- Email: [email protected]
[14] Establishment and culture of insulin-secreting β cell lines
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Acid ethanol: Ethanol (absolute): 750 ml Concentrated HCI: 15 ml Distilled H 2 0 : 2 3 5 ml Keep in a tightly closed bottle at 2 - 4 °. Acknowledgments We thank Dr. Benigna Blondel for invaluable help in the preparation of this manuscript. This work was supported by Grants from the Swiss National Science Foundation Nos. 32-25665.88 (C.B.W.), 31-26625.89 (P.M.), 31-9394.88 (P.A.H.), Grant No. 187384 from the Juvenile Diabetes Foundation International (P.M.), a grant from the Greenwall Foundation (P.A.H.), and Grant No. DK-35292 from the National Institutes of Health (P.A.H.)
[14] E s t a b l i s h m e n t
and Culture of Insulin-Secreting fl C e l l L i n e s
By CLAES B. WOLLHEIM, PAOLO MEDA, and PHILIPPE A. HALBAN
In order to study the molecular mechanism of insulin production a very large number of highly purified pancreatic fl cells is required. The most successful methods for isolating islets from large mammals, including man (see [ 13], this volume) can provide up to 200,000 islets/pancreas. The procedures are, however, time consuming and costly. This number of islets consists of some 5 X l0 s cells, a modest number for detailed biochemical analysis. Only approximately 75% are fl cells, and there is furthermore a significant contamination with exocrine cells. These major obstacles to the study of fl cell function have been overcome in recent years by the use of insulin-producing cell lines. The aim of this chapter is to describe the various types of lines available, as well as their functional characteristics. Lines Derived from Naturally Occurring or Induced Insulinomas
Naturally Occurringlnsulinomas Only a limited number of cell fines have been derived from spontaneously occurring insulinomas of either animal or human sources. Typically, the tumor is dissected out of the pancreas and then digested with collagenase with (or without) the addition of trypsin. The tumor cells are then METHODS IN ENZYMOL(X3Y, VOL. 192
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allowed to grow in tissue culture. The major problem encountered in virtually all attempts to obtain a permanent insulin-producing line in this way is that the cells dedifferentiate (as manifested by a dramatic decrease in cellular insulin content) rather rapidly. There appears to be an inverse relationship between proliferative capacity and differentiated status. There is no reproducible method available for selecting cells with maintained, differentiated function, although approaches including the use of extracellular matrices ',2 have been tried. It is, however, possible on occasion to maintain or restore, at least in part, differentiated function by inducing tumor growth during the in vivo passage of the cells. 3,4 The general view remains, however, that obtaining a well-differentiated line with useful in vitro proliferative capacity depends largely upon chance. Indeed, to our knowledge no human cell line fulfilling these criteria on a long-term basis exists. Despite these reservations, various groups have used insulinoma cells of both human 5-7 and animal3 ,s-'° origin for their studies. Induced Tumors
Insulinomas have been induced in the rat by either radiation" or by the combination of streptozotocin and nicotinamide.'2.'3 The tumor cells have then been used to establish cell lines. It must again be stressed that, as with
C. H. Thivolet, P. Chatelain, P. Nicoloso, A. Durand, and J. Bertrand, Exp. Cell Res. 159, 313 (1985). 2 R. Muschel, G. Khoury, and L. M. Reid, Mol. Cell. Biol. 6, 337 (1986). 3 p. A. Rae, C. C. Yip, and B. P. Schimmer, Can. J. Physiol. Pharmacol. 57, 819 (1979). 4 p. R. Hart, M. G. DeSilva, S. K. Swanston-Flau, C. J. Powdl, and V. Marks, J. Endocrinol. 118, 429 (1988). 5 W. L. Chick, V. Lauds, J. S. Soeldner, M. H. Tan, and M. Gfinsberg, Metabolism 22, 1217 (1973). 6 K. Adcock, M. Austin, W. C. Duckworth, S. S. Solomon, and L. R. Murrell, Diabetologia 11,527 (1975). 7 C. H. Thivolet, A. Demidem, M. Haftek, A. Durand, and J. Bertrand, Diabetes 37, 1279 (1988). s A. F. Gazdar, W. L. Chick, H. K. Oie, H. L. Sims, D. L. King, G. C. Weir, and V. Lauds, Proc. Natl. Acad. Sci. U.S.A. 77, 3519 (1980). 9 C. A. Carrington, E. D. Rubery, E. C. Pearson, and C. N. Hales, J. Endocrinol. 109, 193, (1986). 1oG. A. Praz, P. A. Halban, C. B. Wollheim, B. Blondel, A. J. Strauss, and A. E. Renold, Biochem. J. 210, 345 (1983). '~ W. L. Chick, W. Shields, R. N. Chute, A. A. Like, V. Lauds, and K. C. Kitchen, Proc. Natl. Acad. Sci. U.S.A. 74, 628 (1977). ,2 W. L. Chick, M. C. Appel, G. C. Weir, A. A. Like, V. Lauds, J. G. Porter, and R. N. Chute, Endocrinology 107, 954 (1980). ,3 p. Masiello, C. B. Wollheim, B. Blondel, and A. E. Renold, Diabetologia 24, 30 (1983). '
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the use of spontaneously occurring insulinomas (see above), there is no guarantee that a given tumor will provide a useful line. The main, but not the only, 12 exception to this rule has been an X-ray-induced rat insulinoma. 8 This tumor can be maintained by in vivo passage (serial transplantation) in inbred (NEDH) rats. H Several tumors derived from the original one now exist. They differ in their rate of growth and, typically, those which grow the slowest display the highest degree of differentiation. There has also been a suggestion that growing the tumor under the kidney capsule, rather than subcutaneously (the more conventional method), may further favor differentiation. 14 The first cell lines derived from this insulinoma were the RIN cells. These lines were established by Chick and associates by growing isolated tumor cells in culture. 8 These investigators deserve credit for their perseverance since their initial attempts were unsuccessful. The RIN cells have indeed provided many groups with a useful source of insulin-producing cells for a variety of purposes. The most commonly used subline is RINm5F. This line was initially selected since it had a relatively high insulin content, with nondetectable amounts of glucagon and somatostatin.S, 15
Culture and Characteristics of RINm5F Cells and Derived Sublines These cells are grown in RPMI 1640 (11 mMgiueose) medium supplemented with 10% fetal calf serum (FCS). Typically, cells are seeded into plastic culture vessels at a concentration of 2 X 105 cells/ml. The medium is changed after 3 and 6 days, and the cells trypsinized after l week. For this purpose the cells are rinsed with Ca+/Mg2+-free phosphate-buffered saline and then exposed to 0.025% (w/v) trypsin (1:250; Difco, Detroit, MI) and 0.27 m M EDTA in the same buffer for 1 - 3 rain at 37 °. The cells are dislodged from the culture surface by tapping the side of the vessel, and are then washed in medium before being reseeded. In order to maintain the functional status of RIN cells, it is essential that they be exposed to no more than 15-20 tissue culture transfers. Beyond this number there is an ever-increasing chance of the cells suddenly and unpredictably showing a dramatic loss of insulin content and responsiveness to secretagogues. It is unclear what causes this undesirable loss of function, but this phenomenon is by no means unique to RIN cells. Because of this limitation in passage number, it is important to have an adequate reserve of cells in storage. For ~4M. Hoenig and G. W. G. Sharp, Endocrinology119, 2502 (1986). ~5H. K. Oie, A. F. Gazdar, J. D. Minna, G. C. Weir, and S. B. Baylin, Endocrinology112, 1070 0983).
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this purpose, cells are suspended in phosphate-buffered saline or complete culture medium (10% FCS), with the addition of 10% dimethyl sulfoxide (DMSO) (4-6 X 107 cells/ml) and the temperature then progressively decreased before storage in liquid nitrogen. The cells are thawed by rapidly raising the temperature to 37 °, and then transferring them to RPMI 1640 medium supplemented with 20% FCS (the seeding density should be -106 cells/ml). As soon as the cells have attached to the culture surface, the FCS concentration may be decreased to 10%. When handled as described, the subclone of RINm5F used in our laboratories (RINm5F-2A) has maintained apparently unchanged function for over 7 years/°,16 This subline was established by cloning RINm5F cells using the limiting dilution method (i.e., by allowing colony formation fro.m a single cell). For RIN-r cells, another RIN cell clone, culture in chemically defined medium in the absence of serum was found to sustain cell proliferation? 7
Lins Derivedfrom Tumors in Transgenic Mice Transgenic mice expressing simian virus 40 (SV40) T antigen uniquely in the pancreatic p-cell have been derived by injection into a mouse embryonic pronucleus of a fusion gene construct consisting of the structural region of the SV40 T antigen gene driven by the insulin promoter? 8 These animals develop insulinomas and a cell line (B-TC cells) has been derived from such an insulinoma? 9 An alternative approach has been to use a gene construct allowing for random tissue expression of T antigen in the transgenic mice. In this case an insulinoma was also found (although not all mice had such tumors) and a cell line again prepared (IgSV195 cells).2° Such mouse fl cell lines have been less extensively studied than the RIN (see above) or HIT (see below) cell lines. This is due in part to the fact that they have only recently been described. They may be grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS and 25 m M glucose, using the same basic culture techniques described for RIN cells.
16C. B. Woltheim, M. J. Dunne, B. Peter-Riesch, R. Bruzzone, T. Pozzan, and O. H. Petersen, EMBO J. 7, 2443 (1988). 17H. K. W. Fong, W. L. Chick, and G. H. Sato, Diabetes 30, 1022 (1981). is D. Hanahan, Nature (London) 315, 115 (1985). 19S. Efrat, S. Linde, H. Kofold, D. Spector, M. Delannoy, S. Grant, D. Hanahan, and S. Baekkeskov, Proc. Natl. Acad. S¢i. U.S.A. 85, 9037, 1988. 2oA. GiUigan, L. Jewett, D. Simon, I. Damjanov, F. M. Matschinsky, H. Weik, C. Pinkert, and B. B. Knowles, Diabetes 38, 1056 (1989).
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Lines Obtained by Viral Transformation o f t Cells Primary cultures of pancreatic endocrine cells (see [13], this volume) have been transformed with SV40 and clonal cell lines obtained. 21,22One line in particular, HIT cells, derived from SV40 infection and transformation of Syrian hamster t-cells, 22 has proved useful for studies on fl cell function. HIT cells (and in particular the T 15 subclone), are maintained and grown in tissue culture in much the same way as RIN cells (see above) with the following differences. The culture medium originally recommended was F-12 containing 15% horse serum and 2.5% FCS, with the addition of l0 gg/ml glutathione, 0.1 g M selenous acid. 22 We now use RPMI 1640 (ll m M glucose) supplemented with 10% FCS, and again containing 0.1 # M selenous acid. As for the other cell lines described, HIT cells display a marked phenotypic alteration with time in culture,23 and we therefore strongly recommend that an adequate stock of early passage number cells be stored in liquid nitrogen and that the cells not be exposed to more than 10-20 tissue culture passages. Transfection of Secretory Cells with the Insulin Gene Transformed secretory cells have been transfected with the insulin gene driven by a viral promoter,u-2~ In order to facilitate the selection of stable transfectants, we transfect cells with a construct (DOL vector) carrying the insulin gene driven by the murine leukemia virus long terminal repeat, colinear with the neomycin resistance gene.25 The transfected cells are grown in G418 (geneticin) at a concentration toxic 075 gg/ml) to cells not expressing the neomycin resistance gene. Of these G418-resistant clones, approximately 30% were found to produce detectable levels of immunoreactive insulin. The transfection of noninsulin-producing cells with the insulin gene is, typically, useful for studying the mechanism and regulation of insulin expression. It furthermore allows for the expression of mutant insulin
21 E. J. Niesor, C. B. Wollheim, D. H. Mintz, B. Blondel, A. E. Renold, and R. Weft, Biochem. J. 178, 559 (1979). 22R. F. Santerre, R. A. Cook, R. M. D. Crisel, J. D. Sharp, R. J. Schmidt, D. C. Williams, and C. P. Wilson, Proc. Natl. Acad. Sci. U.S.A. 78, 4339 (1981). 23H.-J. Zhang, T. F. Walseth, and R. P. Robertson, Diabetes 38, 44 (1989). 24H.-P. Moore, M. D. Walker, F. Lee, and R. B. Kelly, Ce1135, 531 0983). 25D. J. Gross, P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Komaroff, Proc. Natl. Acad. Sci. U.S.A. 86, 4107 (1989). 26S. K. Powell, L. Orci, C. S. Craik, and H.-P. H. Moore, J. CellBiol. 106, 1843 0988).
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The cell fine most frequently used for such studies is the AtT20 (mouse pituitary corticotroph) transformed fine. These cells may be grown in F-10 medium supplemented with 15% horse serum and 2.5% FCS. We have found that cell growth is favored by raising the glucose concentration to 25 mM. Other cell types have been used for transfection studies, and these include HIT cells.2s g e n e s . 2s-27
Other Approaches to Obtaining Insulin-Producing Cell Lines There have been attempts to obtain cell fines from cultures of B cells of neonatal29 or fetal 3° origin. This approach depends upon the random and spontaneous outgrowth of cells displaying peculiarly elevated proliferative capacity (possibly protoditferentiated stem cells). No stable cell fine has yet become available using this approach. Analytical Methods for Studying Insulin-Producing Cell Lines
Morphology of fl Cell Clones The identification of insulin-containing cells in a clone or fine cannot be made by the mere observation of living cultures since permanent cells vary in shape and size and, usually, do not contain sufficient amounts of secretory granules for a consistent detection under phase contrast. Granules are much less abundant in the fl cell clones presently available than in control primary fl cells and in the tumors from which permanent fl cell lines have been derived (Fig. 1). Conventional electron microscopy is also of limited value since the secretory granules of most cloned fl cells vary in morphology and are often rather different from typical fl granules (Fig. 3). 27 D. J. Gross, L. Villa-Komaroff, C. R. Kahn, G. C. Weir, and P. A. Halban, J. Biol. Chem. 264, 21486 (1989). 2s G. Gold, M. D. Walker, D. L. Edwards, and G. M. Grodsky, Diabetes 37, 1509 0988). 29 K. W. N& P. R. Gummer, B. L. Grills, V. P. Michelangeli, and M. E. Dunlop, J. Endocrinol. 113, 3 0987). 30 I. Matsuba, M. Narimiya, H. Yamada, Y. Ikcda, T. Tanesc, M. Abe, and H. Ishikawa, JikeikaiMed. J. 28, 257 (1981).
FIo. 1. Identification of insulin-containing cells in a transplantable islet cell tumor, induced by X-ray irradiation of NEDH rats. Immunostaining of the tumor reveals the presence of numerous insulin-containing cells which occur in clusters or as scattered single cells among abundant connective tissue (a). At the electron microscope level, most of these cells contain numerous typical fl secretory granules (b) and show an ultrastructural organization analogous to that of normal fl cells. [(a) Bar = 100/~m; (b) bar ffi 1/~m.]
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G A S T R O I N T E S T I NSYSTEM AL
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Flo. 2. Phase-contrast (a) and scanning electron microscopy(b) showingcharacteristic clusters of insulin-producingcells from the RIN-SF clone, under conventionalculture conditions. [(a) Bar -- 100/lm; (b) barffi 20/~m.]
Furthermore, since these few granules are heterogeneously distributed among cells, a large number of sections should be screened to obtain a fair evaluation of the actual granularity of the whole cell population. I m m u n o labeling for insulin is instrumental in addressing this question morphologically. However, due to the poor granularity o f f l cell clones, this approach
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FIG. 3. Ultrastructural organization of an RIN-5F cell. (a) Secretory granules are scarce in these cells and rarely (arrowheads) show the characteristic clear halo of typical p granules. (b) Insulin is immunolocalized by gold particles in a population of small, halo-devoid secretory granules of an RIN-5F cell. (Bars = 0.5 gm.)
inconsistently labels only a few cells at the fight microscope level and, thus, should be performed at the ultrastructural level (Fig. 3). The growth rate of cloned fl cells is usually higher than that of primary fl cells. Thus, periodic morphological control shows readily the progressive growth of cloned fl cells in clusters (Fig. 2) and a high frequency of mitosis. The growth rate offl cell clones varies quite extensively, depending on the culture substratum, 1,2which also appears able to modify cell secretion.
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Biochemistry Short-Term Insulin Release. Immunoreactive insulin release can be studied from insulin-producing cell lines just as from native fl cells in monolayer cultures. The procedures are similar and the reader is referred to Chapter [13], this volume. Note, however, that the cellular insulin content of insulin-producing cell lines is usually less than 1% that of native p cells and that fractional insulin release is often much higher (basal release of 10% of cellular insulin content/hr is not unusual). A typical protocoP ° for RINm5F cells would be as follows (for,buffer recipes, see Appendix, Chapter [ 13], this volume). RIN cells are seeded into 24-well test plates at a density of 2 × 105 cells/ml in 1 ml RPMI 1640, 10% fetal calf serum. The medium is changed after 3 days and insulin release studied 1 day later. The cells are washed three times with 1 ml KRB-HEPES-BSA, 2.8 mMglucose, and then preincubated in this buffer for 15 min at 37*. Insulin release is then followed by incubating the cells in 1 ml KRB-HEPES-BSA, with selected addition of secretagogues, for 30-60 min at 37 °. The incubation buffer is taken and centrifuged at 2-4* for 10 rain at 150 g to pellet any cells which may have detached from the wells during the incubation. This supernantant is kept for radioimmunoassay of insulin. Acid ethanol (1 ml) is added to the cells to extract cellular insulin (leave at 2-4* overnight, then decant the acid ethanol and store it at -20* in stoppered tubes). Cellular DNA may be measured after extracting insulin in this way. Insulin secretion can also be measured from cells in suspension by performing either static incubations or perifusions. In both cases the cells are detached from the culture vessel with trypsin as for routine passaging, washed, and transferred to a 100-ml volume spinner culture flask. We use RPMI 1640 medium with the same composition as for culture, except for the use of 1% newborn calf serum to minimize cell dumping. The usual cell concentration is around 1-2 × 106 cells/ml. The spinner culture is performed at 37 ° for 3 hr, a time considered sufficient to allow the recovery from trypsin treatment of cell surface structures. For static incubation the cells are then washed and resuspended in an appropriate volume of KRB-HEPES-BSA buffer and distributed into siliconized 3-ml glass or plastic tubes. The concentration must be adapted to the cellular content of insulin, but for RINm5F cells we use approximately 0.5 × 106 cells/ml and an incubation volume of 1 ml. Usually the cells are preincubated for 15- 30 min under basal conditions followed by centrifugation at room temperature (150 g, 5 min). The supernatant is discarded. Alternatively only 0.8 ml is removed and kept at - 2 0 * for measurement of immunoreactive insulin. The cells are then resuspended in the various test
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solutions and reincubated at 37* for 10-30 min. The incubation is stopped by chilling the tubes, followed by centrifugation in the cold, decantation, and addition of 1 ml of acid ethanol to the pellet for the assessment of cellular hormone content. The partial removal of the preincubation medium is less perturbing to the cells, but requires the correction of the secretion during the incubation by subtraction of the hormone remaining in the tube from the preincubation,al For perifusion, the cells are resuspended in KRB-HEPES-BSA to yield about 2 - 1 0 × 6 cells/ml. They are placed in the perifusion chamber (chamber volume 700 #1). Usually the cells are perifused for 30-45 min under basal conditions followed by a 15- to 30-min stimulation period. The basal buffer is then reintroduced to verify that no drift in baseline secretion has occurred. 32 This procedure is very similar to that described for the perifusion of isolated islets. 33 It should be stressed that it is essential to keep the temperature of the incubations and perifusions at 37* for optimal secretory responsiveness. Alternative methods, including the perifusion of cells in small BioGel P 2 columns34 (Bio-Rad, Richmond, CA) or perifusion of cells attached to glass coverslips,a5 have been reported. Insulin Biosynthesis. Proinsulin biosynthesis and the conversion of proinsulin to insulin may be studied in cell lines in much the same way as in native islet cells (see [13], this volume). The major difference lies in the amount of proinsulin synthesized per unit time relative to total, noninsulin-related, proteins. For native fl cells this value can reach -50% over a short pulse-label period (i.e., not exceeding 30 min) at high glucose. Such is not the case even for the best differentiated cell lines, where values of 1% or less would be typical. This means that cell extracts can normally not be analyzed directly by reversed-phase high-pressure liquid chromatography (HPLC). Rather, it is customary to first immunoprecipitate radioactively labeled products using anti-insulin serum and Protein A-Sepharose as described in detail in Ref. 36. The precipitated products are then displaced from the immune complex and analyzed by HPLC. 25,27,37 31S. Ullrich and C. B. Wollheim, Mol. Pharmacol. 28, 100 (1985). 32C. B. Wollheim, S. Ullrich, and T. Pozzan, FEBSLett. 177, 17 (1984). 33M. Kikuchi, A. Rabinovitch, W. G. Blackard, and A. E. Renold, Diabetes 23, 550 (1974). 34p. Knudsen, H. Kofod, A. Lemmark, and C. J. Hedeskov, Endocrinol. Metab. 8, E338 (1983). 35R. S. Hill and A. E. Boyd III, Diabetes 34, 115 (1985). P. A. Halban and C. B. Wollheim, J. Biol. Chem. 255, 6003 (1980). 37D. Gross, A. Skvorak, G. Hendrick, G. C. Weir, L. Villa-Komaroff, and P. Halban, FEBS Lett. 241, 205 (1988).
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Differentiated Function of/~ Cell Lines Despite the obvious advantages of having an unlimited source of insulin-producing cells, there are a number of limitations to the use of such lines as a valid model for native p cell function. It is clearly beyond the scope of this chapter to list all of the similarities and differences found between the various lines available and their native p cell counterparts. The individual investigator will always be obliged to make the appropriate comparison using his or her particular line and analytical approaches. In general terms, however, it is perhaps useful to note the following. The native p cell from most adult animals (but not from ruminants) responds to a rise in ambient glucose with a marked increase in insulin release. This stimulation displays an apparent Km of 7 - 8 m M glucose, and reaches a maximum at - 1 5 raM. Such is not the case for any known insulin-producing cell line. Thus, the RIN cell responds (if at all) with increased secretion only when glucose is raised from 0 to 2 raM. 1° The HIT cell originally responded in the glucose range of 2 - 7 m M 22 but seems to have changed its characteristics in many laboratories where a pronounced shift of the glucose dose-response curve to the left has been reported. 3s Furthermore, it has become necessary to preincubate HIT cells in the absence of glucose (e.g., 30 rain) in order to observe a subsequent stimulation of insulin release. 3s Another striking difference between p cell lines and native ~ cells is their insulin content. The highest values observed (250-1000 ng/106 cells) in RIN and HIT cells correspond to - 0 . 5 - 2 % that of native cells. At the other extreme, some less-well-differentiated lines have been studied and insulin cell contents as low as 0.01% of a native ~ cell have been reported. We have found that monitoring cellular insulin content at regular intervals is a useful way of ensuring maintained differentiated function. Although least marked in RIN cells (and most pronounced in HIT and B-TC cells), all lines studied by us have shown sudden and precipitous declines in insulin content as a function of tissue culture passage number. One possible use o f t cell lines is as a target for measuring anti-islet cell antibodies in the serum of diabetic patients. 39 Here again it is important to note that there are considerable differences in the surface features of these cells compared with native cells4° and, as for insulin content and sensitivity to secretagogues, these features also change with time in culture. 39 3s M. D. Meglasson, C. D. Manning, H. Najafi, and F. M. Matschinsky, Diabetes 36, 477
(1987). 39 j. W. Thomas, V. J. Virta, and L. J. Nell, J. Immunol. 138, 2896 (1987). 4o p. A. Halban, S. L. Powers, K. L. George, and S. Bonner-Weir, Endocrinology 123, 113 (1988).
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Finally, even apparently well-defined lines (such as the RIN lines) display remarkable plasticity in differentiated function. Indeed, there are RINm5F sublines which produce more glucagon than insulin, despite their having been selected initially8 for their preferential insulin production. Attempts to improve the differentiated status of RIN cells by exposing them to sodium butyrate have met with limited success. Thus, some RIN cells respond to butyrate with increased levels of insulin production4~ and with better expression of typical fl cell surface antigens42 whereas other sublines do not? 3 It would appear as though only poorly differentiated RIN cell sublines will respond to any extent to this particular agent.43
Acknowledgments We thank Dr. Benigna Blondel for invaluable help in the preparation of this manuscript. This work was supported by Grants from the Swiss National Science Foundation Nos. 32.25665.88 (C.B.W.), 31-26625.89 (P.M.), 31-9394.88 (P.A.H.), and Grant No. 187384 from the Juvenile Diabetes Foundation International (P.M.), a grant from the Greenwall Foundation (P.A.H.), and Grant No. DK-35292 from the National Institutes of Health (P.A.H.). 41 j. Philippe, D. J. Drucker, W. L. Chick, and J. F. Habener, Mol. Cell. Biol. 7, 560 (1987). 42 R. K. Bartholomensz, I. L. Campbell, and L. C. Harrison, Endocrinology 124, 2680 (1989). 43 S. L. Gardner, F. Dotta, R. C. Nayak, K. L. George, G. S. Eisenbarth, and P. A. Halban, Diabetes Res. 12, 93 (1989).
[ 15] M e m b r a n e P o t e n t i a l M e a s u r e m e n t s in P a n c r e a t i c fl C e l l s w i t h I n t r a c e l l u l a r M i c r o e l e c t r o d e s B y HANS PETER MEISSNER
Introduction In 1968 it was reported for the first time that glucose and other insulin secretagogues induce membrane depolarization and trigger electrical activity in pancreatic fl cells, mIn early electrophysiological studies the membrane potential offl cells was found to be low. ~ Later, with the improvement of the microelectrode technique more negative membrane potentials were measured, similar to those known from nerve or muscle.3,4 P. M. Dean and E. K. Matthews, Nature (London) 219, 389 (1968). 2 p. M. Dean and E. IC Matthews, J. Physiol. (London) 210, 255 (1970).
METHODS IN ENZYMOI.L~Y, VOL. 192
Copyright © 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.
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Acid ethanol: Ethanol (absolute): 750 ml Concentrated HCI: 15 ml Distilled H 2 0 : 2 3 5 ml Keep in a tightly closed bottle at 2 - 4 °. Acknowledgments We thank Dr. Benigna Blondel for invaluable help in the preparation of this manuscript. This work was supported by Grants from the Swiss National Science Foundation Nos. 32-25665.88 (C.B.W.), 31-26625.89 (P.M.), 31-9394.88 (P.A.H.), Grant No. 187384 from the Juvenile Diabetes Foundation International (P.M.), a grant from the Greenwall Foundation (P.A.H.), and Grant No. DK-35292 from the National Institutes of Health (P.A.H.)
[14] E s t a b l i s h m e n t
and Culture of Insulin-Secreting fl C e l l L i n e s
By CLAES B. WOLLHEIM, PAOLO MEDA, and PHILIPPE A. HALBAN
In order to study the molecular mechanism of insulin production a very large number of highly purified pancreatic fl cells is required. The most successful methods for isolating islets from large mammals, including man (see [ 13], this volume) can provide up to 200,000 islets/pancreas. The procedures are, however, time consuming and costly. This number of islets consists of some 5 X l0 s cells, a modest number for detailed biochemical analysis. Only approximately 75% are fl cells, and there is furthermore a significant contamination with exocrine cells. These major obstacles to the study of fl cell function have been overcome in recent years by the use of insulin-producing cell lines. The aim of this chapter is to describe the various types of lines available, as well as their functional characteristics. Lines Derived from Naturally Occurring or Induced Insulinomas
Naturally Occurringlnsulinomas Only a limited number of cell fines have been derived from spontaneously occurring insulinomas of either animal or human sources. Typically, the tumor is dissected out of the pancreas and then digested with collagenase with (or without) the addition of trypsin. The tumor cells are then METHODS IN ENZYMOL(X3Y, VOL. 192
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allowed to grow in tissue culture. The major problem encountered in virtually all attempts to obtain a permanent insulin-producing line in this way is that the cells dedifferentiate (as manifested by a dramatic decrease in cellular insulin content) rather rapidly. There appears to be an inverse relationship between proliferative capacity and differentiated status. There is no reproducible method available for selecting cells with maintained, differentiated function, although approaches including the use of extracellular matrices ',2 have been tried. It is, however, possible on occasion to maintain or restore, at least in part, differentiated function by inducing tumor growth during the in vivo passage of the cells. 3,4 The general view remains, however, that obtaining a well-differentiated line with useful in vitro proliferative capacity depends largely upon chance. Indeed, to our knowledge no human cell line fulfilling these criteria on a long-term basis exists. Despite these reservations, various groups have used insulinoma cells of both human 5-7 and animal3 ,s-'° origin for their studies. Induced Tumors
Insulinomas have been induced in the rat by either radiation" or by the combination of streptozotocin and nicotinamide.'2.'3 The tumor cells have then been used to establish cell lines. It must again be stressed that, as with
C. H. Thivolet, P. Chatelain, P. Nicoloso, A. Durand, and J. Bertrand, Exp. Cell Res. 159, 313 (1985). 2 R. Muschel, G. Khoury, and L. M. Reid, Mol. Cell. Biol. 6, 337 (1986). 3 p. A. Rae, C. C. Yip, and B. P. Schimmer, Can. J. Physiol. Pharmacol. 57, 819 (1979). 4 p. R. Hart, M. G. DeSilva, S. K. Swanston-Flau, C. J. Powdl, and V. Marks, J. Endocrinol. 118, 429 (1988). 5 W. L. Chick, V. Lauds, J. S. Soeldner, M. H. Tan, and M. Gfinsberg, Metabolism 22, 1217 (1973). 6 K. Adcock, M. Austin, W. C. Duckworth, S. S. Solomon, and L. R. Murrell, Diabetologia 11,527 (1975). 7 C. H. Thivolet, A. Demidem, M. Haftek, A. Durand, and J. Bertrand, Diabetes 37, 1279 (1988). s A. F. Gazdar, W. L. Chick, H. K. Oie, H. L. Sims, D. L. King, G. C. Weir, and V. Lauds, Proc. Natl. Acad. Sci. U.S.A. 77, 3519 (1980). 9 C. A. Carrington, E. D. Rubery, E. C. Pearson, and C. N. Hales, J. Endocrinol. 109, 193, (1986). 1oG. A. Praz, P. A. Halban, C. B. Wollheim, B. Blondel, A. J. Strauss, and A. E. Renold, Biochem. J. 210, 345 (1983). '~ W. L. Chick, W. Shields, R. N. Chute, A. A. Like, V. Lauds, and K. C. Kitchen, Proc. Natl. Acad. Sci. U.S.A. 74, 628 (1977). ,2 W. L. Chick, M. C. Appel, G. C. Weir, A. A. Like, V. Lauds, J. G. Porter, and R. N. Chute, Endocrinology 107, 954 (1980). ,3 p. Masiello, C. B. Wollheim, B. Blondel, and A. E. Renold, Diabetologia 24, 30 (1983). '
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the use of spontaneously occurring insulinomas (see above), there is no guarantee that a given tumor will provide a useful line. The main, but not the only, 12 exception to this rule has been an X-ray-induced rat insulinoma. 8 This tumor can be maintained by in vivo passage (serial transplantation) in inbred (NEDH) rats. H Several tumors derived from the original one now exist. They differ in their rate of growth and, typically, those which grow the slowest display the highest degree of differentiation. There has also been a suggestion that growing the tumor under the kidney capsule, rather than subcutaneously (the more conventional method), may further favor differentiation. 14 The first cell lines derived from this insulinoma were the RIN cells. These lines were established by Chick and associates by growing isolated tumor cells in culture. 8 These investigators deserve credit for their perseverance since their initial attempts were unsuccessful. The RIN cells have indeed provided many groups with a useful source of insulin-producing cells for a variety of purposes. The most commonly used subline is RINm5F. This line was initially selected since it had a relatively high insulin content, with nondetectable amounts of glucagon and somatostatin.S, 15
Culture and Characteristics of RINm5F Cells and Derived Sublines These cells are grown in RPMI 1640 (11 mMgiueose) medium supplemented with 10% fetal calf serum (FCS). Typically, cells are seeded into plastic culture vessels at a concentration of 2 X 105 cells/ml. The medium is changed after 3 and 6 days, and the cells trypsinized after l week. For this purpose the cells are rinsed with Ca+/Mg2+-free phosphate-buffered saline and then exposed to 0.025% (w/v) trypsin (1:250; Difco, Detroit, MI) and 0.27 m M EDTA in the same buffer for 1 - 3 rain at 37 °. The cells are dislodged from the culture surface by tapping the side of the vessel, and are then washed in medium before being reseeded. In order to maintain the functional status of RIN cells, it is essential that they be exposed to no more than 15-20 tissue culture transfers. Beyond this number there is an ever-increasing chance of the cells suddenly and unpredictably showing a dramatic loss of insulin content and responsiveness to secretagogues. It is unclear what causes this undesirable loss of function, but this phenomenon is by no means unique to RIN cells. Because of this limitation in passage number, it is important to have an adequate reserve of cells in storage. For ~4M. Hoenig and G. W. G. Sharp, Endocrinology119, 2502 (1986). ~5H. K. Oie, A. F. Gazdar, J. D. Minna, G. C. Weir, and S. B. Baylin, Endocrinology112, 1070 0983).
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this purpose, cells are suspended in phosphate-buffered saline or complete culture medium (10% FCS), with the addition of 10% dimethyl sulfoxide (DMSO) (4-6 X 107 cells/ml) and the temperature then progressively decreased before storage in liquid nitrogen. The cells are thawed by rapidly raising the temperature to 37 °, and then transferring them to RPMI 1640 medium supplemented with 20% FCS (the seeding density should be -106 cells/ml). As soon as the cells have attached to the culture surface, the FCS concentration may be decreased to 10%. When handled as described, the subclone of RINm5F used in our laboratories (RINm5F-2A) has maintained apparently unchanged function for over 7 years/°,16 This subline was established by cloning RINm5F cells using the limiting dilution method (i.e., by allowing colony formation fro.m a single cell). For RIN-r cells, another RIN cell clone, culture in chemically defined medium in the absence of serum was found to sustain cell proliferation? 7
Lins Derivedfrom Tumors in Transgenic Mice Transgenic mice expressing simian virus 40 (SV40) T antigen uniquely in the pancreatic p-cell have been derived by injection into a mouse embryonic pronucleus of a fusion gene construct consisting of the structural region of the SV40 T antigen gene driven by the insulin promoter? 8 These animals develop insulinomas and a cell line (B-TC cells) has been derived from such an insulinoma? 9 An alternative approach has been to use a gene construct allowing for random tissue expression of T antigen in the transgenic mice. In this case an insulinoma was also found (although not all mice had such tumors) and a cell line again prepared (IgSV195 cells).2° Such mouse fl cell lines have been less extensively studied than the RIN (see above) or HIT (see below) cell lines. This is due in part to the fact that they have only recently been described. They may be grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS and 25 m M glucose, using the same basic culture techniques described for RIN cells.
16C. B. Woltheim, M. J. Dunne, B. Peter-Riesch, R. Bruzzone, T. Pozzan, and O. H. Petersen, EMBO J. 7, 2443 (1988). 17H. K. W. Fong, W. L. Chick, and G. H. Sato, Diabetes 30, 1022 (1981). is D. Hanahan, Nature (London) 315, 115 (1985). 19S. Efrat, S. Linde, H. Kofold, D. Spector, M. Delannoy, S. Grant, D. Hanahan, and S. Baekkeskov, Proc. Natl. Acad. S¢i. U.S.A. 85, 9037, 1988. 2oA. GiUigan, L. Jewett, D. Simon, I. Damjanov, F. M. Matschinsky, H. Weik, C. Pinkert, and B. B. Knowles, Diabetes 38, 1056 (1989).
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Lines Obtained by Viral Transformation o f t Cells Primary cultures of pancreatic endocrine cells (see [13], this volume) have been transformed with SV40 and clonal cell lines obtained. 21,22One line in particular, HIT cells, derived from SV40 infection and transformation of Syrian hamster t-cells, 22 has proved useful for studies on fl cell function. HIT cells (and in particular the T 15 subclone), are maintained and grown in tissue culture in much the same way as RIN cells (see above) with the following differences. The culture medium originally recommended was F-12 containing 15% horse serum and 2.5% FCS, with the addition of l0 gg/ml glutathione, 0.1 g M selenous acid. 22 We now use RPMI 1640 (ll m M glucose) supplemented with 10% FCS, and again containing 0.1 # M selenous acid. As for the other cell lines described, HIT cells display a marked phenotypic alteration with time in culture,23 and we therefore strongly recommend that an adequate stock of early passage number cells be stored in liquid nitrogen and that the cells not be exposed to more than 10-20 tissue culture passages. Transfection of Secretory Cells with the Insulin Gene Transformed secretory cells have been transfected with the insulin gene driven by a viral promoter,u-2~ In order to facilitate the selection of stable transfectants, we transfect cells with a construct (DOL vector) carrying the insulin gene driven by the murine leukemia virus long terminal repeat, colinear with the neomycin resistance gene.25 The transfected cells are grown in G418 (geneticin) at a concentration toxic 075 gg/ml) to cells not expressing the neomycin resistance gene. Of these G418-resistant clones, approximately 30% were found to produce detectable levels of immunoreactive insulin. The transfection of noninsulin-producing cells with the insulin gene is, typically, useful for studying the mechanism and regulation of insulin expression. It furthermore allows for the expression of mutant insulin
21 E. J. Niesor, C. B. Wollheim, D. H. Mintz, B. Blondel, A. E. Renold, and R. Weft, Biochem. J. 178, 559 (1979). 22R. F. Santerre, R. A. Cook, R. M. D. Crisel, J. D. Sharp, R. J. Schmidt, D. C. Williams, and C. P. Wilson, Proc. Natl. Acad. Sci. U.S.A. 78, 4339 (1981). 23H.-J. Zhang, T. F. Walseth, and R. P. Robertson, Diabetes 38, 44 (1989). 24H.-P. Moore, M. D. Walker, F. Lee, and R. B. Kelly, Ce1135, 531 0983). 25D. J. Gross, P. A. Halban, C. R. Kahn, G. C. Weir, and L. Villa-Komaroff, Proc. Natl. Acad. Sci. U.S.A. 86, 4107 (1989). 26S. K. Powell, L. Orci, C. S. Craik, and H.-P. H. Moore, J. CellBiol. 106, 1843 0988).
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The cell fine most frequently used for such studies is the AtT20 (mouse pituitary corticotroph) transformed fine. These cells may be grown in F-10 medium supplemented with 15% horse serum and 2.5% FCS. We have found that cell growth is favored by raising the glucose concentration to 25 mM. Other cell types have been used for transfection studies, and these include HIT cells.2s g e n e s . 2s-27
Other Approaches to Obtaining Insulin-Producing Cell Lines There have been attempts to obtain cell fines from cultures of B cells of neonatal29 or fetal 3° origin. This approach depends upon the random and spontaneous outgrowth of cells displaying peculiarly elevated proliferative capacity (possibly protoditferentiated stem cells). No stable cell fine has yet become available using this approach. Analytical Methods for Studying Insulin-Producing Cell Lines
Morphology of fl Cell Clones The identification of insulin-containing cells in a clone or fine cannot be made by the mere observation of living cultures since permanent cells vary in shape and size and, usually, do not contain sufficient amounts of secretory granules for a consistent detection under phase contrast. Granules are much less abundant in the fl cell clones presently available than in control primary fl cells and in the tumors from which permanent fl cell lines have been derived (Fig. 1). Conventional electron microscopy is also of limited value since the secretory granules of most cloned fl cells vary in morphology and are often rather different from typical fl granules (Fig. 3). 27 D. J. Gross, L. Villa-Komaroff, C. R. Kahn, G. C. Weir, and P. A. Halban, J. Biol. Chem. 264, 21486 (1989). 2s G. Gold, M. D. Walker, D. L. Edwards, and G. M. Grodsky, Diabetes 37, 1509 0988). 29 K. W. N& P. R. Gummer, B. L. Grills, V. P. Michelangeli, and M. E. Dunlop, J. Endocrinol. 113, 3 0987). 30 I. Matsuba, M. Narimiya, H. Yamada, Y. Ikcda, T. Tanesc, M. Abe, and H. Ishikawa, JikeikaiMed. J. 28, 257 (1981).
FIo. 1. Identification of insulin-containing cells in a transplantable islet cell tumor, induced by X-ray irradiation of NEDH rats. Immunostaining of the tumor reveals the presence of numerous insulin-containing cells which occur in clusters or as scattered single cells among abundant connective tissue (a). At the electron microscope level, most of these cells contain numerous typical fl secretory granules (b) and show an ultrastructural organization analogous to that of normal fl cells. [(a) Bar = 100/~m; (b) bar ffi 1/~m.]
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230
G A S T R O I N T E S T I NSYSTEM AL
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Flo. 2. Phase-contrast (a) and scanning electron microscopy(b) showingcharacteristic clusters of insulin-producingcells from the RIN-SF clone, under conventionalculture conditions. [(a) Bar -- 100/lm; (b) barffi 20/~m.]
Furthermore, since these few granules are heterogeneously distributed among cells, a large number of sections should be screened to obtain a fair evaluation of the actual granularity of the whole cell population. I m m u n o labeling for insulin is instrumental in addressing this question morphologically. However, due to the poor granularity o f f l cell clones, this approach
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FIG. 3. Ultrastructural organization of an RIN-5F cell. (a) Secretory granules are scarce in these cells and rarely (arrowheads) show the characteristic clear halo of typical p granules. (b) Insulin is immunolocalized by gold particles in a population of small, halo-devoid secretory granules of an RIN-5F cell. (Bars = 0.5 gm.)
inconsistently labels only a few cells at the fight microscope level and, thus, should be performed at the ultrastructural level (Fig. 3). The growth rate of cloned fl cells is usually higher than that of primary fl cells. Thus, periodic morphological control shows readily the progressive growth of cloned fl cells in clusters (Fig. 2) and a high frequency of mitosis. The growth rate offl cell clones varies quite extensively, depending on the culture substratum, 1,2which also appears able to modify cell secretion.
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Biochemistry Short-Term Insulin Release. Immunoreactive insulin release can be studied from insulin-producing cell lines just as from native fl cells in monolayer cultures. The procedures are similar and the reader is referred to Chapter [13], this volume. Note, however, that the cellular insulin content of insulin-producing cell lines is usually less than 1% that of native p cells and that fractional insulin release is often much higher (basal release of 10% of cellular insulin content/hr is not unusual). A typical protocoP ° for RINm5F cells would be as follows (for,buffer recipes, see Appendix, Chapter [ 13], this volume). RIN cells are seeded into 24-well test plates at a density of 2 × 105 cells/ml in 1 ml RPMI 1640, 10% fetal calf serum. The medium is changed after 3 days and insulin release studied 1 day later. The cells are washed three times with 1 ml KRB-HEPES-BSA, 2.8 mMglucose, and then preincubated in this buffer for 15 min at 37*. Insulin release is then followed by incubating the cells in 1 ml KRB-HEPES-BSA, with selected addition of secretagogues, for 30-60 min at 37 °. The incubation buffer is taken and centrifuged at 2-4* for 10 rain at 150 g to pellet any cells which may have detached from the wells during the incubation. This supernantant is kept for radioimmunoassay of insulin. Acid ethanol (1 ml) is added to the cells to extract cellular insulin (leave at 2-4* overnight, then decant the acid ethanol and store it at -20* in stoppered tubes). Cellular DNA may be measured after extracting insulin in this way. Insulin secretion can also be measured from cells in suspension by performing either static incubations or perifusions. In both cases the cells are detached from the culture vessel with trypsin as for routine passaging, washed, and transferred to a 100-ml volume spinner culture flask. We use RPMI 1640 medium with the same composition as for culture, except for the use of 1% newborn calf serum to minimize cell dumping. The usual cell concentration is around 1-2 × 106 cells/ml. The spinner culture is performed at 37 ° for 3 hr, a time considered sufficient to allow the recovery from trypsin treatment of cell surface structures. For static incubation the cells are then washed and resuspended in an appropriate volume of KRB-HEPES-BSA buffer and distributed into siliconized 3-ml glass or plastic tubes. The concentration must be adapted to the cellular content of insulin, but for RINm5F cells we use approximately 0.5 × 106 cells/ml and an incubation volume of 1 ml. Usually the cells are preincubated for 15- 30 min under basal conditions followed by centrifugation at room temperature (150 g, 5 min). The supernatant is discarded. Alternatively only 0.8 ml is removed and kept at - 2 0 * for measurement of immunoreactive insulin. The cells are then resuspended in the various test
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solutions and reincubated at 37* for 10-30 min. The incubation is stopped by chilling the tubes, followed by centrifugation in the cold, decantation, and addition of 1 ml of acid ethanol to the pellet for the assessment of cellular hormone content. The partial removal of the preincubation medium is less perturbing to the cells, but requires the correction of the secretion during the incubation by subtraction of the hormone remaining in the tube from the preincubation,al For perifusion, the cells are resuspended in KRB-HEPES-BSA to yield about 2 - 1 0 × 6 cells/ml. They are placed in the perifusion chamber (chamber volume 700 #1). Usually the cells are perifused for 30-45 min under basal conditions followed by a 15- to 30-min stimulation period. The basal buffer is then reintroduced to verify that no drift in baseline secretion has occurred. 32 This procedure is very similar to that described for the perifusion of isolated islets. 33 It should be stressed that it is essential to keep the temperature of the incubations and perifusions at 37* for optimal secretory responsiveness. Alternative methods, including the perifusion of cells in small BioGel P 2 columns34 (Bio-Rad, Richmond, CA) or perifusion of cells attached to glass coverslips,a5 have been reported. Insulin Biosynthesis. Proinsulin biosynthesis and the conversion of proinsulin to insulin may be studied in cell lines in much the same way as in native islet cells (see [13], this volume). The major difference lies in the amount of proinsulin synthesized per unit time relative to total, noninsulin-related, proteins. For native fl cells this value can reach -50% over a short pulse-label period (i.e., not exceeding 30 min) at high glucose. Such is not the case even for the best differentiated cell lines, where values of 1% or less would be typical. This means that cell extracts can normally not be analyzed directly by reversed-phase high-pressure liquid chromatography (HPLC). Rather, it is customary to first immunoprecipitate radioactively labeled products using anti-insulin serum and Protein A-Sepharose as described in detail in Ref. 36. The precipitated products are then displaced from the immune complex and analyzed by HPLC. 25,27,37 31S. Ullrich and C. B. Wollheim, Mol. Pharmacol. 28, 100 (1985). 32C. B. Wollheim, S. Ullrich, and T. Pozzan, FEBSLett. 177, 17 (1984). 33M. Kikuchi, A. Rabinovitch, W. G. Blackard, and A. E. Renold, Diabetes 23, 550 (1974). 34p. Knudsen, H. Kofod, A. Lemmark, and C. J. Hedeskov, Endocrinol. Metab. 8, E338 (1983). 35R. S. Hill and A. E. Boyd III, Diabetes 34, 115 (1985). P. A. Halban and C. B. Wollheim, J. Biol. Chem. 255, 6003 (1980). 37D. Gross, A. Skvorak, G. Hendrick, G. C. Weir, L. Villa-Komaroff, and P. Halban, FEBS Lett. 241, 205 (1988).
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Differentiated Function of/~ Cell Lines Despite the obvious advantages of having an unlimited source of insulin-producing cells, there are a number of limitations to the use of such lines as a valid model for native p cell function. It is clearly beyond the scope of this chapter to list all of the similarities and differences found between the various lines available and their native p cell counterparts. The individual investigator will always be obliged to make the appropriate comparison using his or her particular line and analytical approaches. In general terms, however, it is perhaps useful to note the following. The native p cell from most adult animals (but not from ruminants) responds to a rise in ambient glucose with a marked increase in insulin release. This stimulation displays an apparent Km of 7 - 8 m M glucose, and reaches a maximum at - 1 5 raM. Such is not the case for any known insulin-producing cell line. Thus, the RIN cell responds (if at all) with increased secretion only when glucose is raised from 0 to 2 raM. 1° The HIT cell originally responded in the glucose range of 2 - 7 m M 22 but seems to have changed its characteristics in many laboratories where a pronounced shift of the glucose dose-response curve to the left has been reported. 3s Furthermore, it has become necessary to preincubate HIT cells in the absence of glucose (e.g., 30 rain) in order to observe a subsequent stimulation of insulin release. 3s Another striking difference between p cell lines and native ~ cells is their insulin content. The highest values observed (250-1000 ng/106 cells) in RIN and HIT cells correspond to - 0 . 5 - 2 % that of native cells. At the other extreme, some less-well-differentiated lines have been studied and insulin cell contents as low as 0.01% of a native ~ cell have been reported. We have found that monitoring cellular insulin content at regular intervals is a useful way of ensuring maintained differentiated function. Although least marked in RIN cells (and most pronounced in HIT and B-TC cells), all lines studied by us have shown sudden and precipitous declines in insulin content as a function of tissue culture passage number. One possible use o f t cell lines is as a target for measuring anti-islet cell antibodies in the serum of diabetic patients. 39 Here again it is important to note that there are considerable differences in the surface features of these cells compared with native cells4° and, as for insulin content and sensitivity to secretagogues, these features also change with time in culture. 39 3s M. D. Meglasson, C. D. Manning, H. Najafi, and F. M. Matschinsky, Diabetes 36, 477
(1987). 39 j. W. Thomas, V. J. Virta, and L. J. Nell, J. Immunol. 138, 2896 (1987). 4o p. A. Halban, S. L. Powers, K. L. George, and S. Bonner-Weir, Endocrinology 123, 113 (1988).
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Finally, even apparently well-defined lines (such as the RIN lines) display remarkable plasticity in differentiated function. Indeed, there are RINm5F sublines which produce more glucagon than insulin, despite their having been selected initially8 for their preferential insulin production. Attempts to improve the differentiated status of RIN cells by exposing them to sodium butyrate have met with limited success. Thus, some RIN cells respond to butyrate with increased levels of insulin production4~ and with better expression of typical fl cell surface antigens42 whereas other sublines do not? 3 It would appear as though only poorly differentiated RIN cell sublines will respond to any extent to this particular agent.43
Acknowledgments We thank Dr. Benigna Blondel for invaluable help in the preparation of this manuscript. This work was supported by Grants from the Swiss National Science Foundation Nos. 32.25665.88 (C.B.W.), 31-26625.89 (P.M.), 31-9394.88 (P.A.H.), and Grant No. 187384 from the Juvenile Diabetes Foundation International (P.M.), a grant from the Greenwall Foundation (P.A.H.), and Grant No. DK-35292 from the National Institutes of Health (P.A.H.). 41 j. Philippe, D. J. Drucker, W. L. Chick, and J. F. Habener, Mol. Cell. Biol. 7, 560 (1987). 42 R. K. Bartholomensz, I. L. Campbell, and L. C. Harrison, Endocrinology 124, 2680 (1989). 43 S. L. Gardner, F. Dotta, R. C. Nayak, K. L. George, G. S. Eisenbarth, and P. A. Halban, Diabetes Res. 12, 93 (1989).
[ 15] M e m b r a n e P o t e n t i a l M e a s u r e m e n t s in P a n c r e a t i c fl C e l l s w i t h I n t r a c e l l u l a r M i c r o e l e c t r o d e s B y HANS PETER MEISSNER
Introduction In 1968 it was reported for the first time that glucose and other insulin secretagogues induce membrane depolarization and trigger electrical activity in pancreatic fl cells, mIn early electrophysiological studies the membrane potential offl cells was found to be low. ~ Later, with the improvement of the microelectrode technique more negative membrane potentials were measured, similar to those known from nerve or muscle.3,4 P. M. Dean and E. K. Matthews, Nature (London) 219, 389 (1968). 2 p. M. Dean and E. IC Matthews, J. Physiol. (London) 210, 255 (1970).
METHODS IN ENZYMOI.L~Y, VOL. 192
Copyright © 1990 by Academic Press, Inc. All rights ofreproduction in any form reserved.