Ruth M. Shepherd

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.

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.

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.

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 β 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.

Genetics and Pathophysiology of Hyperinsulinism in Infancy

Hyperinsulinism in infancy (HI) is a condition of neonates and early childhood. For many years the pathophysiology of this potentially lethal disorder was unknown. Advances in the genetics, histopathology and molecular physiology of this disease have now provided insights into the causes of β-cell dysfunction and revealed levels of diversity far in excess of our previous knowledge. These include defects in ion channel subunit genes and mutations in several enzymes associated with β-cell metabolism and anaplerosis. In most cases, β-cell pathophysiology leads to an alteration in the function of ATP-sensitive K+ channels. This can manifest as ‘channelopathies’ of KATP channels through gene defects in ABCC8 and KCNJ11 (Ch.11p15); or as a result of ‘metabolopathies’ through defects in the genes encoding glucokinase (GCK, Ch.7p15–p13), glutamate dehydrogenase (GLUD1, Ch.10q23.3) and short-chain L-3-hydroxyacyl-CoA dehydrogenase (HADHSC, Ch.4q22–q26). This review focuses upon the relationship between the causes of HI and therapeutic options.

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.

Causes and therapy of hyperinsulinism in infancy

AbstractHyperinsulinism in infancy is a potentially lethal condition of neonates and occurs during early childhood as well. From defects in ion channel subunit genes to lesions in the control of β-cell metabolism and anaplerosis, the causes of hyperinsulinism in infancy are both varied and numerous. However, in all cases they appear to share a common target protein: the ATP-sensitive K+ channel. This review focuses on the relation between causes of hyperinsulinism in infancy and current treatment options.

Unregulated Insulin Release in Neonatal Hyperinsulinism; an Overview of Functional Studies Reporting the Loss of Depolarization-Response Coupling Events |[bull]| 997

In insulin-secreting β-cells, depolarization-response coupling is dependent upon the operation of function of ion selective channels. In response to glucose, a depolarization of the cell membrane results from ATP-sensitive K+ (KATP) channel closure. This in turn generates openings from voltage-dependent Ca2+ channels, and the appearance of action potentials associated with the accelerated influx of Ca2+. The subsequent rise in cytosolic Ca2+ then leads to insulin release. Diazoxide is an agonist of KATP channels and thus inhibits the release of insulin in normal β-cells. In persistent hyperinsulinaemic hypoglycaemia of infancy (PHHI) gene variations in the subunits of KATP channels have been linked to the disease. In our studies, we have investigated the functional properties of β-cells isolated from 29 patients with PHHI following pancreatectomy. 5/29 patients were from families with a previous history of PHHI, and 28/29 patients had a severe form of the disease that was either unresponsive or incompletely responsive to medical therapy with either diazoxide or somatostatin, used in combination with an elevated glucose infusion rate. In 8/29 subjects defects in the SUR1 gene have been identified. Studies of ion channel function and cytosolic Ca2+ signalling events were performed on isolated β-cells using patch-clamp techniques and cell microfluorimetry proceedures, respectively. The results of these experiments were correlated to insulin secretion studies in vitro and in vivo. These investigations revealed that loss of KATP channel function prevails inβ-cells isolated from PHHI patients. In many cases there was a direct correlation between defects in KATP channel activity and a spontaneous depolarisation of the cell membrane, leading to the appearance of action potentials. This inappropriate influx of Ca2+ is suggested to underpin the uncontrolled release of insulin from PHHI β-cells and is supported by[1] direct measurements of Ca2+-dependent exocytosis (through capacitance changes in isolated cells) and [2] by showing that removal of external Ca2+ or the addition of Ni2+ to the isolated cells, both lowers cytosolic Ca2+ levels and inhibits insulin secretion. These novel studies of isolated β-cells from patients with neonatal hyperinsulinsm [1] document the condition as a novel ion channel disorder and[2] provide important clues to the development of new lines of medical therapy with which to treat this condition.