Engineering a Glucose-responsive Human Insulin-secreting Cell Line from Islets of Langerhans Isolated from a Patient with Persistent Hyperinsulinemic Hypoglycemia of Infancy

Abstract

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 celllike 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. Persistent hyperinsulinemic hypoglycemia of infancy (PHHI)1 is a potentially lethal disease of the newborn. It is characterized by inappropriate insulin release in relation to the corresponding levels of glycemia (1, 2). Affected children run the risk of severe neurological damage unless immediate and adequate steps are taken to avoid profound hypoglycemia. Treatment involves administration of glucose along with drugs such as diazoxide and somatostatin that inhibit insulin secretion. However, in many cases this is not effective, and within the first few weeks of birth a near total (ϳ95%) pancreatectomy is required to control blood glucose levels.Recently, it has been shown that PHHI arises from defects in the regulation of insulin secretion. This is due principally to the loss of function of ATP-regulated potassium (K ATP ) channels. Genetic linkage has identified a susceptibility locus for PHHI within a region of chromosome 11 that encodes subunits of these channels (3, 4), while direct recordings of beta cells isolated from PHHI patients (following pancreatectomy) have documented the absence of K ATP channels (5). In beta cells these channels are composed of at least two subunits as follows: a K ϩ channel pore, Kir6.2, and an ATP-binding cassette protein, SUR1 (6, 7). Open K ATP channels set the resting membrane potential for the beta cell and a change in the intracellular ATP/ADP ratio following glucose metabolism results in their closure and the initiation of a depolarization of the cell membrane. This in turn activates voltage-dependent calcium channels and the ensuing influx of calcium stimulates membrane docking and fusion of preformed insulin granules resulting ...

Altered β-Cell Distribution of pdx-1 and GLUT-2 After a Short-Term Challenge With a High-Fat Diet in C57BL/6J Mice

“…Activation of pdx-1 seems to be induced by nutrient stimulation (21) and, as shown by Macfarlane et al (22), high glucose induces nuclear translocation of pdx-1 in rat islets by a mechanism governed by SAPK2. Furthermore, pdx-1 expression has been shown to be impaired in a patient with persistent hyperinsulinemic hypoglycemia of infancy (23). We also found that the translocation of GLUT2 to the plasma membrane was compromised in mice fed the high-fat diet, which would perturb the islets' capacity to adequately respond to glucose.…”

Hyperinsulinism induced by targeted suppression of beta cell K ATP channels

“…In the least severe forms of the disease, there may be a slower onset of hyperinsulinemia, and it may be chronically treatable without progression to beta cell failure. Importantly, although a complete lack of K ATP activity has been reported in human PHHI beta cells (34)(35)(36), active K ATP channels have been observed in other patient samples (36,37), consistent with the reduced channel activity that is the result of many disease mutations when they are expressed in recombinant systems (3, 38, 39 ‡). ‡Cosgrove, K. E., Gonzalez, A. M., Lee, A. T., Barnes, P. J., Hussaim, K., Aynsley-Green, A., Lindley, K. J., Pirotte, B., Lebrun We are very grateful to Dr. Stan Misler for use of his Ca 2ϩ imaging facility and for valuable discussions during the course of this work.…”

Development and Functional Characterization of Insulin-releasing Human Pancreatic Beta Cell Lines Produced by Electrofusion

“…Other investigators have attempted to immortalize fetal or adult human beta cells or non-pancreatic cells using transfection with oncogenes and pancreatic endocrine developmental markers (15,16). The human Persistant Hyperinsulinemic Hypoglycemia of Infancy (PHHI)-derived pancreatic beta cell line, NES2Y, has also been reported to exhibit glucosestimulated insulin secretion after successful transfection to repair defects in expression of K-ATP channel and PDX-1 genes (17). Blox5 is an immortalized cell line produced from a purified population of human beta cells by infection with retroviral vectors expressing the SV40 T antigen, H-ras val12 , and hTERT oncogenes (18).…”