Engineering a Glucose-responsive Human Insulin-secreting Cell Line from Islets of Langerhans Isolated from a Patient with Persistent Hyperinsulinemic Hypoglycemia of Infancy
1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês
10.1074/jbc.274.48.34059
ISSN1083-351X
AutoresWendy M. Macfarlane, Joanna C. Chapman, Ruth M. Shepherd, Molly N. Hashmi, Noritaka Kamimura, Karen E. Cosgrove, Rachel O’Brien, P. D. Barnes, Alan Hart, Hilary M. Docherty, Keith Lindley, A Aynsley‐Green, Roger F.L. James, Kevin Docherty, Mark J. Dunne,
Tópico(s)Diabetes Management and Research
ResumoPersistent 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. 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. persistent hyperinsulinemic hypoglycemia of infancy ATP-sensitive potassium channel intracellular free calcium ion concentration 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 (1Aynsley-Green A. Dev. Med. Child. Neurol. 1981; 23: 372-379Crossref PubMed Scopus (36) Google Scholar,2Aynsley-Green A. Polak J.M. Bloom S.R. Gough M.H. Keeling J. Ashcroft S.J.H. Turner R.C. Baum J.D Arch. Dis. Child. 1981; 56: 496-508Crossref PubMed Scopus (197) Google Scholar). 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 (∼95%) 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 (KATP) channels. Genetic linkage has identified a susceptibility locus for PHHI within a region of chromosome 11 that encodes subunits of these channels (3Glaser B. Chiu K.C. Anker R. Nestorowicz A. Landau H. Ben-Bassat H. Sholmai Z. Kaiser N. Thornton P.S. Stanley C.A. Spielman R.S. Gogolin-Ewens K. Cerasi E. Baker L. Rice J. Donis-Keller H. Permutt M.A. Nat. Genet. 1994; 7: 185-188Crossref PubMed Scopus (113) Google Scholar, 4Thomas P.M. Cote G.J. Wohlik N. Haddad B. Mathew P.M. Rabl W. Aguilar-Bryan L. Gagel R.F. Bryan J. Science. 1995; 268: 426-429Crossref PubMed Scopus (739) Google Scholar), while direct recordings of beta cells isolated from PHHI patients (following pancreatectomy) have documented the absence of KATP channels (5Kane C. Shepherd R.M. Squires P.E. Johnson P.R.V. James R.F.L. Milla P.J. Aynsley-Green A. Lindley K.J. Dunne M.J. Nat. Med. 1996; 2: 1344-1347Crossref PubMed Scopus (225) Google Scholar). In beta cells these channels are composed of at least two subunits as follows: a K+ channel pore, Kir6.2, and an ATP-binding cassette protein, SUR1 (6Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1608) Google Scholar, 7Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Crossref PubMed Scopus (402) Google Scholar). Open KATP 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 in insulin exocytosis (8Ashcroft F.M. Ashcroft S.J.H. Ashcroft F.M. Ashcroft S.J.H. Insulin, Molecular Biology to Pathology. Oxford University Press, Oxford, New York1992: 97-150Google Scholar). A number of mutations in the SUR1 and Kir6.2 genes, which affect KATP channel function in PHHI, have been described (9Dunne M.J. Kane C. Shepherd R.M. Sanchez J.A. James R.F.L. Johnson P.R.V. Aynsley-Green A. Lu S. Clement IV, J.P. Lindley K.J. Seino S. Aguilar-Bryan L. N. Engl. J. Med. 1997; 336: 703-706Crossref PubMed Scopus (228) Google Scholar, 10Nestorowicz A. Glaser B. Wilson B.A. Shyng S.L. Nichols C.G. Stanley C.A. Thornton P.S. Permutt M.A. Hum. Mol. Genet. 1998; 7: 1119-1128Crossref PubMed Scopus (99) Google Scholar). The significance of this loss of channel function is that beta cells can no longer adequately control the regulated entry of Ca2+ ions. Since elevated [Ca2+]iconcentrations have been reported in isolated PHHI beta cells, this unregulated Ca2+ influx has been suggested to stimulate Ca2+-dependent exocytosis, which underpins insulin hypersecretion (11Dunne M.J. Aynsley-Green A. Lindley K.J. News Physiol. Sci. 1997; 12: 197-203Google Scholar). Recently, two related but clinically distinct disorders of familial hyperinsulinemia-induced hypoglycemia have also been linked to defects in beta cell stimulus-response coupling mechanisms. These are associated with gene defects in glucokinase (12Glaser B. Kesavan P. Heyman M. Davis E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (510) Google Scholar), a key component of the beta cell glucose-sensing mechanism (13Randle P.J. Diabetologia. 1993; 36: 269-275Crossref PubMed Scopus (71) Google Scholar), and in glutamate dehydrogenase (14Stanley C.A. Lieu Y.K. Hsu B.Y.L. Burlina A.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Poncz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (610) Google Scholar). However, unlike PHHI, patients with glucokinase-hyperinsulinism of infancy and glutamate dehydrogenase-hyperinsulinism of infancy present with milder symptoms and are responsive to medical therapy.We have recently presented preliminary data on a beta cell-like cell line (NES2Y) that was derived from islets of Langerhans isolated from the pancreas of a patient with PHHI resulting from defective KATP channel activity (15Macfarlane W.M. Cragg H. Docherty H.M. Read M.L. James R.F.L. Aynsley-Green A. Docherty K. FEBS Lett. 1997; 413: 304-308Crossref PubMed Scopus (51) Google Scholar). Here, we now describe how cell engineering can be used to repair the genetic defects within these cells by demonstrating that the restoration of ion channel function leads to the generation of a glucose-responsive human insulin-secreting cell line. The resultant cell line, NISK9, exhibited normal KATP channel activity, intracellular calcium regulation, and insulin output within the normal physiological range of glucose concentrations. This approach may be of value in engineering PHHI-derived islets for autotransplantation to treat the disease following pancreatectomy and for generating beta cell lines for use in the treatment of diabetes mellitus.DISCUSSIONPersistent hyperinsulinemic hypoglycemia of infancy is a rare neonatal disorder with devastating consequences for the newborn. The clinical characteristics of the condition are heterogeneous, with most cases presenting symptoms within the first few hours of life with very severe disease. However, some cases present at several months to 1 year of age and even some adult cases have also been described. Drug responsiveness in PHHI patients is also highly variable, and in the majority of cases medical therapy is of limited use, and this can be directly correlated with a major loss in the control of insulin release (5Kane C. Shepherd R.M. Squires P.E. Johnson P.R.V. James R.F.L. Milla P.J. Aynsley-Green A. Lindley K.J. Dunne M.J. Nat. Med. 1996; 2: 1344-1347Crossref PubMed Scopus (225) Google Scholar). The molecular basis of this syndrome is also heterogeneous. In 1994, familial disease was linked to chromosome 11p15.1 (3Glaser B. Chiu K.C. Anker R. Nestorowicz A. Landau H. Ben-Bassat H. Sholmai Z. Kaiser N. Thornton P.S. Stanley C.A. Spielman R.S. Gogolin-Ewens K. Cerasi E. Baker L. Rice J. Donis-Keller H. Permutt M.A. Nat. Genet. 1994; 7: 185-188Crossref PubMed Scopus (113) Google Scholar) and was later confirmed in multiplex Saudi Arabian families (22Thomas P.M. Cote G.J. Hallman D.M. Mathew P.M. Am. J. Hum. Genet. 1995; 56: 416-421PubMed Google Scholar). This region of Ch.11 encodes both subunits of KATP channels, Kir6.2 and SUR1 (6Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1608) Google Scholar, 7Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Crossref PubMed Scopus (402) Google Scholar). In those cases where mutations have been linked to the disease (23Nestorowicz A. Glaser B. Wilson B.A. Shyng S.L. Nichols C.G. Stanley C.A. Thornton P.S. Permutt M.A. Hum. Mol. Genet. 1998; 7: 1119-1128Crossref PubMed Scopus (107) Google Scholar), defects in the SUR1 (23 mutations reported) and KIR6.2 (2 reported) genes are mainly, but not exclusively, inherited in an autosomally recessive manner. Thus there are reports of non-Mendelian inheritance among discordant identical twins (24Santer R. Hoffmann H. Suttorp M. Simeoni E. Schaub J. J. Pediatr. 1995; 126: 1017Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar) and data to suggest that PHHI can be inherited in an apparent autosomal dominant way (25Thornton P.S. Satin-Smith M.S. Herold K. Glaser B. Chiu K.C. Nestorowicz A. Permutt M.A. Baker L. Stanley C.A. J. Pediatr. 1998; 132: 9-14Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 26Kukuvitis A. Deal C. Arbour L. Polychronakos C. J. Clin. Endocrinol. & Metab. 1997; 82: 1192-1194PubMed Google Scholar). Loss of maternally-imprinted genes in PHHI has recently been described. In these cases, the patient inherits a single paternal recessive SUR1 gene mutation, and a portion of the pancreas is reduced to hemizygosity because of the loss of maternal imprinted genes (27Ryan F. Devaney D. Joyce C. Nestorowicz A. Permutt M.A. Glaser B. Barton D.E. Thornton P.S. Arch. Dis. Child. 1998; 79: 445-447Crossref PubMed Scopus (62) Google Scholar, 28Verkarre V. Fournet J.-C. deLonlay P. Gross-Morand M.S. Devillers M. Rahier J. Brunelle F. Robert J.-J. Nihoul-Fekute C. Saudubray J.M. Junien C. J. Clin. Invest. 1998; 102: 1286-1291Crossref PubMed Scopus (259) Google Scholar). Loss of heterozygosity in the affected beta cells results in insulin hypersecretion, but because of the loss of other maternally-expressed imprinting genes (such as the tumor suppressor genes H19 and p57KIP2), the pancreata of patients with this condition also have the morphologically distinct appearance of focal regions of beta cells hyperproliferation. This has given rise to the terminology of “focal PHHI” (Fo-PHHI) as distinct from “diffuse PHHI” which is not associated with loss of imprinted genes. In addition, spontaneous SUR1 gene mutations also give rise to PHHI, and each of these factors has hindered attempts to provide a concise genotype-phenotype relationship in the field. In previous publications (5Kane C. Shepherd R.M. Squires P.E. Johnson P.R.V. James R.F.L. Milla P.J. Aynsley-Green A. Lindley K.J. Dunne M.J. Nat. Med. 1996; 2: 1344-1347Crossref PubMed Scopus (225) Google Scholar, 15Macfarlane W.M. Cragg H. Docherty H.M. Read M.L. James R.F.L. Aynsley-Green A. Docherty K. FEBS Lett. 1997; 413: 304-308Crossref PubMed Scopus (51) Google Scholar), we have described how the NES2Y cells were derived from a patient with diffuse PHHI and that the disease was manifest in association with the loss of beta cell KATP channels. As a result of the complexities of inherited KATP channel gene defects, it has still not been possible to identify the genetic lesions in this particular patient, but this is perhaps not too unexpected since an estimated 60% of all patients with PHHI remain anonymous in terms of the genetic basis of the condition even following pancreatectomy (29Glaser B. Landau H. Permutt M.A. Trends Endocrinol. Metab. 1999; 10: 55-61Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).The NES2Y cell is a beta cell-like insulin-secreting cell line derived without modification from post-operative PHHI tissue. Although a number of rodent and hamster beta cell-like lines have been generated through viral transformation (HIT-T15) (30Santerre R.F. Cook R.A. Cristel R.M.D. Shar J.D. Schmidt R.J. Williams D.C. Wilson C.P. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 4339-4343Crossref PubMed Scopus (305) Google Scholar), X-irradiation (RIN/Ins-1) (31Gazdar A.F. Chick W.L. Oie H.K. Sims H.L. King D.L. Weir G.C. Lauris V. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3519-3523Crossref PubMed Scopus (443) Google Scholar), transgenic expression of tumor-promoting proteins such as SV40 large T antigen in beta cells (betaTC and MIN6) (17Miyazaki J.-I. Araki K. Yamamoto E. Ikegami H. Asano T. Shibasaki Y. Oka Y. Yamamura K.-I. Endocrinology. 1990; 127: 126-132Crossref PubMed Scopus (1043) Google Scholar, 32Efrat S. Linde S. Kofod H. Spector D. Delannoy M. Grant S. Hanahan D. Baekkeskov S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 9037-9041Crossref PubMed Scopus (473) Google Scholar) or electrofusion (BRIN BD11) (33McClenaghan N.H. Barnett C.R. Ah-Sing E. Abdel-Wahab Y.H. O'Harte F.P. Yoon T.W. Swanston-Flatt S.K. Flatt P.R. Diabetes. 1996; 45: 1132-1140Crossref PubMed Google Scholar), attempts to generate human beta cell lines have proved unsuccessful. It is unclear as to why the NES2Y cells proliferate in culture. As described previously (15Macfarlane W.M. Cragg H. Docherty H.M. Read M.L. James R.F.L. Aynsley-Green A. Docherty K. FEBS Lett. 1997; 413: 304-308Crossref PubMed Scopus (51) Google Scholar), it may be a consequence of the impaired expression of the homeodomain transcription factor PDX1, a major islet cell differentiation and lineage determination factor (19Jonsson J. Carlsson J. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1552) Google Scholar). Thus the NES2Y cells may represent a stage in islet cell development at which the cells have retained the ability to replicate while attaining a beta cell-like phenotype. Alternatively, the ability to proliferate may be a general property of neonatal human islet tissue, which has not been well studied because of the paucity of available tissue. On the other hand, the requirement for pancreatectomy for PHHI has made available for experimental purposes tissue from this source. Further studies will address the potential applications of the NES2Y as a model for beta cell replication. The present paper has focused on the properties of the cell line as they relate to the insulin secretory dysfunction in PHHI. A major finding was that the secretory defects could be repaired at the molecular level by cell engineering.The NES2Y cells were shown to reproduce the properties and key features of acutely-isolated insulin-secreting cells from patients with PHHI (5Kane C. Shepherd R.M. Squires P.E. Johnson P.R.V. James R.F.L. Milla P.J. Aynsley-Green A. Lindley K.J. Dunne M.J. Nat. Med. 1996; 2: 1344-1347Crossref PubMed Scopus (225) Google Scholar). Thus NES2Y cells (i) lack operational KATP channels, (ii) have markedly impaired cytosolic Ca2+ signaling mechanisms, (iii) constitutively release insulin at an elevated rate in the absence of stimuli, and (iv) do not respond to depolarization-dependent agonists through the release of insulin (Figs. 1 and 2). These same characteristics have also been recently described in transgenic mice that express a “dominant-negative” form of KATP channels in pancreatic beta cells (34Miki T. Tashiro F. Iwanaga T. Nagashima K. Yoshitomi H. Aihara H. Nitta Y. Gonoi T. Inagaki N. Miyazaki J. Seino S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11969-11973Crossref PubMed Scopus (169) Google Scholar) but not in “Kir6.2 knock-out” animals (35Miki T. Nagashima K. Tashiro F. Kotake K. Yoshitomi H. Tamamoto A. Gonoi T. Iwanaga T. Miyazaki J. Seino S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10402-10406Crossref PubMed Scopus (435) Google Scholar). Thus, as PHHI is a rare condition, the availability of the NES2Y beta cell is an important asset to ongoing studies of the molecular pathophysiology of the condition.The NES2Y cells retained some capacity to secrete insulin in response to elevated glucose (Fig. 2 B). This finding can be explained through the “KATP channel-independent” pathway of glucose-induced secretion. Glucose “augmentation” routes have been described in rodent (36Sato Y. Aizawa T. Komatsu M. Okada N. Yamada T. Diabetes. 1992; 41: 438-443Crossref PubMed Scopus (175) Google Scholar, 37Gembal M. Gilon P. Henquin J.C. J. Clin. Invest. 1992; 89: 1288-1295Crossref PubMed Scopus (421) Google Scholar, 38Best L. Yates A.P. Tomlinson S. Biochem. Pharmacol. 1992; 43: 2483-2485Crossref PubMed Scopus (49) Google Scholar) and human insulin-secreting cells (39Straub S.G. James R.F.L. Dunne M.J. Sharp G.W.G. Diabetes. 1998; 47: 758-764Crossref PubMed Scopus (72) Google Scholar,40Straub S.G. James R.F.L. Dunne M.J. Sharp G.W.G. Diabetes. 1998; 47: 1053-1057Crossref PubMed Scopus (39) Google Scholar). These pathways are uncovered in normal beta cells by using pharmacological agents that eliminate the contribution of KATP channels to the operation of beta cells and are now recognized as accounting for the second phase of insulin release. As second phase secretion is dependent upon glucose metabolism and the concomitant entry of Ca2+, the lack of operational KATP channels in NES2Y cells coupled with the unregulated influx of Ca2+ will fuel glucose-induced secretion in these cells (Fig. 2 B). Similar findings have also been recently observed in acutely isolated beta cells from a patient with PHHI. 4W. M. Macfarlane, K. Docherty, and M. J. Dunne, manuscript in preparation. Defects in NES2Y cells were overcome following a triple transfection with cDNAs encoding SUR1, Kir6.2, and PDX1. The properties of KATP channels expressed in the NISK9 beta cells were strikingly similar to those reported in native tissue (41Dunne M.J. Petersen O.H. Biochim. Biophys. Acta. 1991; 1071: 67-82Crossref PubMed Scopus (151) Google Scholar). The recombinant channels were inwardly rectifying, inhibited by cytosolic ATP in a concentration-dependent manner, activated by ADP in the presence of ATP, underwent spontaneous run-down in isolated patches, and were modulated by diazoxide, tolbutamide, and efaroxan (Figs. Figure 3, Figure 4, Figure 5, Figure 6). The operation of these KATP channels is also clearly important to the function of the NISK9 beta cells. Not only did the transfection event substantiate the development of glucose responsiveness within a physiologically-relevant concentration range (Fig. 7 C), but it also governed both KCl- and tolbutamide-induced increases in the cytosolic Ca2+concentration and insulin release (Fig. 7, A-C) and controlled the inhibition of glucose-induced rises in cytosolic Ca2+ by diazoxide (Fig. 7 A). Experiments are currently in progress to determine the contribution of each of the transfected cDNAs to these properties. In cells stably transfected with PDX1 alone (NES-PDX1 cells), we know that insulin mRNA levels are increased following incubation in high glucose, whereas in NES2Y cells they are not. 5W. M. MacFarlane and H. M. Docherty, unpublished observations. This provides the first clear evidence for the pivotal role of PDX1 in regulating not only insulin promoter activity (as measured by transfected reporter constructs) but also insulin gene expression in response to glucose. These results suggest that PDX1 would be necessary when engineering PHHI-derived islet cells in order to provide the capacity to replenish insulin stores following secretory stimuli. Interestingly, NISK9 and NES2Y-PDX1 cells proliferated in culture with a cell doubling time roughly similar to that of NES2Y. This would indicate that PDX1 contributes only partially, if at all, to the ability of the PHHI-derived islet cells to proliferate. NES2Y cells stably transfected with SUR1 and Kir6.2 (but not PDX1) also express fully-operational KATP channels and secrete insulin in response to glucose stimulation. However, unlike NISK9 beta cells these cells fail to exhibit glucose-dependent insulin gene promoter activity because of impaired PDX1 function.NES2Y cells represent a key in vitro model system for the study of beta cells in the absence of functional KATPchannels. Our data have established proof of concept that in vitro gene therapy could be used to successfully reverse a metabolically related disorder. These results allude to the possibility that in the future, following pancreatectomy, acutely-isolated beta cells from PHHI patients could be similarly engineered for subsequent autotransplantation. By transgenic manipulation of the NES2Y cells, we have generated the first fully glucose-responsive human insulin-secreting cell line. We believe that these, and other PHHI-derived islet cell lines, are of major importance for in vitro studies of human beta cell function and potentially valuable in transplantation-based therapies for both diabetes mellitus and PHHI. 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 (1Aynsley-Green A. Dev. Med. Child. Neurol. 1981; 23: 372-379Crossref PubMed Scopus (36) Google Scholar,2Aynsley-Green A. Polak J.M. Bloom S.R. Gough M.H. Keeling J. Ashcroft S.J.H. Turner R.C. Baum J.D Arch. Dis. Child. 1981; 56: 496-508Crossref PubMed Scopus (197) Google Scholar). 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 (∼95%) 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 (KATP) channels. Genetic linkage has identified a susceptibility locus for PHHI within a region of chromosome 11 that encodes subunits of these channels (3Glaser B. Chiu K.C. Anker R. Nestorowicz A. Landau H. Ben-Bassat H. Sholmai Z. Kaiser N. Thornton P.S. Stanley C.A. Spielman R.S. Gogolin-Ewens K. Cerasi E. Baker L. Rice J. Donis-Keller H. Permutt M.A. Nat. Genet. 1994; 7: 185-188Crossref PubMed Scopus (113) Google Scholar, 4Thomas P.M. Cote G.J. Wohlik N. Haddad B. Mathew P.M. Rabl W. Aguilar-Bryan L. Gagel R.F. Bryan J. Science. 1995; 268: 426-429Crossref PubMed Scopus (739) Google Scholar), while direct recordings of beta cells isolated from PHHI patients (following pancreatectomy) have documented the absence of KATP channels (5Kane C. Shepherd R.M. Squires P.E. Johnson P.R.V. James R.F.L. Milla P.J. Aynsley-Green A. Lindley K.J. Dunne M.J. Nat. Med. 1996; 2: 1344-1347Crossref PubMed Scopus (225) Google Scholar). In beta cells these channels are composed of at least two subunits as follows: a K+ channel pore, Kir6.2, and an ATP-binding cassette protein, SUR1 (6Inagaki N. Gonoi T. Clement IV, J.P. Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1608) Google Scholar, 7Sakura H. Ämmälä C. Smith P.A. Gribble F.M. Ashcroft F.M. FEBS Lett. 1995; 377: 338-344Crossref PubMed Scopus (402) Google Scholar). Open KATP 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 in insulin exocytosis (8Ashcroft F.M. Ashcroft S.J.H. Ashcroft F.M. Ashcroft S.J.H. Insulin, Molecular Biology to Pathology. Oxford University Press, Oxford, New York1992: 97-150Google Scholar). A number of mutations in the SUR1 and Kir6.2 genes, which affect KATP channel function in PHHI, have been described (9Dunne M.J. Kane C. Shepherd R.M. Sanchez J.A. James R.F.L. Johnson P.R.V. Aynsley-Green A. Lu S. Clement IV, J.P. Lindley K.J. Seino S. Aguilar-Bryan L. N. Engl. J. Med. 1997; 336: 703-706Crossref PubMed Scopus (228) Google Scholar, 10Nestorowicz A. Glaser B. Wilson B.A. Shyng S.L. Nichols C.G. Stanley C.A. Thornton P.S. Permutt M.A. Hum. Mol. Genet. 1998; 7: 1119-1128Crossref PubMed Scopus (99) Google Scholar). The significance of this loss of channel function is that beta cells can no longer adequately control the regulated entry of Ca2+ ions. Since elevated [Ca2+]iconcentrations have been reported in isolated PHHI beta cells, this unregulated Ca2+ influx has been suggested to stimulate Ca2+-dependent exocytosis, which underpins insulin hypersecretion (11Dunne M.J. Aynsley-Green A. Lindley K.J. News Physiol. Sci. 1997; 12: 197-203Google Scholar). Recently, two related but clinically distinct disorders of familial hyperinsulinemia-induced hypoglycemia have also been linked to defects in beta cell stimulus-response coupling mechanisms. These are associated with gene defects in glucokinase (12Glaser B. Kesavan P. Heyman M. Davis E. Cuesta A. Buchs A. Stanley C.A. Thornton P.S. Permutt M.A. Matschinsky F.M. Herold K.C. N. Engl. J. Med. 1998; 338: 226-230Crossref PubMed Scopus (510) Google Scholar), a key component of the beta cell glucose-sensing mechanism (13Randle P.J. Diabetologia. 1993; 36: 269-275Crossref PubMed Scopus (71) Google Scholar), and in glutamate dehydrogenase (14Stanley C.A. Lieu Y.K. Hsu B.Y.L. Burlina A.B. Greenberg C.R. Hopwood N.J. Perlman K. Rich B.H. Zammarchi E. Poncz M. N. Engl. J. Med. 1998; 338: 1352-1357Crossref PubMed Scopus (610) Google Scholar). However, unlike PHHI, patients with glucokinase-hyperinsulinism of infancy and glutamate dehydrogenase-hyperinsulinism of infancy present with milder symptoms and are responsive to medical therapy. We have recently presented preliminary data on a beta cell-like cell line (NES2Y) that was derived from islets of Langerhans isolated from the pancreas of a patient with PHHI resulting from defective KATP channel activity (15Macfarlane W.M. Cragg H. Docherty H.M. Read M.L. James R.F.L. Aynsley-Green A. Docherty K. FEBS Lett. 1997; 413: 304-308Crossref PubMed Scopus (51) Google Scholar). Here, we now describe how cell engineering can be used to repair the genetic defects within these cells by demonstrating that the restoration of ion channel function leads to the generation of a glucose-responsive human insulin-secreting cell line. The resultant cell line, NISK9, exhibited normal KATP channel activity, intracellular calcium regulation, and insulin output within the normal physiological range of glucose concentrations. This approach may be of value in engineering PHHI-derived islets for autotransplantation to treat the disease following pancreatectomy and for generating beta cell lines for use in the treatment of diabetes mellitus. DISCUSSIONPersistent hyperinsulinemic hypoglycemia of infancy is a rare neonatal disorder with devastating consequences for the newborn. The clinical characteristics of the condition are heterogeneous, with most cases presenting symptoms within the first few hours of life with very severe disease. However, some cases present at several months to 1 year of age and even some adult cases have also been described. Drug responsiveness in PHHI patients is also hi
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