Glonal Insulinoma Cell Line That Stably Maintains Correct ... - Diabetes

Glonal Insulinoma Cell Line That Stably Maintains Correct Glucose Responsiveness David Knaack, Deborah M. Fiore, Manju Surana, Margarita Leiser, Megan Laurance, David Fusco-DeMane, Orion D. Hegre, Norman Fleischer, and Shimon Efrat

A number of pancreatic P-tumor cell (PTC) lines have been derived from insulinomas arising in transgenic mice expressing the SV40 T antigen gene under control of the insulin promoter. Some of these lines secrete insulin in response to physiological glucose concentrations. However, this phenotype is unstable. After propagation in culture, these nonclonal lines become responsive to subphysiological glucose levels and/or manifest reduced insulin release. Here we report the use of soft-agar cloning to isolate single-cell clones from a PTC line, which give rise to sublines that maintain correct glucose responsiveness and high insulin production and secretion for >55 passages (over a year) in culture. One of these clonal lines, denoted PTC6-F7, was characterized in detail. (JTC6-F7 cells expressed high glucokinase and low hexokinase activity, similarly to normal islets. In addition, they expressed mRNA for the GLUT2 glucose transporter isotype and no detectable GLUT1 mRNA, as is characteristic of normal P-cells. These results demonstrate that transformed P-cells can maintain a highly differentiated phenotype during prolonged propagation in culture, which has implications for the development of continuous P-cell lines for transplantation therapy of diabetes. Diabetes 1413-1417, 1994

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ancreatic p-cell lines derived from rodents have been extensively used to study the regulation of insulin production and secretion. A number of continuous P-cell lines have been generated from insulinomas and hyperplastic islets arising in mice expressing a transgene encoding the SV40 T antigen oncogene under control of the insulin promoter (RIP-Tag) (1-6). Several of these lines displayed insulin secretion characteristics similar to those observed in intact adult islets, in particular the response to glucose concentrations in the physiological range (5-15 mmol/1). However, a common problem encountered with all these cell lines is their phenotypic instability. After prolonged propagation in culture, these cells become

From CytoTherapeutics (D.K., D.M.F., M. Laurance, O.D.H.), Providence, Rhode Island, and the Departments of Medicine (M.S., M. Leiser, N.F.) and Molecular Pharmacology (D.F.-D., S.E.), Albert Einstein College of Medicine, Bronx, New York. Address correspondence and reprint requests to Dr. Shimon Efrat, Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Present addresses: M. Laurance, Oregon Health Sciences University, Portland, OR; O.D.H., Neocrin Inc., 31 Technology Drive, Irvine, CA. Received for publication 9 April 1994 and accepted in revised form 21 July 1994. PTC, p-tumor cell; KRB, Krebs-Ringer buffer; RIA, radioimmunoassay; IBMX, isobutylmethylxanthine; BSA, bovine serum albumin; PCR, polymerase chain reaction. DIABETES, VOL. 43, DECEMBER 1994

responsive to subphysiological concentrations of glucose and/or manifest diminished insulin output (4,6-9). A similar instability has been observed with P-cell lines derived by other methods (10-12). The triggering of insulin secretion by glucose requires glucose metabolism in p-cells (13). It has been proposed that glucose phosphorylation to glucose-6-phosphate, which constitutes the rate-limiting step in glycolysis, represents the "glucose sensor" of P-cells (14). This reaction is catalyzed in P-cells primarily by the high-ifm enzyme glucokinase (15), while most other cells use \ow-Km hexokinases (16). In the case of one insulinoma cell line, P-tumor cell (PTC)7, the development of responsiveness to subphysiological glucose concentrations has been clearly associated with an increase in hexokinase activity (6). Like most other reported p-cell lines, the PTC7 line is a nonclonal cell population. Therefore, it remains unclear whether the observed changes represent an adaptation of the entire cell population to growth in culture or a selection of a subpopulation. A phenotypically stable cell line manifesting correct glucose responsiveness would be of considerable value for the study of P-cell function and regulation. Here we report the use of soft-agar cloning to establish correctly regulated p-cell clonal sublines from a poorly regulated parental PTC cell population. One of these sublines was studied in detail during prolonged propagation in culture. It has maintained high insulin output and correct glucose regulation for >55 passages. RESEARCH DESIGN AND METHODS Cell culture. (3TC cells were grown according to Efrat et al. (2) in Dulbecco's modified Eagle's medium (GIBCO) containing 25 mmol/1 glucose and supplemented with 15% horse serum and 2.5% fetal bovine serum (GIBCO) (growth medium). Cells were passaged twice a week using a brief trypsinization in calcium-free Hank's balanced salt solution containing 0.05% trypsin and 0.53 mmol/1 EDTA. Except in the case of expanding cultures, cells were plated at 50,000 cells/ml in Falcon tissue culture dishes. Soft-agar cloning. Approximately 60,000 PTC6 cells at passage 18 were suspended in 9 ml prewarmed growth medium with 10% conditioned medium from (3TC3 cells (complete medium). The cell suspension was examined to assure that it consisted of well-dispersed individual cells; 0.5 ml of Matrigel (Collaborative Research) and 1 ml 3% agar (Batco) were added to the cell suspension, and 1.5 ml of the mixture was then placed in each well of a six-well plate. Cells were fed with complete medium twice a week and were allowed to grow in agar for 3 weeks. Individual cell clusters were then harvested from the agar by pipette, and each cell cluster was placed in a single well of a 96-well plate. Clusters were disrupted mechanically, fed three times a week with complete medium, and split as required. Each clone was tested in a single well for insulin secretory response to glucose. The selected clones were then expanded in complete medium. Insulin release. Initial screening of clones for glucose responsiveness was performed in 24-well plates. Cells were rinsed several times with 1413

CORRECTLY REGULATED STABLE |i-CELL LINE

glucose-free Krebs-Ringer buffer (KRB: 119 mmol/1 NaCl, 4.74 mmol/1 KC1, 2.54 mmol/1 CaCl2, 1.19 mmol/1 MgSO4) 1.19 mmol/1 KH2PO4) 25 mmol/1 NaHCO3) containing 10% horse serum and 20 mmol/1 HEPES, pH 7.4 (assay medium). One milliliter of the same medium was then added to the cultures for a 30-min preincubation; 0.5 ml of medium was then removed and replaced with 0.5 ml of fresh medium containing 6.4 mmol/1 glucose to give a final glucose concentration of 3.2 mmol/1. Cells were then incubated for 30 min, at which time 0.5 ml was sampled, and 0.5 ml of fresh medium was added to give a final concentration of 8 mmol/1 glucose. The incubation period was repeated, and a third sampling was performed, at which time the glucose concentration was adjusted to 16 mmol/1. All samples were frozen and subsequently assayed for insulin by radioimmunoassay (RIA) (1). The amount of insulin released during a given incubation period was calculated by subtracting the insulin amount secreted during the respective previous incubation period from the total insulin amount in the sample. After further clonal expansion, cells were assayed in replicate wells for response to a range of glucose concentrations (experiments in Fig. 2); 50,000 cells/well were plated in 24-well Falcon tissue culture plates. Three days later the growth medium was removed, and the cells were rinsed 2-3 times with assay medium and preincubated for 30 min in 1.5 ml assay medium containing 0.5 mmol/1 isobutylmethylxanthine (IBMX). We then removed 0.5 ml of medium, and the glucose concentration of the remaining medium was adjusted to the indicated concentration (in quadruplicate wells) using a 30 mg/ml glucose solution. After a 45-min incubation, the medium was removed and frozen for insulin RIA. The amount of insulin released during the incubation period was calculated by subtracting the amount in the preincubation sample from the amount in the incubation sample. For the insulin release assays in Figs. 1 and 4 and Table 1, the procedure described previously was followed (6). Cells were preincubated for 1 h in HEPES-buffered KRB containing 0.1% bovine serum albumin (BSA), followed by a 2-h incubation in fresh medium containing the indicated secretagogues. The medium was removed for insulin RIA, and the cells were lysed to determine insulin and total protein contents. Electron microscopy. Cells in four-well slides were fixed in Karnovsky's fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/1 sodium cacodylate buffer, pH 7.3). The sample was postfixed for 1 h in 1.0% osmium tetroxide in 0.1 mol/1 sodium cacodylate buffer, dehydrated through a graded series of ethanol solutions and three changes of propylene oxide, and embedded in Epon resin. Sections (0.06 p,m) were stained with methanolic uranyl acetate and lead citrate and were studied by transmission electron microscopy on a Phillips 300 electron microscope. Glucose phosphorylation. Glucose phosphorylation assays were performed with cell lysates using a fluorescent method previously described (6). RNA analysis. Cellular RNA was extracted, reverse-transcribed, and amplified in 40 cycles of polymerase chain reaction (PCR) previously described (17). The following oligo nucleotides were used: for GLUT2, sense 5'-GAGGCATCGACTGAGCAGAAGGTC-3', antisense 5'CACACTC TCTGAAGACGCCAGGA-3'; for GLUT1, sense 5'-GAGCAGTGCTCGGAT CACTGCAGTTC-3', antisense 5'-GGAACAGCTCCAAGATGGTGACCTT C-3'; for (3-actin, sense 5'-GATGACGATATCGCTGCGCTGGTCGTC-3', antisense 5'-GAGCCTCAGGGCATCGGAACCGCTCG-3'. The amplified fragments were fractionated on a 1.25% agarose gel, visualized with ethidium bromide, and photographed under ultraviolet light. RESULTS

(3TC6 cells (1) were serially passaged, and their insulin secretory responses to glucose were assessed as a function of passage number (Fig. 1). At passages 5 and 12, insulin release occurred only at glucose concentrations >5 mmol/1. By passage 18, pTC6 cells acquired a secretory response to lower glucose levels. After further propagation, the cells ceased to secrete insulin in response to glucose in the static incubation assay, although their insulin content remained unaltered. These deviations from the original phenotype could result from changes in the entire cell population, or from a selection of a poorly regulated subpopulation. To distinguish between these two possibilities, we attempted to derive clonal sublines of (3TC6 cells. (3TC6 cells at passage 18 were dispersed into single-cell suspension and plated in soft 1414

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P22

0 .1 .3

1

10 30

0 .1 .3

1

3

10 30

Glucose (mM) FIG. 1. Glucose-induced insulin release in (JTC6 cells. Cells at the indicated passages were preincubated for 1 h in HEPES-buffered KRB containing 0.1% BSA, followed by a 2-h incubation in fresh medium containing the indicated glucose concentrations and 0.5 mmol/1 IBMX. The incubation medium and cell extracts were analyzed for insulin by RIA. Values are means ± SE of triplicates from representative experiments.

agar in the presence of 0.1% Matrigel and 10% conditioned medium from (3TC3 (2) cultures. Individual colonies grew as clusters within the agar. After 3 weeks, clusters were harvested with a pipette, dispersed, and plated in 96-well tissue culture plates. In the absence of agar, the clonal cell populations continued to grow in clusters. The clusters attached to the plate loosely and could be easily removed by mechanical agitation. Thirty-five clones derived by this procedure were assayed for insulin secretory response to three glucose concentrations. Glucose concentration was adjusted using serial medium changes, initially in a single well and then, as cell numbers for a given clone increased, on replicate wells. In the serial assay, a clone was considered to be a candidate for further study if the insulin output doubled between 8 and 16 mmol/1 glucose. Using this criterion, 4 of the 35 clones were selected for further study. Their doubling times were ~3 days, which was similar to that of the parental cell line. After expansion, the four clones were assayed for insulin release at several glucose concentrations in replicate wells. All four clones manifested a progressive increase in insulin release in response to glucose concentrations between 5 and 16.7 mmol/1 (Fig. 2). One of the clones, PTC6-F7, was selected for detailed characterization over a prolonged culture period. These cells had an insulin content of 3 |xg/106 cells. Transmission electron microscopy revealed an abundance of dense core vesicles (Fig. 3), as well as well-developed mitochondria and golgi. (3TC6-F7 cultures manifested a stable phenotype of insulin secretion in response to physiological glucose concentrations (Fig. 4). Constitutive release over a 2-h period was low, averaging 1.83 ± 0.18% of their content (Table 1). Glucose levels 55 passages (Fig. 4). No significant change in this response has been noted to date as a function of passage number. The majority of glucose phosphorylation activity in PTC6-F7 cells, 73% of the total activity, was of the high-# m enzyme glucokinase. Only 27% of the activity was low-Km phosphorylation by hexokinases. The Vmax values and the proportion between glucokinase and hexokinase activities were similar to those of normal islets (6). (3TC6-F7 cells stably maintained this normal glucose phosphorylation pattern when last examined at passage 42 (Table 2). RNA analysis of glucose transporter transcripts in PTC6-F7 cells by reverse-transcriptase-PCR revealed the expression of GLUT2 (Fig. 5), the normal isotype of P-cells. No GLUT1 mRNA could be detected by this sensitive assay. For comparison, (3TC3 mRNA analyzed in parallel showed the presence of transcripts for both glucose transporter isotypes. DISCUSSION

These results demonstrate that a clonal population of {3TC cells can be propagated in culture for >55 passages without significant changes in its phenotype of high insulin production and correct glucose responsiveness. These results sug-

FIG. 3. Electron micrograph of (JTC6-F7 cells. Arrows point to mature insulin secretory granules; n, cell nucleus. Bar represents 340 nm. DIABETES, VOL. 43, DECEMBER 1994

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CORRECTLY REGULATED STABLE |i-CELL LINE

TABLE 2 Glucose phosphorylation activity in (3TC6-F7 cells Glucokinase Passage 34 42

o O

o 30 25

1.16 1.48

iirm(mmol/l)

ymax(U/g)

# ni (mmol/l)

8.6 12.3

0.56 0.56

0.03 0.06

Cell homogenates from the indicated passages were assayed for glucose phosphorylation activity. Apparent Vmax and Km values were determined by Eadie-Hofstee plots, with the best-fitted lines drawn using the method of least-squares. One unit is defined as the amount of enzyme that transforms 1 ixmol/min of substrate at 25°C.

P46

20 15 10

0 0.1 0.3 1

3

10 30

0 0.10.3 1

Glucose (mM)

FIG. 4. Glucose-induced insulin release in PTC6-F7 cells. Cells at the indicated passages were preincubated as described in Fig. 1, followed by incubation with the indicated glucose concentrations and 0.5 mmol/1 IBMX for 2 h. The incubation medium and cell extracts were analyzed for insulin by RIA. Values are means ± SE of triplicates from representative experiments. gest that the parental BTC6 line used in this study represents a heterogeneous population of cells with respect to the insulin secretory response to glucose. Thus the glucose response phenotype of the nonclonal line is determined by the relative proportion of cells with varying glucose sensitivities present within the population at a given passage. This suggests that the drift in glucose response characteristics observed in the parental BTC6 line and other nonclonal BTC cell lines results from a selection of abnormally regulated subpopulations, rather than from changes associated with the adaptation of the entire population to growth in culture. Such cells may have a selective advantage because of the elevated hexokinase activity, which provides them with the ability to metabolize glucose at much lower concentrations. It is likely that BTC6 cells are representative of other B-cell lines derived from RIP-Tag transgenic mouse insulinomas, and therefore it should be possible to routinely establish stable, correctly regulated lines in the future using cellcloning techniques. These findings have implications for the development of B-cell lines for transplantation therapy of diabetes. TABLE 1 Insulin release from (3TC6-F7 cells in response to various secretagogues Insulin released (% of cell content) Glucose (mmol/1)

No additive

0 1.67 5 30

1.83 ± 0.18 1.84 ± 0.23 1.85 ± 0.24 8.48 ± 0.55

IBMX (0.5 mmol/1)

Carbachol (2.0 |xmol/l)

Glipizide (1.0 |xmol/l)

1.33 ± 1.62 ± 4.31 ± 21.17 ±

1.86 ± 0.12 ND ND 21.04 ± 3.20

10.57 ± 1.99 ND ND 19.41 ± 2.40

0.14 0.18 0.18 1.44

Data are means ± SE. n = 10. Cells at passages 35-50 were incubated for 2 h with the indicated glucose concentrations in the absence or presence of additional secretagogues. The incubation medium and cell extracts were analyzed for insulin by RIA. ND, not determined. 1416

vmax(v/g)

Hexokinase

The phenotype of insulin secretion in response to glucose concentrations in the physiological range was associated in the BTC6-F7 clone with the presence of high glucokinase activity, low hexokinase activity, expression of GLUT2, and absence of GLUT1, as observed in normal B-cells. This pattern has remained stable during prolonged propagation in culture. These findings demonstrate that a population of transformed B-cells actively proliferating in culture is capable of stably maintaining the expression of specialized proteins normally associated with differentiated nonproliferating B-cells in vivo. In addition to the soft-agar method, we were able to derive sublines enriched for cells with physiological glucose responsiveness by a fluorescence-activated cell sorting technique. Cells were labeled with the vital dye Fluo-3-AM, which increases its fluorescence intensity when bound to cellular calcium (manuscript in preparation). The method relies on detection of the increase in intracellular calcium levels in cells stimulated by glucose and allows enrichment for correctly regulated cells. One subline derived from passage 21 3TC6 cells by this method, denoted SB-1, maintained correct glucose responsiveness for ~ 5 months after sorting (~20 passages). Subsequently, its insulin output dropped, suggesting that, unlike the clonal populations derived by the softagar cloning procedure, the sorted population contained residual poorly regulated cells that ultimately prevailed over the majority of correctly regulated cells. This finding underscores the importance of cell cloning for maintaining a stable phenotype. The cell phenotypes observed in the course of this study varied in both insulin output level and the degree and quality of glucose regulation. These varying B-cell phenotypes may represent actual physiologically relevant subpopulations normally present in mature or developing islets. The availability of clonally derived, phenotypically stable cell lines CM

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FIG. 5. RT-PCR analysis of glucose transporter transcripts in PTC6-F7 cells. Total cellular RNA was reverse-transcribed and amplified by PCR with oligonucleotides to the indicated transcripts. The amplified fragments were fractionated on a 1.25% agarose gel, visualized with ethidium bromide, and photographed under ultraviolet light. I , (JTC6-F7 cells at passage 42; 2, (JTC3 cells at passage 36. DIABETES, VOL. 43, DECEMBER 1994

D. KNAACK AND ASSOCIATES

with a variety of defined phenotypes may provide insights into possible physiological and developmental roles of these (3-cell subpopulations. 8.

ACKNOWLEDGMENTS We thank Jack Harvey of CytoTherapeutics for advice in the development of cloning conditions and Alice Gardiner, Keith Dionne, and Joe Hammang for providing assistance with various aspects of this study and Louise Flnnegan for manuscript preparation.

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REFERENCES

11.

1. D'Ambra D, Surana M, Efrat S, Starr RG, Fleischer N: Regulation of insulin secretion from (3-cell lines derived from transgenic mice insulinomas resembles that of normal (3-cells. Endocrinology 126:2815-2822, 1990 2. Efrat S, Linde S, Kofod H, Spector D, Delannoy M, Grant S, Hanahan D, Baekkeskov, S: Beta-cell lines derived from transgenic mice expressing a hybrid insulin gene-oncogene. Proc Natl Acad Sci USA 85:9037-9041, 1988 3. Miyazaki J-I, Araki K, Yamato E, Ikegami H, Asano T, ShibasaW Y, Oka Y, Yamaura K-I: Establishment of a pancreatic (3 cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms. Endocrinology 127:126-132, 1990 4. Radvanyi F, Christgau S, Baekkeskov S, Jolicoeur C, Hanahan D: Pancreatic (3 cells cultured from individual preneoplastic foci in a multistage tumorigenesis pathway: a potentially general technique for isolating physiologically representative cell lines. Mol Cell Biol 13:4223-4232,1993 5. Hamaguchi K, Gaskins HR, Leiter EH: NIT-1, a pancreatic |3-cell line established from a transgenic NOD/Lt mouse. Diabetes 40:842-849, 1991 6. Efrat S, Leiser M, Surana M, Tal M, Fusco-DeMane D, Fleischer N: Murine insulinoma cell line with normal glucose-regulated insulin secretion. Diabetes 42:901-907, 1993 7. Ishihara H, Asano T, Tsukuda K, Katagiri H, Inukai K, Anai M, Kikuchi M,

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Yazaki Y, Miyazaki J-I, Oka Y: Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 36:1139-1145, 1993 Sakurada M, Kanatsuka A, Saitoh T, Makino H, Yamamura K, Miyazaki J-I, Kikuchi M, Yoshida S: Regulation between glucose-stimulated insulin secretion and intracellular calcium accumulation studied with a superfusion system of a glucose-responsive pancreatic (B-cell line MIN 6. Endocrinology 132:2659-2665, 1993 Tal M, Thorens B, Surana M, Fleischer M, Lodish HL, Hanahan D, Efrat S: Glucose transporter isotypes switch in T-antigen-transformed pancreatic (3 cells growing in culture and in mice. Mol Cell Biol 12:422^132, 1992 Asfari M, Jaryic D, Meda P, Li G, Halban PA, Wollheim CB: Establishment of 2-mercaptoethanol-dependent differentiated insulin-secreting cell lines. Endocrinology 130:167-178, 1992 Gazdar AF, Chick WL, Oie HK, Sims HL, King DL, Weir GC, Lauris V: Continuous, clonal, insulin- and somatostatin-secreting cell lines established from a transplantable rat islet cell tumor. Proc Natl Acad Sci USA 77:3519-3523, 1980 Nielsen DA, Welsh M, Casadaban MJ, Steiner DF: Control of insulin gene expression in pancreatic (3-cells and in an insulin-producing cell line, RIN-5F cells. J Biol Chem 260:13585-13589, 1985 Meglasson MD, Matschinsky FM: Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 2:163-214, 1986 Meglasson MD, Matschinsky FM: New perspectives on pancreatic islet glucokinase. AmJPhysiol 246:E1-E13, 1984 Matschinsky FM, Liang Y, Kesavan P, Wang L, Frougel P, Velho G, Cohen D, Permutt MA, Tanizawa Y, Jetton, TL, Niswender K, Magnuson MA: Glucokinase as pancreatic (3 cell glucose sensor and diabetes gene. J Clin Invest 92:2092-2098, 1993 Wilson JE: Regulation of mammalian hexokinase activity. In Regulation of Carbohydrate Metabolism. Beitner R, Ed. CRC, Boca Raton, FL, 1984, p. 45-85 Efrat S, Leiser M, Wu Y-J, Fusco-DeMane D, Emran OA, Surana M, Jetton T, Magnuson MA, Weir G, Fleischer N: Ribozyme-mediated attenuation of pancreatic (3-cell glucokinase expression in transgenic mice results in impaired glucose-induced insulin secretion. Proc Natl Acad Sci USA 91:2051-2055, 1994

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