HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MacFarlane, W. M. Articles by Dunne, M. J. Search for Related Content PubMed PubMed Citation Articles by MacFarlane, W. M. Articles by Dunne, M. J. Social Bookmarking                      What's this?

J Biol Chem, Vol. 274, Issue 48, 34059-34066, November 26, 1999

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

Wendy M. MacFarlane, Joanna C. Chapman^§, Ruth M. Shepherd^§, Molly N. Hashmi^§, Noritaka Kamimura^§, Karen E. Cosgrove^§, Rachel E. O'Brien^§, Philippa D. Barnes^§, Alan W. Hart, Hilary M. Docherty, Keith J. Lindley^¶, Albert Aynsley-Green^¶, Roger F. L. James, Kevin Docherty, and Mark J. Dunne^§**

From the  Department of Molecular and Cell Biology, University of Aberdeen, Institute of Medical Sciences, Aberdeen AB25 2ZD, the ^§ Institute of Molecular Physiology and Department of Biomedical Science, Sheffield University, Western Bank, Sheffield S10 2TN, the ^¶ Institute of Child Health, University of London, 30 Guilford Street, London WC1N 1EH, and  Department of Surgery, University of Leicester, Leicester Royal Infirmary, Leicester LE2 7LX, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (~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 (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⁺ 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 in insulin exocytosis (8). A number of mutations in the SUR1 and Kir6.2 genes, which affect K_ATP channel function in PHHI, have been described (9, 10). 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⁺]_i concentrations have been reported in isolated PHHI beta cells, this unregulated Ca²⁺ influx has been suggested to stimulate Ca²⁺-dependent exocytosis, which underpins insulin hypersecretion (11). 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 (12), a key component of the beta cell glucose-sensing mechanism (13), and in glutamate dehydrogenase (14). 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 K_ATP channel activity (15). 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 K_ATP 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- NES2Y cells were derived from islets of Langerhans isolated from the pancreas of a patient with PHHI as described previously (15). Isolated intact human or mouse islets and islet cells were prepared as described previously (5, 16). MIN6 cells (17) were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 5 mM D-glucose, and 2 mM L-glutamine. All experiments were performed with MIN6 cells between passage 27 and 33.

Plasmids-- pBK-Kir6.2 and pSK-SUR1 were kind gifts from Professor Frances Ashcroft, University of Oxford, UK. The SUR1 cDNA was removed from the pSK-SUR1 plasmid, blunt-ended, and subcloned into pIRES_neo (CLONTECH, Chicago, IL). Reporter constructs pGL-LUC and pGL-LUC200 and the pCR3-PDX1 construct were used as described previously (15). Plasmid DNA was prepared using the Qiagen Endotoxin-free Maxiprep method and quantitated spectrophotometrically.

Transfections-- NES2Y cells were grown to 80% confluence in 6-well plates, and transfected by mixing 4 µg of DNA with 54 µl of a 1 nM lipid suspension containing a 2:1 mixture of dioleoyl-L-phosphatidylethanolamine (Sigma) and dimethyl-dioctadecylammonium bromide (Fluka, Gillingham, UK) in 1 ml of serum-free Opti-MEM (Life Technologies, Inc.). The lipid-DNA complexes were allowed to form for 20 min at room temperature before being added to the washed cells. Following 5 h incubation, 1 ml of complete medium containing 30% heat-inactivated myoclone fetal calf serum was added to the cells. After 12 h, the medium-DNA complexes were replaced by complete medium. Forty eight hours later cells were split to 10% cell density and cultured in 10-cm Petri dishes, in the presence of 800 µg/ml G418 (Fermentas, UK). Surviving individual colonies of G418-resistant cells were isolated and transferred to 96-well plates. Individual clones of cells were then transferred to 6-well plates, and from there to standard 10-cm tissue culture plates. 192 clonal lines were assayed at this stage for insulin secretion in response to glucose at 3 and 16 mM. Glucose-responsive clonal lines were then selected and maintained in culture (800 µg/ml G418).

Electrophysiology-- Standard KCl- and NaCl-rich solutions were used in all patch clamp recordings to provide quasi-physiological cationic gradients, and the procedures used for data collection and analysis were as described previously (5, 9). All current traces are displayed with upward deflections representing outward current events.

Ca²⁺ Microfluorimetry-- Experiments were performed with fura-2-loaded cells and intact islets as described previously (18).

Insulin Measurements-- Insulin was measured by radioimmunoassay using an anti-human insulin antibody (Linco, UK), and ¹²⁵I-human insulin.

Northern Blotting-- Total RNA was prepared using the Qiagen RNeasy system, according to the manufacturer's protocols. 3 µg per sample of total RNA was denatured and separated on a 1.5% agarose/formaldehyde gel and transferred to Hybond-N⁺ nitrocellulose membrane (Amersham Pharmacia Biotech) in 20× SSC (3 M NaCl, 0.3 M sodium citrate). Filters were baked at 80 °C for 2 h, and then prehybridized in 5× SSC, 5× Denhardt's solution, 0.5% SDS, and 20 mg/ml sonicated salmon sperm DNA, at 65 °C for 1 h. Following the addition of the appropriate probe, filters were hybridized overnight at 65 °C. cDNA probes were labeled using the Prime-a-Gene system (Promega, UK), according to the manufacturer's instructions using 50 Ci of [³²P]dCTP (Amersham Pharmacia Biotech). Following hybridization, filters were washed in 2× SSC, 0.1% SDS at room temperature for 10 min (twice), twice in 1× SSC, 0.1% SDS at 65 °C for 15 min, and twice in 0.1× SSC, 0.1% SDS at 65 °C for 15 min. Filters were then wrapped in Saran wrap and autoradiographed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Properties of the Human Beta Cell Line, NES2Y-- The NES2Y beta cell line was derived in 1995 from a patient with PHHI (clinical details and the in vitro function of isolated beta cells from this patient have been described previously (5)). NES2Y cells are normally used between passage 15 and 20. The cells appear to have a limited capacity to store insulin (<0.95 ng/10⁶ cells), and this is a consistent feature of isolated PHHI islets of Langerhans that typically have insulin contents <5% of control tissue.² The NES2Y cells were found to secrete 12.23 ng/10⁶ cells/24 h at passage 10 and 11.13 ng/10⁶ cells/24 h at passage 15. We have previously shown that the insulin mRNA content of NES2Y is similar to that of one of the more differentiated beta cell lines, MIN6 (15). Karyotype analysis demonstrated that the cells were aneuploid and that all the chromosomes were human. Sequencing of selected gene products further confirmed that the cells were of human origin. In the present study, we describe the mechanisms that govern the ionic control of insulin release in NES2Y beta cells since these are compromised in PHHI. NES2Y cells were characterized by examining the functional properties of the cells by patch clamp methods, measurements of cytosolic Ca²⁺ levels using fura-2, and by the dynamics of insulin release by radioimmunoassay.

In normal human beta cells K_ATP channels are spontaneously active in resting unstimulated cells and are activated by diazoxide and inhibited by the sulfonylurea tolbutamide, as demonstrated by recordings of electrical activity in intact cells (Fig. 1A). In intact NES2Y cells there was no spontaneous K_ATP channel activity and no response of the cells to the potent K_ATP channel agonist diazoxide (Fig. 1A). Similarly, in cell-free patches of membrane isolated from NES2Y beta cells, there was no spontaneous K_ATP channel activity (Fig. 1B) and no actions of an "activation mixture" consisting of nucleotides, potassium fluoride (KF), and diazoxide (Fig. 1B). In sharp contrast to these findings, the simultaneous addition of 0.1 mM ADP, 0.5 mM GDP, 0.2 mM diazoxide, 0.1 mM UDP, and 10 mM KF led to a 6 ± 1.7 (n = 5)-fold increase in K_ATP channel activity in control human beta cells (Fig. 1B).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   The absence of KATP channel activity in NES2Y beta cells. All data were obtained using the patch clamp technique in the cell-attached configuration, A, or the "cell-free" inside-out configuration; B, see cartoons for recording configurations. Similar data were obtained in 4 (A) and 18 (B) other control experiments, and in a total of n = 10 (A) and n = 10 (B) NES2Y beta cell experiments. The same ionic gradients were used for each of the experimental protocols described, and all data are plotted to the same scales within each panel. Note in B how the addition of a "mixture" of nucleotides, potassium fluoride, and diazoxide results in the activation of at least 25 KATP channels. In this and all other electrophysiological recordings upward deflections represent outward current events, and the dashed horizontal lines indicate the zero current level, which corresponds to closure of all K+ channels.

We next investigated the effect of glucose and non-nutrient insulin secretagogues on intracellular calcium concentrations in fura-2-loaded NES2Y cells and intact islets (Fig. 2). Unlike control cells, the NES2Y cells failed to respond with a rise in [Ca²⁺]_i when challenged with either glucose (20 mM) or tolbutamide (0.1-0.2 mM) (Fig. 2A). In addition, there was no action of high external KCl (40 mM) on [Ca²⁺]_i, indicating that NES2Y beta cells, as has been shown for PHHI beta cells (5), are unable to regulate voltage-dependent Ca²⁺ influx (Fig. 2A). In acutely isolated PHHI islet cells, we have previously shown that there is a causal relationship between the loss of the ionic control of beta cell function and insulin hypersecretion (5). This was similarly observed in NES2Y cells, which, in comparison to control beta cells, have elevated rates of insulin release in the absence of stimuli (Fig. 2B). The control beta cells displayed strong (25-fold) insulin secretory response to glucose. The NES2Y cells also exhibited a response to glucose, albeit weak, within the range (6-11 mM). However, in NES2Y cells both KCl (40 mM) and tolbutamide (0.2 mM) failed to stimulate insulin release above basal rates of secretion (Fig. 2B).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Changes in intracellular calcium and insulin secretion in response to secretagogues in NES2Y cells. A, all experiments were carried out by exposing NES2Y cells or control (mouse) islets to medium containing glucose (20 mM), tolbutamide (Tol; 0.2 mM), or KCl (40 mM). Changes in the free intracellular calcium concentration were estimated by microfluorimetry procedures using fura-2. The number of replications of these experiments has been indicated. B NES2Y cells or MIN6 cells (control) were treated with increasing concentrations of glucose or with KCl (40 mM) or tolbutamide (0.2 mM). Media were collected, and insulin was measured by radioimmunoassay. The results represent the mean ± S.E. (n = 4). The data shown are representative of experiments performed on three separate occasions.

Cell Engineering to Repair the Defect in NES2Y Cells-- We have previously shown impaired expression of the transcription factor PDX1 in NES2Y beta cells by immunoblotting and quantitative RT-PCR (15). PDX1 is involved in islet cell differentiation (19) and in mediating the regulation of insulin gene transcription by nutrients (20, 21). Its absence from NES2Y cells resulted in a loss of glucose-sensitive insulin promoter activity as measured by activation of the pGL-LUC200 reporter gene construct, which contains the 60 to 260 region of the human insulin gene promoter (15). However, transient co-transfection of PDX1 and the PGL-LUC200 construct fully restored the stimulatory response of this reporter construct to glucose. We therefore hypothesized that by transfecting NES2Y cells with PDX1 and the two components of the K_ATP channel (SUR1 and Kir6.2), we would restore an insulin secretory response to glucose as well as the ability to stimulate insulin gene expression. The transfected cells were selected on the basis of resistance to the antibiotic G418. Of 192 G418-resistant clones, 3 exhibited a secretory response between 3 and 15 mM glucose. One of these lines was designated NISK9. Northern blot analysis confirmed the overexpression of the three transgenes. PDX1 mRNA was absent from NES2Y cells but present in NISK9 cells; SUR1 mRNA could be detected in NES2Y cells, but in addition to this transcript, NISK9 cells had approximately 10-fold higher levels of a larger mRNA species, which corresponded to the transfected bicistronic (SUR1/neo) mRNA; and Kir6.2 mRNA was present in NES2Y cells, whereas NISK9 cells contained a similar transcript expressed at almost 10-fold higher levels (data not shown). These results confirmed that the transfected genes were stably overexpressed in the NISK9 cells. Immunocytochemistry using SUR1 and PDX1 antibodies indicated that all cells expressed both proteins (not shown). This suggests that the efficiency of transfection was near 100%.

Patch clamp analysis demonstrated that NISK9 cells expressed spontaneous K⁺ channel activity in isolated patches (Fig. 3A) which was similar in overall magnitude to the K_ATP channel current recorded from control cells (Fig. 3B). Immunoblotting using an antibody against SUR1 was used to examine SUR1 protein expression. In NES2Y cells the presence of SUR1 protein was detected as a sharp band of approximately 175 kDa, whereas in both control and NISK9 beta cells SUR1 appears as a broader band (data not shown). This implies that the SUR1 gene defect in NES2Y cells leads to the production of an inactive precursor form of the protein that is not properly glycosylated. Taken together these data indicate that loss of K_ATP channels in NES2Y cells may result from either inappropriate trafficking or incorrect assembly of SUR1 with Kir6.2 subunits into an active channel complex at the plasma membrane and that manipulation of these cells has established an active form of SUR1.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   KATP channel activity of NISK9 cells. A shows typical data obtained from human beta cells, NES2Y beta cells, and NISK9 beta cells under exactly the same experimental conditions. In each experiment, isolated patches of membrane were formed from intact beta cells using the patch clamp technique (see cartoons). In control and NISK9 cells, inside-out patch formation (indicated by vertical dotted line) leads to the appearance of KATP channels due to wash-out of intracellular ATP and the resulting disinhibition of channel openings (5). No KATP channels were seen in NES2Y cell recordings. B, shown average data (mean ± S.E.) from 202 similar experiments.

K_ATP channels in NISK9 beta cells have identical properties to K_ATP channels in native human beta cells (Figs. 4 and 5). The recombinant ion channel has a non-linear unitary current voltage-relationship profile, with an estimated inward current conductance of approximately 70 pS (Fig. 4A). This was similar to the value measured in control human beta cells, i.e. 66 pS (data not shown). K_ATP channels were inhibited in a dose-dependent manner by elevated concentrations of ATP (0.005-1 mM) (Fig. 5B), with 50% inhibition occurring between 5 and 10 µM, and were activated by ADP in the presence of inhibitory concentrations of ATP (n = 6/6; Figs. 4B and 5A). In NISK9 beta cells K_ATP channels also underwent run-down in the isolated inside-out patch configuration (Fig. 3). The pharmacological properties of recombinant channels were also identical to native K_ATP channels, i.e. we saw activation (either in intact cells or isolated patches) by the agonist diazoxide and inhibition by tolbutamide (Fig. 6). In addition to reversing the actions of diazoxide, both tolbutamide and the imidazoline efaroxan also directly inhibited K_ATP channels in NISK9 cells. Thus 250 µM tolbutamide reduced the channel open probability to 40.8 ± 8.5% (n = 3) of the immediate control value (100%), whereas efaroxan (200 µM) inhibited channels to 8.5 ± 5.6% (n = 3) of the control value (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Recombinant KATP channels in NISK9 cells. A shows a typical current (I)-voltage (V) relationship plot for the recombinant KATP channel expressed in NISK9 beta cells. When recorded using symmetrical 140 mM KCl-rich solutions on either side of the cell membrane, the I-V plot is non-linear and inwardly rectifying. These channels have an estimated inward current conductance of approximately 70 pS. B shows representative sequences of data from the same inside-out patch recording. Spontaneously open channels are inhibited by ATP added to the internal domain of the membrane and activated by ADP in the presence of ATP and ADP. Similar findings were found in n = 6/6 patches.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Nucleotide-dependent gating of recombinant KATP channels in NISK9 cells. All data were obtained using the inside-out patch configuration under the conditions shown in Fig. 4B. A summarizes the actions of ADP (0.5 mM) on ATP (0.5 mM)-inhibited KATP channels. Changes in KATP channel open state probability (Po) were obtained from n = 4 separate patches and document the activation of ATP-inhibited channels by ADP. B shows the relationship between Po and elevating concentrations of ATP from four separate inside-out patch recordings. Note that 50% inhibition of KATP channels occurs between 5 and 10 µM ATP. Similar data were also obtained in control human beta cell recordings (not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Pharmacological properties of KATP channels in NISK9 cells. Data shown in A (cell-attached patch recording) and B (inside-out patch recording) were obtained under the same conditions as illustrated in Fig. 1. Both panels show that recombinant KATP channels are activated by diazoxide (Dia) and inhibited by tolbutamide (tol) (n = 4, A; n = 5 B). In B tolbutamide is shown to inhibit diazoxide (Diaz)-activated KATP channels in an inside-out patch; note that when this configuration is used, ATP must be added to the inside face of the cell membrane to facilitate diazoxide-induced K+ channel activation (41). Mean data from several experiments are also illustrated in A and B.

Fig. 7 shows the causal relationship between reconstitution of K_ATP channels and restoration of depolarization-response coupling. In NISK9 cells glucose, tolbutamide, and KCl each caused a marked increase in cytosolic Ca²⁺ signaling (Fig. 7, A and B) and insulin release (Fig. 7C). In comparison to NES2Y cells, NISK9 cells did not constitutively release insulin at elevated rates under non-stimulated conditions (0.9 ± 0.1 ng/10⁶ cells/h versus 0.1 ± 0.05 ng/10⁶ cells/h, respectively), a finding that was directly correlated to a significant decrease in the basal [Ca²⁺]_i concentration in NISK cells; 97 ± 10 nM (n = 29) versus 78 ± 4 nM (n = 68). In addition, the engineered NISK9 cells displayed a highly reproducible response to glucose within the physiological range (3-11 mM) and were fully responsive to KCl and tolbutamide (Fig. 7C).

View larger version (34K): [in this window] [in a new window]   Fig. 7.   Intracellular calcium and insulin secretory properties of NISK9 cells. A and B show that, unlike NES2Y beta cells (see Fig. 2A), NISK9 beta cells will respond to glucose stimulation through an elevation of the cytosolic Ca2+ concentration. In these experiments changes in [Ca2+]i were estimated using microfluorimetry techniques with fura-2 loaded cells. In A note how glucose induces a marked rise in [Ca2+]i in NISK9 cells so that the pattern of [Ca2+]i signaling is oscillatory and is inhibited by the hyperpolarizing KATP channel agonist diazoxide (n = 5). Like glucose (Glu.) (n = 8), both tolbutamide (Tol.) (A and B; 0.1-0.2 mM, n = 22) and KCl (B; 40 mM, n = 22) also raise [Ca2+]i. C, the consequences of KATP channel operation and the restoration of Ca2+ signaling are shown to be linked to the development of regulated insulin secretion in response to glucose (0.5-16 mM), KCl (40 mM), and tolbutamide (0.2 mM). Note how the basal rates of secretion are markedly lower than in the NES2Y beta cells (see Fig. 2). Mean ± S.E. values (n = 4) are shown, and the results are reproduced in three other separate experiments.

We examined the consequences of K_ATP channel restoration and PDX1 expression on insulin gene promoter activity in NISK9 cells. The control construct pGL-LUC displayed a weak response to glucose in control beta cells, NISK9 cells (Fig. 8), and in all other cells so far tested (data not shown). The construct pGL-LUC200, which contains a 200-base pair fragment from the human insulin promoter, exhibited a 5-fold response to glucose in both control beta cells and NISK9 cells (Fig. 8). This is in contrast to NES2Y cells, which show no significant effect on the pGL-LUC200 construct above that seen with the control plasmid pGL-LUC.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Insulin promoter activity in NISK9 cells. MIN6, NES2Y, or NISK9 cells were transfected with control construct pGL-LUC (LUC) or with a construct containing the 60 to 260 region of the human insulin gene promoter, pGL-LUC200 (LUC200), as indicated. Forty-eight hours post-transfection, cells were preincubated in 0.5 mM glucose for 3 h and then incubated for a further 3 h in low (0.5 mM) or high (16 mM) glucose as indicated. Values are shown as a percentage increase over basal (luciferase activity of the control construct at 0.5 mM glucose). Values represent an average from six replicates, and error bars represent standard deviation. Each set of values has been reproduced in three separate experiments.

Finally, we also found similar ion channel data when NES2Y beta cells were transfected with both SUR1 and Kir6.2 independently of PDX1, designated NESK beta cells. Insulin release from these cells was glucose-dependent, but because of impaired PDX1 expression, glucose had no effect on insulin gene promoter activity.³

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Persistent 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 (5). The molecular basis of this syndrome is also heterogeneous. In 1994, familial disease was linked to chromosome 11p15.1 (3) and was later confirmed in multiplex Saudi Arabian families (22). This region of Ch.11 encodes both subunits of K_ATP channels, Kir6.2 and SUR1 (6-7). In those cases where mutations have been linked to the disease (23), 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 (24) and data to suggest that PHHI can be inherited in an apparent autosomal dominant way (25, 26). 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 (27, 28). 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 p57^KIP2), 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 (5, 15), 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 K_ATP channels. As a result of the complexities of inherited K_ATP 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 (29).

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) (30), X-irradiation (RIN/Ins-1) (31), transgenic expression of tumor-promoting proteins such as SV40 large T antigen in beta cells (betaTC and MIN6) (17, 32) or electrofusion (BRIN BD11) (33), 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 (15), it may be a consequence of the impaired expression of the homeodomain transcription factor PDX1, a major islet cell differentiation and lineage determination factor (19). 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 (5). Thus NES2Y cells (i) lack operational K_ATP 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 K_ATP channels in pancreatic beta cells (34) but not in "Kir6.2 knock-out" animals (35). 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. 2B). This finding can be explained through the "K_ATP channel-independent" pathway of glucose-induced secretion. Glucose "augmentation" routes have been described in rodent (36-38) and human insulin-secreting cells (39, 40). These pathways are uncovered in normal beta cells by using pharmacological agents that eliminate the contribution of K_ATP 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 K_ATP channels in NES2Y cells coupled with the unregulated influx of Ca²⁺ will fuel glucose-induced secretion in these cells (Fig. 2B). Similar findings have also been recently observed in acutely isolated beta cells from a patient with PHHI.⁴

Defects in NES2Y cells were overcome following a triple transfection with cDNAs encoding SUR1, Kir6.2, and PDX1. The properties of K_ATP channels expressed in the NISK9 beta cells were strikingly similar to those reported in native tissue (41). 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. 3-6). The operation of these K_ATP 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. 7C), 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. 7A). 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.⁵ 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 K_ATP 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 K_ATP channels. 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.

	FOOTNOTES

^* This work was supported by grants from the Wellcome Trust, Medical Research Council, and the British Diabetic Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Institute of Molecular Physiology and Dept. of Biomedical Science, Sheffield University, Western Bank, Sheffield, S10 2TN, UK. Tel.: 114-222-4636; Fax: 114-276-5413; E-mail: m.j.dunne@sheffield.ac.uk.

² M. J. Dunne and A. Aynsley-Green, unpublished observations.

³ W. M. MacFarlane and K. Docherty, unpublished observations.

⁴ W. M. Macfarlane, K. Docherty, and M. J. Dunne, manuscript in preparation.

⁵ W. M. MacFarlane and H. M. Docherty, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: PHHI, persistent hyperinsulinemic hypoglycemia of infancy; K_ATP channel, ATP-sensitive potassium channel; [Ca²⁺]_i, intracellular free calcium ion concentration.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. L. Mosley, J. A. Corbett, and S. Ozcan Glucose Regulation of Insulin Gene Expression Requires the Recruitment of p300 by the {beta}-Cell-Specific Transcription Factor Pdx-1 Mol. Endocrinol., September 1, 2004; 18(9): 2279 - 2290. [Abstract] [Full Text] [PDF]

				J. C. Koster, M. S. Remedi, T. P. Flagg, J. D. Johnson, K. P. Markova, B. A. Marshall, and C. G. Nichols Hyperinsulinism induced by targeted suppression of beta cell KATP channels PNAS, December 24, 2002; 99(26): 16992 - 16997. [Abstract] [Full Text] [PDF]

				C. Shiota, O. Larsson, K. D. Shelton, M. Shiota, A. M. Efanov, M. Hoy, J. Lindner, S. Kooptiwut, L. Juntti-Berggren, J. Gromada, et al. Sulfonylurea Receptor Type 1 Knock-out Mice Have Intact Feeding-stimulated Insulin Secretion despite Marked Impairment in Their Response to Glucose J. Biol. Chem., September 27, 2002; 277(40): 37176 - 37183. [Abstract] [Full Text] [PDF]

				M. K. Reimer and B. Ahren Altered {beta}-Cell Distribution of pdx-1 and GLUT-2 After a Short-Term Challenge With a High-Fat Diet in C57BL/6J Mice Diabetes, February 1, 2002; 51(90001): S138 - 143. [Abstract] [Full Text] [PDF]

				K. Heald, T. Jay, and R. Downing Human islet transplantation: expectation versus reality The British Journal of Diabetes & Vascular Disease, November 1, 2001; 1(2): 128 - 130. [Abstract] [PDF]

				S. G. Straub, K. E. Cosgrove, C. Ämmälä, R. M. Shepherd, R. E. O'Brien, P. D. Barnes, N.'a. Kuchinski, J. C. Chapman, M. Schaeppi, B. Glaser, et al. Hyperinsulinism of Infancy: The Regulated Release of Insulin by KATP Channel--Independent Pathways Diabetes, February 1, 2001; 50(2): 329 - 339. [Abstract] [Full Text]

				R. M Shepherd, K. E Cosgrove, R. E O'Brien, P. D Barnes, C. Ämmälä, and M. J Dunne Hyperinsulinism of infancy: towards an understanding of unregulated insulin release Arch. Dis. Child. Fetal Neonatal Ed., March 1, 2000; 82(2): 87F - 97. [Abstract] [Full Text]

				A Aynsley-Green, K Hussain, J Hall, J M Saudubray, C Nihoul-Fékété, P De Lonlay-Debeney, F Brunelle, T Otonkoski, P Thornton, and K J Lindley Practical management of hyperinsulinism in infancy Arch. Dis. Child. Fetal Neonatal Ed., March 1, 2000; 82(2): 98F - 107. [Abstract] [Full Text]

				W. M. Macfarlane, S. C. Campbell, L. J. Elrick, V. Oates, G. Bermano, K. J. Lindley, A. Aynsley-Green, M. J. Dunne, R. F. L. James, and K. Docherty Glucose Regulates Islet Amyloid Polypeptide Gene Transcription in a PDX1- and Calcium-dependent Manner J. Biol. Chem., May 12, 2000; 275(20): 15330 - 15335. [Abstract] [Full Text] [PDF]

				S. Vaulont, M. Vasseur-Cognet, and A. Kahn Glucose Regulation of Gene Transcription J. Biol. Chem., October 6, 2000; 275(41): 31555 - 31558. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by MacFarlane, W. M. Articles by Dunne, M. J. Search for Related Content PubMed PubMed Citation Articles by MacFarlane, W. M. Articles by Dunne, M. J. Social Bookmarking                      What's this?