Glucokinase activators: molecular tools for studying the physiology of insulin-secreting cells.

GK (glucokinase) catalyses the phosphorylation of glucose to glucose 6-phosphate in glucosensitive cells. In pancreatic beta-cells, this reaction is the rate-limiting step of insulin release. Recent work has led to the discovery of synthetic small-molecule activators of GK that stimulate beta-cell physiology and subsequently enhance the glucose-dependent release of insulin. It is currently recognized that these compounds may represent a significant advance in the development of new agents in the treatment of diabetes. In addition, GKAs (GK activators) are emerging as reagents that are useful tools with which to probe the function of pancreatic beta-cells and other glucosensitive cells. This includes providing insights into the physiology of the beta-cell by helping to elucidate the kinetic cycle of GK, confirming the central role of glucose metabolism to the beta-cell and highlighting subtle species-dependent differences in insulin secretion between rodent and human islets of Langerhans.

Hyperinsulinemic hypoglycemia in Beckwith-Wiedemann syndrome due to defects in the function of pancreatic beta-cell adenosine triphosphate-sensitive potassium channels.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome that is clinically and genetically heterogeneous. Hyperinsulinemic hypoglycemia occurs in about 50% of children with BWS and, in the majority of infants, it resolves spontaneously. However, in a small group of patients the hypoglycemia can be persistent and may require pancreatectomy. The mechanism of persistent hyperinsulinemic hypoglycemia in this group of patients is unclear. PATIENTS AND METHODS: Using patch-clamp techniques on pancreatic tissue obtained at the time of surgery, we investigated the electrophysiological properties of ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells in a patient with BWS and severe medically-unresponsive hyperinsulinemic hypoglycemia. RESULTS: Persistent hyperinsulinism was found to be caused by abnormalities in K(ATP) channels of the pancreatic beta-cell. Immunofluorescence studies using a SUR1 antibody revealed perinuclear pattern of staining in the BWS cells, suggesting a trafficking defect of the SUR1 protein. No mutations were found in the genes ABCC8 and KCNJ11 encoding for the two subunits, SUR1 and KIR6.2, respectively, of the K(ATP) channel. Genetic analysis of this patients BWS showed evidence of mosaic paternal isodisomy. CONCLUSIONS: In this novel case of BWS with mosaic paternal uniparental disomy for 11p15, persistent hyperinsulinism was due to abnormalities in K(ATP) channels of the pancreatic beta-cell. The mechanism/s by which mosaic paternal uniparental disomy for 11p15 causes a trafficking defect in the SUR1 protein of the K(ATP) channel remains to be elucidated.

Uncontrolled insulin secretion from a childhood pancreatic beta-cell adenoma is not due to the functional loss of ATP-sensitive potassium channels.

We report the case of an 8-year-old child who presented with severe hyperinsulinaemic hypoglycaemia due to a pancreatic islet cell adenoma. In vivo, there was no beneficial response to the hyperglycaemia-inducing agent diazoxide and as a consequence the child underwent a subtotal pancreatectomy. In vitro studies of adenomatous beta-cells revealed no operational defects in ATP-sensitive potassium channel activity and appropriate responses to diazoxide. In comparison with patients with focal adenomatous hyperplasia, genetic analysis of the isolated adenoma showed no loss of heterozygosity for chromosome 11p15 and expression of the cyclin-dependent kinase inhibitor p57(kip2). This case illustrates that the excess insulin secretion from an infantile adenoma has an aetiology different from that observed in hyperinsulinism in infancy.

Hyperinsulinism of infancy: the regulated release of insulin by KATP channel-independent pathways.

Hyperinsulinism of infancy (HI) is a congenital defect in the regulated release of insulin from pancreatic beta-cells. Here we describe stimulus-secretion coupling mechanisms in beta-cells and intact islets of Langerhans isolated from three patients with a novel SUR1 gene defect. 2154+3 A to G SUR1 (GenBank accession number L78207) is the first report of familial HI among nonconsanguineous Caucasians identified in the U.K. Using patch-clamp methodologies, we have shown that this mutation is associated with both a decrease in the number of operational ATP-sensitive K+ channels (KATP channels) in beta-cells and impaired ADP-dependent regulation. There were no apparent defects in the regulation of Ca2+- and voltage-gated K+ channels or delayed rectifier K+ channels. Intact HI beta-cells were spontaneously electrically active and generating Ca2+ action currents that were largely insensitive to diazoxide and somatostatin. As a consequence, when intact HI islets were challenged with glucose and tolbutamide, there was no rise in intracellular free calcium ion concentration ([Ca2+]i) over basal values. Capacitance measurements used to monitor exocytosis in control and HI beta-cells revealed that there were no defects in Ca2+-dependent exocytotic events. Finally, insulin release studies documented that whereas tolbutamide failed to cause insulin secretion as a consequence of impaired [Ca2+]i signaling, glucose readily promoted insulin release. Glucose was also found to augment the actions of protein kinase C- and protein kinase A-dependent agonists in the absence of extracellular Ca2+. These findings document the relationship between SUR1 gene defects and insulin secretion in vivo and in vitro and describe for the first time KATP channel-independent pathways of regulated insulin secretion in diseased human beta-cells.

A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene.

Usher syndrome type 1 describes the association of profound, congenital sensorineural deafness, vestibular hypofunction and childhood onset retinitis pigmentosa. It is an autosomal recessive condition and is subdivided on the basis of linkage analysis into types 1A through 1E. Usher type 1C maps to the region containing the genes ABCC8 and KCNJ11 (encoding components of ATP-sensitive K + (KATP) channels), which may be mutated in patients with hyperinsulinism. We identified three individuals from two consanguineous families with severe hyperinsulinism, profound congenital sensorineural deafness, enteropathy and renal tubular dysfunction. The molecular basis of the disorder is a homozygous 122-kb deletion of 11p14-15, which includes part of ABCC8 and overlaps with the locus for Usher syndrome type 1C and DFNB18. The centromeric boundary of this deletion includes part of a gene shown to be mutated in families with type 1C Usher syndrome, and is hence assigned the name USH1C. The pattern of expression of the USH1C protein is consistent with the clinical features exhibited by individuals with the contiguous gene deletion and with isolated Usher type 1C.

Sulfonylurea receptor 1 and Kir6.2 expression in the novel human insulin-secreting cell line NES2Y.

NES2Y is a proliferating human insulin-secreting cell line that we have derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy. This disease is characterized by unregulated insulin release despite profound hypoglycemia. NES2Y cells, like beta-cells isolated from the patient of origin, lack functional ATP-sensitive potassium channels (KATP) and also carry a defect in the insulin gene-regulatory transcription factor PDX1. Here, we report that the NES2Y beta-cells that are transfected with the genes encoding the components of KATP channels in beta-cells, sulfonylurea receptor (SUR) 1 and Kir6.2, have operational KATP channels and show normal intracellular Ca2+ and secretory responses to glucose. However, these cells, designated NESK beta-cells, have impaired insulin gene transcription responses to glucose. NES2Y beta-cells that are transfected with either Kir6.2 or SUR1 alone do not express functional KATP channels and have impaired intracellular free Ca2+ concentration-signaling responses to depolarization-dependent beta-cell agonists. These findings document that in NES2Y beta-cells, coexpression of both subunits is critically required for fully operational KATP channels and KATP channel-dependent signaling events. This article further characterizes the properties of the novel human beta-cell line, NES2Y, and documents the usefulness of these cells in diabetes-related research.

Glucose modulation of insulin mRNA levels is dependent on transcription factor PDX-1 and occurs independently of changes in intracellular Ca2+.

Glucose regulates insulin production in pancreatic beta-cells in the long term by stimulating insulin gene transcription. These effects are partially mediated through the activity of a homeodomain transcription factor, PDX-1, which binds to four sites within the human insulin gene promoter. The availability of a human beta-like cell line, NES2Y, which lacks PDX-1 but expresses the insulin gene, allowed us to determine whether PDX-1 was essential for the stimulatory effect of glucose on insulin mRNA levels. In NES2Y cells, glucose had no effect on the insulin gene promoter linked to a firefly luciferase reporter or on endogenous insulin mRNA levels. However, in NES2Y cells stably transfected with PDX-1 (NES-PDX-1), glucose exhibited a marked stimulatory effect on both the insulin promoter (5+/-0.2-fold, n = 6) and insulin mRNA levels (4.8+/-0.5-fold, n = 4). NES2Y cells were derived from a patient with persistent hyperinsulinemic hypoglycemia of infancy; the cells therefore lacked operational ATP-sensitive potassium channels, which results in the failure to control depolarization-dependent intracellular Ca2+ signaling. Despite the loss of control of Ca2+ channel activity, NES-PDX-1 cells maintained normal glucose-responsive insulin gene regulation. These results demonstrate that glucose modulation of insulin mRNA levels is dependent on the activity of PDX-1 and that these effects are independent of changes in intracellular Ca2+ concentrations.

Hyperinsulinism of infancy: towards an understanding of unregulated insulin release. European Network for Research into Hyperinsulinism in Infancy.

Insulin is synthesised, stored, and secreted from pancreatic beta cells. These are located within the islets of Langerhans, which are distributed throughout the pancreas. Less than 2% of the total pancreas is devoted to an endocrine function. When the mechanisms that control insulin release are compromised, potentially lethal diseases such as diabetes and neonatal hypoglycaemia are manifest. This article reviews the physiology of insulin release and illustrates how defects in these processes will result in the pathophysiology of hyperinsulinism of infancy.

ATP-sensitive potassium channels and efaroxan-induced insulin release in the electrofusion-derived BRIN-BD11 beta-cell line.

The properties of ATP-sensitive K+ (K(ATP)) channels were explored in the electrofusion-derived, glucose-responsive, insulin-secreting cell line BRIN-BD11 using patch-clamp techniques. In intact cells, K(ATP) channels were inhibited by glucose, the sulfonylurea tolbutamide, and the imidazoline compounds efaroxan and phentolamine. Each of these agents initiated insulin secretion and potentiated the actions of glucose. K(ATP) channels were blocked by ATP in a concentration-dependent manner and activated by ADP in the presence of ATP. In both intact cells and excised inside-out patches, the K(ATP) channel agonists diazoxide and pinacidil activated channels, and both compounds inhibited insulin secretion evoked by glucose, tolbutamide, and imidazolines. The mechanisms of action of imidazolines were examined in more detail. Pre-exposure of BRIN-BD11 cells to either efaroxan or phentolamine selectively inhibited imidazoline-induced insulin secretion but not the secretory responses of cells to glucose, tolbutamide, or a depolarizing concentration of KCl. These conditions did not result in the loss of depolarization-dependent rises in intracellular Ca2+ ([Ca2+]i), K(ATP) channel operation, or the actions of either ATP or efaroxan on K(ATP) channels. Desensitization of the imidazoline receptor following exposure to high concentrations of efaroxan, however, was found to result in an increase in SUR1 protein expression and, as a consequence, an upregulation of K(ATP) channel density. Our data provide 1) the first characterization of K(ATP) channels in BRIN-BD11 cells, a novel insulin-secreting cell line produced by electrofusion techniques, and 2) a further analysis of the role of imidazolines in the control of insulin release.

Engineering a glucose-responsive human insulin-secreting cell line from islets of Langerhans isolated from a patient with persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a neonatal disease characterized by dysregulation of insulin secretion accompanied by profound hypoglycemia. We have discovered that islet cells, isolated from the pancreas of a PHHI patient, proliferate in culture while maintaining a beta cell-like phenotype. The PHHI-derived cell line (NES2Y) exhibits insulin secretory characteristics typical of islet cells derived from these patients, i.e. they have no K(ATP) channel activity and as a consequence secrete insulin at constitutively high levels in the absence of glucose. In addition, they exhibit impaired expression of the homeodomain transcription factor PDX1, which is a key component of the signaling pathway linking nutrient metabolism to the regulation of insulin gene expression. To repair these defects NES2Y cells were triple-transfected with cDNAs encoding the two components of the K(ATP) channel (SUR1 and Kir6.2) and PDX1. One selected clonal cell line (NISK9) had normal K(ATP) channel activity, and as a result of changes in intracellular Ca(2+) homeostasis ([Ca(2+)](i)) secreted insulin within the physiological range of glucose concentrations. This approach to engineering PHHI-derived islet cells may be of use in gene therapy for PHHI and in cell engineering techniques for administering insulin for the treatment of diabetes mellitus.

Enhancing the identification of excessive drinkers on medical wards: a 1-year follow-up study.

This paper describes a 1-year follow-up study examining whether hospital ward doctors and nurses continue to take quantitative alcohol histories and provide brief intervention to problem drinkers on general medical wards after the introduction of a simple protocol. Regular training in the use of this protocol was stipulated in the annual service contract between the Health Authority and the Hospital Trusts. Improvements in staff practice persisted at 1-year follow-up, although it fell from a peak at an earlier phase of the study. The positive role of state purchasers of health services in sustaining improvements in clinical practice is discussed.

Intra-arterial calcium stimulation test in the investigation of hyperinsulinaemic hypoglycaemia.

OBJECTIVE: To investigate the use of a calcium infusion test in the diagnosis and localisation of insulin secreting tumours in children. PATIENTS: Three patients with persistent hypoglycaemia of infancy (PHHI). PROCEDURE: During planned selective coeliac and mesenteric arteriography, serial samples were taken from a catheter in the right hepatic vein for insulin measurement following the injection of calcium gluconate. RESULTS: In all three children, selective intra-arterial calcium stimulation produced a significant rise in plasma insulin and was of value in localising the pancreatic abnormality in one child. In vitro studies on islets of Langerhans isolated from this patient following partial pancreatectomy showed unresponsive intracellular calcium signalling of the cells when stimulated with high extracellular concentrations of glucose and potassium or with sulphonylurea drugs (tolbutamide), but normal responsiveness to increasing extracellular calcium concentrations. CONCLUSIONS: The findings suggest a functional abnormality of the calcium channel in PHHI and provide a rationale for the reported efficacy of channel blocking drugs in this condition. The role of selective intra-arterial calcium stimulation in the diagnosis of hyperinsulinaemic hypoglycaemia in childhood warrants further investigation.

Loss of functional KATP channels in pancreatic beta-cells causes persistent hyperinsulinemic hypoglycemia of infancy.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a disorder of childhood associated with inappropriate hypersecretion of insulin by the pancreas. The pathogenesis of the condition has hitherto remained controversial. We show here that insulin-secreting cells from a homogeneous group of five infants with PHHI lack ATP-sensitive K+ channel (KATP) activity. As a consequence, PHHI beta-cells are spontaneously electrically active with high basal cytosolic Ca2+ concentrations due to Ca2+ influx. Our findings define the pathogenesis of this disease as a novel K+ channel disorder.

Elevation of cytosolic calcium by imidazolines in mouse islets of Langerhans: implications for stimulus-response coupling of insulin release.

1. Microfluorimetry techniques with fura-2 were used to characterize the effects of efaroxan (200 microM), phenotolamine (200-500 microM) and idazoxan (200-500 microM) on the intracellular free Ca2+ concentration ([Ca2+]i) in mouse isolated islets of Langerhans. 2. The imidazoline receptor agonists efaroxan and phentolamine consistently elevated cytosolic Ca2+ by mechanisms that were dependent upon Ca2+ influx across the plasma membrane; there was no rise in [Ca2+]i when Ca2+ was removed from outside of the islets and diazoxide (100-250 microM) attenuated the responses. 3. Modulation of cytosolic [Ca2+]i by efaroxan and phentolamine was augmented by glucose (5-10 mM) which both potentiated the magnitude of the response and reduced the onset time of imidazoline-induced rises in [Ca2+]i. 4. Efaroxan- and phentolamine-evoked increases in [Ca2+]i were unaffected by overnight pretreatment of islets with the imidazolines. Idazoxan failed to increase [Ca2+]i under any experimental condition tested. 5. The putative endogenous ligand of imidazoline receptors, agmatine (1 microM-1 mM), blocked KATP channels in isolated patches of beta-cell membrane, but effects upon [Ca2+]i could not be further investigated since agmatine disrupts fura-2 fluorescence. 6. In conclusion, the present study shows that imidazolines will evoke rises in [Ca2+]i in intact islets, and this provides an explanation to account for the previously described effects of imidazolines on KATP channels, the cell membrane potential and insulin secretion in pancreatic beta-cells.

Ionic control of beta cell function in nesidioblastosis. A possible therapeutic role for calcium channel blockade.

A preterm female infant presented with intractable hypoglycaemia within 10 minutes of delivery. Normoglycaemia could be maintained only by the intravenous infusion of glucose at a rate of 20-22 mg/kg/min. Persistent hyperinsulinaemic hypoglycaemia of infancy was diagnosed from an inappropriately raised plasma insulin concentration (33 mU/l) at the time of hypoglycaemia (blood glucose < 0.5 mmol/l). Medical treatment with glucagon, somatostatin, and diazoxide led to only a modest reduction in the intravenous glucose requirement; a 95% pancreatectomy was performed and histological 'nesidioblastosis' confirmed. In vitro electrophysiological studies using patch clamp techniques on isolated pancreatic beta cells characterised the ionic basis for insulin secretion in nesidioblastosis. The beta cells were depolarised in low ambient glucose concentrations with persistently firing action potentials; these were blocked reversibly by the calcium channel blocking agent verapamil. Persistent postoperative hyperinsulinaemic hypoglycaemia was treated with oral nifedipine. This increased median blood glucose concentrations from 3.5 to 4.8 mmol/l and increased in duration the child's tolerance to fasting from 3 to 10.5 hours. These data allude to an abnormality in the ionic control of insulin release in nesidioblastosis and offer a new logical approach to treatment which requires further evaluation.

Ketoisocaproic acid and leucine increase cytoplasmic pH in mouse pancreatic B cells: role of cytoplasmic Ca2+ and pH-regulating exchangers.

The effects of nonglucose nutrient insulin secretagogues on cytoplasmic pH (pHi) in pancreatic B cells are unclear. These were studied with intact mouse islets loaded with BCECF and stimulated with ketoisocaproic acid (KIC) or leucine, which, unlike glucose, are exclusively metabolized in mitochondria. The changes in pHi were compared to those in cytoplasmic Ca2+ ([Ca2+]i; islets loaded with fura-2), metabolism [NAD(P)H fluorescence], and insulin release. In HCO3- buffer containing 3 mM glucose, 10 mM KIC produced a rapid and sustained increase in metabolism, [Ca2+]i, pHi, and insulin release. In HEPES buffer, the increase in metabolism, [Ca2+]i, and release were also rapid but not as sustained, whereas the alkalinization was delayed. The changes in release thus follow a time course more similar to that of [Ca2+]i and metabolism than to that of pHi. The role of [Ca2+]i in pHi changes was next examined. A similar rapid rise in pHi was produced by KIC in both HCO3- and HEPES buffers when its effects on [Ca2+]i were prevented, whether [Ca2+]i was kept low (4.8 mM KCl plus diazoxide) or high (30 mM KCl plus diazoxide). When the Na(+):H+ exchanger was blocked by dimethylamiloride, the alkalinizing effect of KIC was unaffected in HCO3- buffer, indicating that it does not result from an activation of this exchanger. In HEPES buffer, however, KIC strongly decreased pHi unless the rise in [Ca2+]i was prevented, in which case KIC increased pHi. When the HCO3-/Cl-exchanger was blocked by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), the effect of KIC in HCO3- buffer became similar to that in HEPES buffer without or with DIDS. The effects of leucine on pHi were similar to those of KIC. In conclusion, the effect of KIC and leucine on islet cell pHi, like that of glucose, is the complex result of an alkalinizing action of their metabolism and an acidifying action of the [Ca2+]i rise that they also produce. Compensation of this acidification is achieved by operation of the pHi-regulating exchangers, of which the HCO3-/Cl- exchanger plays a predominant role.

Clinical obstacles in administrating the South Oaks Gambling Screen in a methadone and alcohol clinic.

The South Oaks Gambling Screen (SOGS) was administered to veterans in both an outpatient methadone and problem drinking clinic. The instrument was given to 93 veterans who represented the poor and homeless. It was anticipated that the SOGS would provide important diagnostic information to the clinicians counseling the substance abusing population since many compulsive gamblers have a history of substance abuse. A number of clinical obstacles were encountered in administrating the SOGS in this environment. Both Client and staff noncompliance during the screening were major concerns in this respect. The clinical obstacles encountered in this study were examined and suggestions to prevent these problems are discussed.