Mark J. Dunne

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 (refs 2–6). 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 (ref. 11). 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.

The α2-adrenoceptor antagonist efaroxan modulates K+ATP channels in insulin-secreting cells

Abstract The actions of efaroxan, a highly selective and potent α 2 -adrenoceptor antagonist, on insulin secretion, cAMP levels, 86 Rb4 + efflux and ATP-regulated potassium (K + ATP ) channels have been studied using isolated pancreatic islets of Langerhans and RINm5F cells. In the absence of an adrenoceptor agonist, efaroxan (1–100 μM) potentiated glucose-induced secretion over the range 4–10 mM glucose, but was without effect upon the maximal rate of secretion induced by 20 mM glucose. Efaroxan did not affect cAMP levels. Suppression of insulin release by the potassium channel opener diazoxide, was partially alleviated by efaroxan and was associated with an inhibition of the diazoxide-induced increase in the rate of 86 Rb + efflux. Using isolated patches of membrane we found efaroxan to be an effective blocker of k + ATP channels, with a K 1 value of 12 μm and a Hill coefficient of approximately 1. These data indicate that efaroxan promotes insulin secretion, in the absence of exogenous agonists, by a mechanism that involves inhibition of ATP-regulated K + channels.

Block of ATP-regulated potassium channels by phentolamine and other α-adrenoceptor antagonists

1 The patch clamp technique has been used to characterize the effects of phentolamine, an unselective blocker of α1 and α2‐adrenoceptors, on the electrical activity of isolated RINm5F insulin‐secreting cells and the gating of ATP‐regulated potassium (K+ATP) channels. 2 Current‐clamp experiments carried out by use of both conventional whole‐cell recordings and nystatin‐perforated cells, have demonstrated that phentolamine (5–20 μm) in the complete absence of α‐adrenoceptor agonists, caused a sharp depolarization of the cell membrane from approximately −66 mV to −42 mV. This depolarization was associated with the generation of calcium action potential‐like spikes. In the continued presence of phentolamine, diazoxide (100 μm) reversed these effects by causing a hyperpolarization of the cell, thereby preventing Ca2+ spikes. 3 Unitary current events from K+ATP channels were recorded from both outside‐out membrane patches and saponin permeabilized or open‐cells. When added to either the inside or the outside of the plasma membrane, phentolamine (0.1–100 μm) blocked openings from these channels. The effects of phentolamine were rapid, sustained and fully reversible. Phentolamine was apparently a more effective blocker of channels from the inside than the outside of the membrane. 4 The K1 value, corresponding to 50% inhibition of channels was estimated to be approximately 0.7 μm when phentolamine was added to the inside of the membrane and the Hill coefficient approximately 1. 5 Yohimbine (1–10 μm) and the chemically 2‐substituted imidazoline α‐adrenoceptor antagonists, antazoline (25 μm) and tolazoline (25 μm) were also found to block K+ATP channels in isolated patches of membrane. 6 In conclusion the present study demonstrates that phentolamine and other imidazoline adrenoceptor antagonists have effects upon ATP‐sensitive K+ channels that are not associated with stimulation of the adrenoceptor.

Glucose-dependent modulation of insulin secretion and intracellular calcium ions by GKA50, a glucokinase activator.

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca2+ homeostasis ([Ca2+]i) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 μmol/l GKA50 was found to stimulate insulin secretion and raise [Ca2+]i in the presence of glucose (2–10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 μmol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC50) values for glucose were shifted by 3 mmol/l in rat islets and ∼10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca2+]i (1 μmol/l GKA50) or to stimulate insulin release (30 nmol/l–10 μmol/l GKA50). At 5 mmol/l glucose, the EC50 for GKA50 in MIN6 cells was ∼0.3 μmol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca2+]i but not the actions of tolbutamide. Finally, the ATP-sensitive K+ channel agonist diazoxide (200 μmol/l) inhibited GKA50-induced insulin release and its elevation of [Ca2+]i. We show that GKA50 is a glucose-like activator of β-cell metabolism in rodent and human islets and a Ca2+-dependent modulator of insulin secretion.

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 KATP 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 KATP channel (SUR1 and Kir6.2) and PDX1. One selected clonal cell line (NISK9) had normal KATPchannel activity, and as a result of changes in intracellular Ca2+ homeostasis ([Ca2+] 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.

ATP-induced intracellular Ca2+ signals in isolated human insulin-secreting cells

Using isolated β-cells from human islets of Langerhans we have demonstrated that purinergic receptor agonists are functionally coupled to rises in the intracellular calcium ion concentration ([Ca2+]i). The effects of ATP, ADP and AMP have been examined over a range of concentrations, 0.5 to 500μM. The actions of ATP were more potent than those of either ADP or AMP suggesting that a P2-type of purinergic receptor operates in these cells. Responses to ATP were concentration-related, but exhibited marked desensitisation at high concentrations (>100μM). Purinergic receptor agonists elevate [Ca2+]i by mechanisms that involve both Ca2+ influx and Ca2+ mobilisation from intracellular stores. The physiological significance of our data has been discussed, and related to previous studies carried out upon rodent and clonal insulin-secreting cells.

PAX4 Enhances Beta-Cell Differentiation of Human Embryonic Stem Cells

Background Human embryonic stem cells (HESC) readily differentiate into an apparently haphazard array of cell types, corresponding to all three germ layers, when their culture conditions are altered, for example by growth in suspension as aggregates known as embryoid bodies (EBs). However, this diversity of differentiation means that the efficiency of producing any one particular cell type is inevitably low. Although pancreatic differentiation has been reported from HESC, practicable applications for the use of β-cells derived from HESC to treat diabetes will only be possible once techniques are developed to promote efficient differentiation along the pancreatic lineages. Methods and Findings Here, we have tested whether the transcription factor, Pax4 can be used to drive the differentiation of HESC to a β-cell fate in vitro. We constitutively over-expressed Pax4 in HESCs by stable transfection, and used Q-PCR analysis, immunocytochemistry, ELISA, Ca2+ microfluorimetry and cell imaging to assess the role of Pax4 in the differentiation and intracellular Ca2+ homeostasis of β-cells developing in embryoid bodies produced from such HESC. Cells expressing key β-cell markers were isolated by fluorescence-activated cell sorting after staining for high zinc content using the vital dye, Newport Green. Conclusion Constitutive expression of Pax4 in HESC substantially enhances their propensity to form putative β-cells. Our findings provide a novel foundation to study the mechanism of pancreatic β-cells differentiation during early human development and to help evaluate strategies for the generation of purified β-cells for future clinical applications.

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 μm), phentolamine (200–500 μm) and idazoxan (200–500 μm) 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 μm) 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 μm‐1 mM), blocked KATP channels in isolated patches of β‐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 β‐cells.

Congenital hyperinsulinism and mosaic abnormalities of the ploidy

Background: Congenital hyperinsulinism and Beckwith-Wiedemann syndrome both lead to β islet hyperplasia and neonatal hypoglycaemia. They may be related to complex genetic/epigenetic abnormalities of the imprinted 11p15 region. The possibility of common pathophysiological determinants has not been thoroughly investigated. Objective: To report abnormalities of the ploidy in two unrelated patients with congenital hyperinsulinism. Methods: Two patients with severe congenital hyperinsulinism, one overlapping with Beckwith-Wiedemann syndrome, had pancreatic histology, ex vivo potassium channel electrophysiological studies, and mutation detection of the encoding genes. The parental genetic contribution was explored using genome-wide polymorphism, fluorescent in situ hybridisation (FISH), and blood group typing studies. Results: Histological findings diverged from those described in focal congenital hyperinsulinism or Beckwith-Wiedemann syndrome. No potassium channel dysfunction and no mutation of its encoding genes (SUR1, KIR6.2) were detected. In patient 1 with congenital hyperinsulinism and Beckwith-Wiedemann syndrome, paternal isodisomy for the whole haploid set was homogeneous in the pancreatic lesion, and mosaic in the leucocytes and skin fibroblasts (hemihypertrophic segment). Blood group typing confirmed the presence of two erythroid populations (bi-parental v paternal only contribution). Patient 2 had two pancreatic lesions, both revealing triploidy with paternal heterodisomy. Karyotype and FISH analyses done on the fibroblasts and leucocytes of both patients were unremarkable (diploidy). Conclusions: Diploid (biparental/paternal-only) mosaicism and diploid/triploid mosaicism were present in two distinct patients with congenital hyperinsulinism. These chromosomal abnormalities led to paternal disomy for the whole haploid set in pancreatic lesions (with isodisomy or heterodisomy), thereby extending the range and complexity of the mechanisms underlying congenital hyperinsulinism, associated or not with Beckwith-Wiedemann syndrome.

Human embryonic stem cells: possibilities for human cell transplantation.

Human embryonic stem (ES) cells serve as a potentially unlimited renewable source for cell transplantation targeted to treat several diseases. One advantage of embryonic stem (ES) cells over other stem cells under research is their apparently indefinite self‐renewal capacity if cultured appropriately, and their ready differentiation into various cell phenotypes of all three germ layers. To date, a number of studies have reported the derivation of specific functional derivatives from human ES cells in vitro. While there have been clinical trials of human embryonal carcinoma (EC) cell‐derived neurons in humans there has been no attempt as yet using human ES cell derivatives. However, the latter have been transplanted into recipient animals. In some cases ES‐derived cells were shown to undergo further maturation, displayed integration with host tissue and even ameliorated the disease condition in the animal model. Recently, it has been reported that human ES cells can be genetically manipulated. Such procedures could be used to direct differentiation to a specific cell type or to reduce graft rejections by the modification of immune responses. This review highlights some of the recent advances in the field and the challenges that lie ahead before clinical trials using ES‐derived cells can be contemplated.