced channel activation (n = 8).

The effects of BPDZ 154 and diazoxide on K_ATP channels in ß-cells isolated from Fo-HI patients was investigated in 22 experiments using three donor preparations in which samples of tissue were available from both the focal lesion and the adjacent portions of the pancreas. In experiments on all control cells, K_ATP channel function was apparently normal (n = 12 of 12), and 10 µmol/liter diazoxide activated 500 µmol/liter ATP-inhibited channels in inside-out recordings (n = 11 of 12). BPDZ 154 (10 µmol/liter) was also effective but evoked a more pronounced activation of channels when applied to the same patches of membrane (n = 12 of 12, Fig. 7, top). On average we found that diazoxide caused a 1.56 ± 0.3-fold increase in channel activity in the presence of ATP (n = 9), whereas the equivalent value for BPDZ 154 was 2.96 ± 0.3 (n = 9, P < 0.001). In contrast, K_ATP channel currents were not recorded from cells isolated from the focal lesions, and there were no responses to either BPDZ 154 or diazoxide (n = 10, Fig. 7, bottom).

View larger version (27K): [in this window] [in a new window]   Figure 7. Activation of KATP channels in ß-cells isolated from the control area of the pancreas of patients with Fo-HI. The upper trace is representative of 12 recordings from control ß-cells of three patients (N88, N90, and N91). Analysis of nine experiments showed that BPDZ 154 produced a more significant increase in channel activity than diazoxide (Diaz) in the same cells. By contrast, in cells isolated from the focal lesions of the same patients, KATP channels were nonfunctional and neither BPDZ 154 nor diazoxide caused the activation of channels. The record illustrated in the bottom panel is typical of 10 recordings from focal lesions.

Although principally associated with ABCC8 or KCNJ11 gene mutations, not all patients with Di-HI have identified gene defects in K_ATP channel subunits. In this series of experiments, ß-cells isolated from two of the four patients with Di-HI had fully operational K_ATP channels in more than 87% of the experiments carried out (patients N80 and N92, 49 of 56 experiments). Our studies were, therefore, undertaken on tissues isolated from both groups of patients. Patients N89 (31) and N93 had typical Di-HI in which loss of K_ATP channel function was directly correlated with ß-cell pathophysiology (Table 1). Figure 8A shows a representative recording of 23 experiments of ionic events in ß-cells isolated from this group of patients; K_ATP channel activity was abnormal (compare Fig. 8A with 8B), and there were no actions of 500 µmol/liter ATP, 10 µmol/liter diazoxide, or 10 µmol/liter BPDZ 154 (n = 7–13). In contrast to these data, when recordings were made from the ß-cells of patients with nontypical Di-HI (patients N80 and N92, Fig. 8B), K_ATP channels were found in the majority of cells, and they were readily inhibited by ATP and activated by either diazoxide or BPDZ 154 (n = 12). On average, BPDZ 154 increased K_ATP channel activity in the presence of ATP by 4.3 ± 0.4-fold, compared with 2.9 ± 0.5-fold for diazoxide (n = 7, P < 0.05). In addition to increasing the degree of K_ATP channel activation, BPDZ 154 was also found to be less readily reversible than diazoxide. The time taken for the peak current to decline to 50% of its maximum value following removal of the drug was 10 ± 4 sec for diazoxide vs. 58 ± 12 sec for BPDZ 154 (six separate recordings, P < 0.01). This was comparable with values obtained using rodent-derived ß-cells under the same conditions (5 ± 3 sec vs. 24 ± 1 sec for diazoxide and BPDZ 154, respectively; n = 4, P < 0.01).

View larger version (21K): [in this window] [in a new window]   Figure 8. Effects of BPDZ 154 on KATP channels in patients with diffuse HI. A, Illustration of typical data from patients with Di-HI as a consequence of KATP channel defects. In this ß-cell, which is representative of 22 other experiments, KATP channel openings were rare, and open events were typically brief in duration, indicated by the arrows, and there were no effects of diazoxide or BPDZ 154 in 7 and 13 experiments, respectively. B, An inside-out patch from a patient with nontypical Di-HI, in which KATP channels were readily recorded. Activation of 500 µmol/liter ATP-inhibited channels by BPDZ 154 was more pronounced than that observed for diazoxide. In this experiment, BPDZ 154 increased Po from 0.001 to 0.47, compared with diazoxide, which increased Po from 0.017 to 0.16.

The molecular pathophysiology of pancreatic INS is incompletely resolved. Here, we found that although all recordings of patient tissues possessed K_ATP channels, there were some differences when compared with controls. First, there was a significant decrease in the magnitude of K_ATP channel currents generated on formation of inside-out patches (12.7 ± 3.8 pA, n = 11 vs. 25.5 ± 1.5 pA, n = 269, in INS3 and human control ß-cells, respectively, P < 0.05). Second, we also found that there was a decrease in the sensitivity of K_ATP channels to ATP in INS ß-cells. In control tissue, 500 µmol/liter ATP typically reduced the P_o to 5 ± 1% of the initial value (100%) in 19 experiments from three cadaver donors, compared with only 74 ± 7% in INS3 ß-cells (n = 6, P < 0.001). Despite this, in INS3 ß-cells we consistently found that both diazoxide and BPDZ 154 readily activated K_ATP channels and that when compared with 10 µmol/liter diazoxide, 10 µmol/liter BPDZ 154 was found to be significantly more effective at increasing K_ATP channel activity in the presence of ATP (4.2 ± 0.5-fold vs. 2.0 ± 0.2-fold for diazoxide, n = 4, P < 0.05, Fig. 9. In INS ß-cells we also found that the actions of BPDZ 154 were less readily reversible than those of diazoxide (39 ± 7 sec vs. 4 ± 1 sec, respectively, n = 4, P < 0.05).

View larger version (17K): [in this window] [in a new window]   Figure 9. Activation of KATP channels by BPDZ 154 in ß-cells from patients with pancreatic insulinoma. Summary of data from four separate inside-out patches in INS3 ß-cells. Compared with 10 µmol/liter diazoxide, 10 µmol/liter BPDZ 154 produced a more significant increase in the activation of KATP channels.

Disorders of glucose homeostasis resulting in hyperinsulinism and hypoglycemia are potentially devastating conditions that present a challenge for clinical management because of the lack of effective and selective inhibitors of insulin secretion. For cases of both HI and INS, activation of K_ATP channels by diazoxide often fails to control the insulin secretion in vivo, and surgical removal of either the discrete lesion or up to 95% of the pancreas is then required to prevent hypoglycemia (14). Here, we investigated the effects of a novel diazoxide analog, BPDZ 154, on rodent tissue and tissues isolated from patients with hyperinsulinism.

BPDZ 154 was found to be a potent inhibitor of insulin release with an EC₅₀ of 0.28 µmol/liter, compared with approximately 20 µmol/liter for diazoxide (17, 23), and caused a sulfonylurea-sensitive increase in ⁸⁶Rb⁺ efflux from preloaded islets. These data suggested that BPDZ 154 was a K_ATP channel agonist, and this was confirmed using patch-clamp techniques that revealed BPDZ 154 (0.1–10 µmol/liter)-induced activation of K_ATP channels and inhibition of these effects by efaroxan and sulfonylureas (32). Consistent with the actions of glibenclamide on BPDZ 154-enhanced ⁸⁶Rb⁺ efflux, we also found that closure of K_ATP channels by glibenclamide in the presence of BPDZ 154 was poorly reversible (Fig. 6). When compared with diazoxide, equivalent concentrations of BPDZ 154 were found to be more effective, and although there were no obvious differences in the time for onset of activation of K_ATP channels by diazoxide or BPDZ 154, the novel agent was consistently found to be less readily reversible than diazoxide. A similar pharmacokinetic profile of slow reversibility was also seen when insulin release, ⁸⁶Rb⁺ efflux, and ⁴⁵Ca²⁺ outflow rates were monitored using perifused rodent islets.

In vitro studies of human ß-cells were also performed. First, in cells isolated from three patients with Fo-HI, we showed that in those regions unaffected by loss of heterozygosity, K_ATP channel activity was normal, and both diazoxide and BPDZ 154 were agonists of the channel. However, when parallel studies were carried out on ß-cells isolated from the focal lesions from the same patients, K_ATP channels were defective, and there were no actions of either agent. Next, the effects of BPDZ 154 were examined on ion channels in ß-cells isolated from two groups of patients with Di-HI. Although a small minority of patients with Di-HI retain functional ß-cell K_ATP channels, approximately 95% of all cases arise through loss of K_ATP channel function (33). In these cases the degree of K_ATP channel deficiency ranges from a total loss of function (13) to abnormal channel operation as a result of defects in nucleotide-sensing (34, 35) or subunit trafficking (36). In this patient group, which was typified by studies involving patients N93 and N89 (31), when K_ATP channels were absent from ß-cells, BPDZ 154 and diazoxide failed to induce channel activity. By contrast, in Di-HI patients with nontypical disease (patients N80 and N92), K_ATP channels were retained and despite the fact that diazoxide was ineffective in vivo, both diazoxide and BPDZ 154 activated K_ATP channels in vitro. Similar findings were also observed in ß-cells from a patient with pancreatic INS. Collectively, these findings suggest that when there is a severe loss of K_ATP channel function, neither diazoxide nor BPDZ 154 are likely to modulate insulin release in vivo. However, in those cases of HI or INS in which K_ATP channel function is preserved, our data show that despite the fact that there were no beneficial effects of diazoxide in vivo, BPDZ 154 was able to activate K_ATP channels in vitro. These findings are potentially relevant to the clinical management of such patients because they imply that increasing the concentrations of diazoxide in vivo may prove beneficial.

Apart from hyperinsulinism, diazoxide-induced inhibition of insulin release has also been implicated in the treatment of both obesity and diabetes. In the Zucker rat (fa/fa), an animal model of hyperinsulinemic obesity (37), the administration of diazoxide was found to prevent glucose-induced insulin release, reduce weight gain, improve glucose uptake, and modify central neuropeptide Y levels of both free-fed and fasted obese animals (38, 39, 40). Diazoxide was also found to prevent type 1 diabetes in diabetes-prone Bio-Breeding rats (41), and short-term administration was shown to improve insulin secretion in diabetic subjects (42), preserve endogenous insulin production in newly diagnosed patients (43), and reduce body fat in hyperinsulinemic obese adults (44). These observations have, therefore, led to the suggestion that by inducing ß-cell rest (45), diazoxide used in combination with intensive insulin therapy may prolong ß-cell survival and preserve endogenous insulin release for several months following diagnosis of diabetes.

In summary, we have presented data related to the actions of a novel antagonist of pancreatic ß-cell function. BPDZ 154 is more effective than diazoxide at both inhibiting insulin release from isolated intact islets and inducing the activation of K_ATP channels. We have also demonstrated that BPDZ 154 will modulate K_ATP channels in ß-cells isolated from a human pancreatic INS and in those cases of HI in which the condition appears not to be directly related to defects in K_ATP channels. Our data suggest that benzothiadiazine 1,1-dioxides, or other diazoxide analogs (46) may prove useful as therapeutic utilities or in the development of new compounds for disorders of glucose homeostasis.

Acknowledgments

We thank Drs. Roger James, Seran Hill, and Sue Swift of the University of Leicester and Dr. Kate Hinchliffe of the University of Sheffield for preparation of control and HI ß-cells. Some of this work involved patient tissues originating from Great Ormond Street Hospital for Children NHS Trust, which received a portion of its funding from the NHS Executive. The views expressed in this publication are those of the authors and are not necessarily those of the NHS Executive.

Footnotes

This work was supported by the Juvenile Diabetes Research Fund (to K.E.C. and M.J.D.), University of Sheffield (to P.D.B. and M.J.D.), Wellcome Trust (to A.T.L. and M.J.D.), and National Fund for Scientific Research, Belgium (to P.DT., B.P., and P.L.). Collaborative initiatives were supported by a European Union-funded Concerted Action Grant (Treatment of Hyperinsulinism in Infancy, THIQLG1-2000-00513).

Abbreviations: Di-HI, Diffuse hyperinsulinism in infancy; DMSO, dimethylsulfoxide; Fo-HI, focal hyperinsulinism in infancy; HI, hyperinsulinism in infancy; INS, insulinoma; K_ATP, ATP-sensitive K⁺; Kir, inwardly rectifying K⁺ channel; P_o, open-state probability; SUR, sulfonylurea receptor.

Received March 20, 2002.

Accepted August 14, 2002.

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