0073-5655/81/0378-0384 $01.50/0 IN VITROVol. 17, No. 5, May 1981 9 1981TissueCulture Association,Inc.

G L U C A G O N P R O D U C T I O N BY C U L T U R E D P A N C R E A T I C I S L E T S : EFFECTS OF DIFFERENT CULTURE CONDITIONS AND MEDIA ARNE ANDERSSON,' ULF ERIKSSON, ANDCLAES-GORAN ~)STENSON

Department of Medical CellBiology, Universityof Uppsala, Uppsala, Sweden (Received May 12, 1980; accepted August 25, 1980}

SUMMARY Various conditions for tissue culture of collagenase-isolated mouse pancreatic islets were studied for their effects on the glucagon production of the cultured specimens. Culture media containing heat-treated bovine calf serum degraded [~2SI]glucagon to a much less extent than those supplemented with untreated serum. Addition of aprotinin to the heattreated serum gave a further reduction of the [~2~I]glucagon degradation in the culture medium. A similar supplementation of Medium 199, used for culture of isolated islets, resulted in the most extensive glucagon accumulation in the culture medium. Islets cultured free-floating or attached to the bottom of the culture dishes contained similar amounts of glucagon. However, the free-floating islets released less glucagon when tested in short-term experiments performed at the end of the 1 wk culture period. A comparison between different culture media showed that islets cultured in RPMI-1640 had the highest glucagon content and released most glucagon to the culture medium. Moreover, these islets responded most actively to an acute arginine challenge at the end of the culture period. The present data suggest that the optimal conditions for culture of isolated islets aimed at studies of glucagon production may be obtained by using a culture medium consisting of RPMI-1640 supplemented with both a proteinase inhibitor and heat-inactivated serum.

Key words: cultured islets; glucagon production; glucagon degradation. INTRODUCTION The application of culture techniques to the study of the islets of Langerhans has greatly expanded our knowledge on the structure and metabolism of the various islet ceils. By this means it has been possible to maintain in vitro the viability and hormone production of the islets over prolonged periods and to study the effects of various secretagogues and drugs on the islet cells (1). Moreover, storage of islets in tissue culture would make possible the collection of a sufficient number of endocrine cells for transplantation into human diabetics (2). Some essential details of the culture methodology, employed in our laboratory for culture of collagenase-isolated islets, have recently been reconsidered in an attempt to optimize the maintenance of insulin production in the cul-

tured specimens (3L It was found that the best condition for long-term storage of collagenaseisolated islets in culture were obtained when the islets were maintained as free-floating explants in a culture medium consisting of RPMI-1640 supplemented with serum. The present study was designed to determine in a similar way, which conditions were the most favorable for the maintenance of glucagon production in cultured islets.

MATERIALS AND METHODS

Isolation and culture of islets. Pancreatic islets, obtained by a collagenase digestion (Worthington Biochemical Corp., Freehold, N J) technique (4) were prepared from male, adult NMRI-mice (Anticimex, Stockholm, Sweden}, that were starved overnight prior to experimentation. The islets were washed in Hanks' solution before being 'To whom all correspondence should be addressed at transferred, by means of a braking pipette, to Department of Medical Cell Biology, Biomedicum, plastic petri dishes containing tissue culture P.O. Box 571, S-751 23 Uppsala, Sweden. 378

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GLUCAGON AND CULTURED ISLETS media as described in detail below. Two kinds of culture dishes were used: Ca} Dishes especially designed for tissue culture (A/S Nunc, Roskilde, Denmark} that promote attachment of islets when cultured in the presence of serum; (b)Dishes made of ordinary plastic (Heger Plastics, Oslo, Norway, or Linbro multidishes model FB-6-S, Flow Laboratories Ltd., Irvine, Ayrshire, Scotland) that do not permit attachment of cultured islets. The dishes were incubated in a humidified atmosphere of 95% air:5% C02 at + 3 7 ~ C. Attached islets were removed from the petri dishes with a rubber policeman. Culture media. All the culture experiments were performed with serum-supplemented media because culture in the absence of serum has been found to decrease the general survival of islet explants rapidly (1,3,5). Unless otherwise stated, the standard medium used was Medium 199 (Statens Bakteriologiska Laboratorium, Stockholm, Sweden} supplemented with 5 . 5 m M glucose, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Other additions included 10% (vol/vol) bovine calf serum (Statens Bakteriologiska Laboratorium) - - treated at + 5 6 ~ C for 60 min or untreated - - and in some experiments 100/~g/ml aprotinin (Novo Research Institute, Bagswaerd, Denmark). All other culture media were from Flow Laboratories Ltd. To determine the approximate rate of glucagon degradation in the medium during the culture period, tracer amounts of [~2SI]glucagon (Novo Research Institute) were added to the culture dishes (6). An estimate of the glucagon degradation was then obtained from the percentage of the radioactivity (contained in 100/~l culture medium), which was precipitated by 5% (wt/vol) TCA after various incubation times. No islets were present in the culture dishes during these experiments. Glucngon secretion and content. For measurements of glucagon accumulation in the culture media, samples of different media were obtained and frozen at each medium change (Days 3 and 5) and at the end of the 7-d culture period. In these experiments, each dish contained approximately 50 islets. Short-term experiments performed at the end of the culture period tested the effects of glucose or arginine, or both, on the rate of glucagon secretion. Groups of 10 cultured islets were incubated for 120 min at + 3 7 ~ C in small glass vials containing 250/~l Krebs-Ringer bicarbonate buffer supplemented with 10 m M HEPES and 2 mg/ml of bovine albumin. Glucose and arginine were added as given below. Prior to incubation,

each vial was gassed for 30 s with Oz:CO~ (95%:5%L At the end of the incubation period the medium was removed and stored frozen at -20 ~ C before glucagon assay. For determination of islet glucagon content, groups of 10 cultured islets were disrupted ultrasonically for 30 s in 500 ~1 acid ethanol [15 ml 12 M HCI in 70% (vol/vol) ethanol], extracted overnight at + 4 ~ C, .~nd then stored at - 2 0 ~ C. Glucagon assay. Glucagon was determined according to the method of Heding (7), utilizing porcine glucagon standards, [~SI]porcine glucagon, and antiporcine glucagon rabbit serum (K 4023) binding to the N-terminal end of the glucagon molecule lall items were obtained from Novo Research Institute). The radioactivity of the ethanol precipitates was recorded on a punch tape and all subsequent calculations were performed on a programmable desk-top calculator (Compucorp 445 Statistician, Compucorp, Los Angeles, CA) with a Teletype (Teletype Corp., Skokie, IL) as input/ output device. Ten standard triplicate samples in the range of 0 to 20 ng/ml were fitted to a curve with the equation y=

(A x ~ + B CI ~Y' + Ct

where x and y denote glucagon concentration and counts per minute, respectively. Analogous curves have been shown to give a very close fit to RIAstandard values (8). The values of the four constants, A, B, C, and D, were determined in an iterative search using unweighed linear regression analysis. This process halted when the optimal absolute value of the correlation coefficient was obtained, usually in the range of 0.97 to 0.99. In particular, for each step in the search procedure, values for the constants A and B were fixed and values for the constants C and D were calculated from linear regression of the standard samples using log x and log (B-y/y-A) as independent and dependent variables. Finally, the estimation of the individual unknown samples was performed with the aid of the equation x~= C

B-y y-A

Statistical methods. Results are expressed as means • SEM. The significance of differences between means was analyzed by using Student's ttest.

380

ANDERSSON, ERIKSSON, AND OSTENSON RESULTS AND DISCUSSION

Glucagon

degradation

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accumulation.

Figure 1 shows clearly that an extensive degradation of ['25I]glucagon occurred shortly after the start of the incubation performed in the presence of untreated bovine calf serum, with no more than one fourth remaining after 24 h. This finding is consistent with earlier reports of the ability of untreated bovine calf serum to degrade ['25I]insulin (6,9). The latter report further showed that heattreated bovine calf serum had a much less degrading effect, a finding confirmed in the present study of glucagon degradation, and could be further reduced by the addition of the proteinase inhibitor, aprotinin. A further indication of the advantages of adding heat-treated serum and aprotinin to Medium 199 was obtained when the accumulation of native glucagon in the different culture media was estimated (Fig. 2). Thus, at the first medium change after 3 d of culture, the medium supplemented with heat-treated serum and aprotinin contained two and a half times more glucagon than those supplemented with just heat-treated or untreated serum ( P < 0.001L This difference, however, tended to decrease toward the end of the

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FIG. 1. Degradation at +37 ~ C, of ['2SI]glucagon added to Medium 199 supplemented with 10% (vol/voD untreated bovine calf serum (open circles), heatinactivated bovine calf serum (closed circles), or heatinactivated bovine calf serum plus aprotinin, 100 #g/ml (open triangles). Values represent radioactivity precipitated with 5% TCA expressed as percentage of total radioactivity. Each point represents the mean -+ SEM of four experiments.

F1G.2. Glucagon accumulation in different culture media [1 = Medium 199 + 10% (v/v) untreated calf serum; 2 = Medium 199 + 10% (v/v) heat-inactivated bovine calf serum; 3 ----Medium 199 + 10% (v/v) heat-inactivated bovine calf serum and 100 #g/ml aprotinin] during culture of either nonattached or attached islets. Each dish contained approximately 50 islets. Cross-hatched bars represent values obtained from media samples taken on Day 3, stippled bars, Day 5, and open bars, Day 7. Means -+ SEM of six experiments.

GLUCAGON AND CULTURED ISLETS culture period, as did the rate of glucagon secretion to the culture medium. Figure 2 also shows no obvious dif|erence in glucagon accumulation when islets cultured as free-floating explants were compared with attached islets. Short-term experiments. Measurements of glucagon accumulation in the culture medium gave information only on the long-term effects of different alterations of the medium composition. A more detailed characterization of the functional capacity of the cultured specimens can be obtained by incubating the cultured islets for short time periods at the end of the culture period. In our previous study on the insulin production of islets cultured under different conditions we tested the insulin response of the islets when challenged with a high glucose concentration (3). In this study, however, no statistically significant differences in the rate of glucagon secretion of the different groups of cultured islets were observed when incubated at a low or a high glucose concentration (Fig. 3). These results further support the view that isolated mouse pancreatic islets, either noncultured or cultured in Medium 199, are refractive to changes of the extraceUular glucose concentration when incubated in a Krebs-Riuger bicarbonate buffer (10). On the other hand, the

381

islets cultured as free-floating explants released, in general, less glucagon, suggesting that either the harvest procedure or the attachment process as such induces an enhancement of the glucagon secretion. This difference could not be explained by differences in the glucagon content of the islets because the average glucagon content (1 ng/islet) was similar in all groups of islets. Moreover, the different additions to Medium 199 had little or no effect on the glucagon release in the short-term experiments. Effect of different culture media. This series of culture experiments was performed with nonattached islets, in different culture media containing heat-treated serum and aprotinin. Measurements of the glucagon accumulation in the media during the entire culture period showed that all media, with one exception (RPMI-1640) contained similar amounts of glucagon, which tended to decrease with the time of culture (Fig. 4). This was not the case during islet culture in R P M I 1640 because the medium samples taken on the 5th and 7th d of culture contained considerably more glucagon than those of the other media. The difference in glucagon accumulation was most accentuated during the 6th and 7th d of culture when the RPMI-1640 medium contained 13 times

F IG. 3. Glucagon content and release o| islets cultured for 1 wk under the conditions described in Fig. 2. The glucagon release was estimated in short-term incubations (120 min) performed at the end of the culture period in either 1.5 mM ~hatched bars} or 15 mM glucose ~stippled bars). Open bars indicate glucagon content of the islets at the end of the culture period. Means +_ SEM of six experiments, with each release and content value based on duplicate and quadruplicate samples, respectively.

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ANDERSSON, ERIKSSON, AND {)STENSON

FIG. 4. Accumulation of glucagon in different culture media during islet culture for 1 wk. The islets were cultured free-floating and 10% ivol/voi) heat-inactivated bovine calf serum and 100 #g/ml aprotinin were added to the media. The medium was changed after 34hatched bars) or 5 ~stippled bars) d of culture and a sample of the medium was taken at the end of the culture period (open bars). Means _+SEM of five experiments.

more glucagon than the M E M Eagle's medium t P < 0.01). Despite this increased secretion of glucagon to the culture medium, RPMI-1640-cultured islets contained almost twice IP < 0.01) as much glucagon as those cultured in the other media (Fig. 5). This may also explain the enhanced release of glucagon from this particular group of islets observed in the short-term experiments performed at the end of the culture period. In response to the acute arginine challenge, the RPMI-1640cultured islets secreted three to seven times more glucagon than any of the other groups of islets. Again, no glucose sensitivity of the islet glucagon release was recorded for any of the culture conditions tested. General remarks. The present data and those of previous studies demonstrate clearly the importance of the composition of the culture medium for maintaining the different specific functions of islet tissue in culture. Moreover, the degradation of the hormones secreted into the culture medium must be considered when presenting data obtained from media samples gathered during the

entire culture period. It is also apparent that Medium RPMI-1640, previously shown to be the most suitable of those media tested for maintaining islet B-ceil function (3), is similarly effective in stimulating glucagon production during the culture period. This may be the result of the high content of glucose ~11 raM) because isolated mouse islets challenged with high glucose have been found to incorporate [~H]tryptophan into newly synthesized glucagon at an increased rate ~11). The lack of effect of adjusting the glucose concentration of Medium 199 to that of RPMI1640 does not, however, support this hypothesis. Alternative factors that may be responsible for the different suitability of the tested media include the concentrations of different amino acids, inorganic salts, and vitamins, some of which are known to influence glucagon secretion. Our suggestion of a glucose refractive glucagon release, irrespective of the culture conditions used, agrees with previous studies of isolated rodent islets (10,12,13}. This may be due partly to the peripheral location of the glucagon-producing As-cells in mouse and rat islets, making them

GLUCAGON AND CULTURED ISLETS

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FIG. 5. Glucagon content and release of islets cultured as described in Fig. 4. The glucagon release was estimated in short-term incubations performed either in 1.5 mM glucose {hatched bars}, 1.5 mM glucose plus 5 mM arginine (stippled bars), or 15 mM glucose (filled bars). Open bars indicate glucagon content of the islets at the end of the 1 wk culture period. Means +_SEM of five experiments with each release and content value being based on duplicate or quadruplicate samples, respectively.

more susceptible to damage during the isolation procedure. Culture of such islets has, however, been shown to restore partly the regulation of hormone secretion (14,15k In this context, note that isolated guinea pig islets respond to an acute glucose challenge with a suppressed glucagon secretion after a short culture period (16k The A2ceils are located throughout the guinea pig islets and therefore may be less exposed to collagenase and mechanical influence during islet isolation.

REFERENCES 1. Andersson, A. Tissue culture of isolated pancreatic islets. Acta Endocrinol. Suppl. (KbhA 205: 283-293; 1976. 2. Andersson, A.; Borg, H.; Groth, C.-G.; HellerstrOm C.; Lundgren, G.; Petersson, B.; Ostman, J. Survival of isolated human islets of Langerhans maintained in tissue culture. J. Clin. Invest. 57: 1295-1301; 1976. 3. Andersson, A. Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 14: 397-404; 1978. 4. Howell, S. L.; Taylor, K. W. Potassium ions and the secretion of insulin by islets of Langerhans incubated in vitro. Biochem. J. 108: 17-24; 1968.

5. Buitrago, A. Preservation of B-cell function during culture with a high molecular serum fraction (abstr. no. 16}. Acta Endocrinol. Suppl. (Kbh.} 209: ]977. 6. Andersson, A.; Westman, J.; HellerstriSm, C. Effects of glucose on the ultrastructure and insulin biosynthesis of isolated mouse pancreatic islets maintained in tissue culture. Diabetologia 10: 743-753; 1974. 7. Heding, L. Radioimmunological determination of pancreatic and gut glucagon in plasma. Diabetologia 7: 10-19; 1971. 8. Davis, S.E.; Munson, P . J . ; Jaffe, M.L.; Rodbard, D. Radioimmunoassay data processing with a small programmable calculator. J. Immunoassay 1: 15-25; 1980. 9. Kedinger, M.; Moody, A.; Lannay, J. F.; Haffen, K. Establishment of serum based medium essentially free of proteolytic activity for the culture of mouse pancreatic islets. Experientia 33: 972-973; 1977. 10. Segerstr6m, K.; Andersson, A.; Lundqvist, G.; Petersson, B.; Hellerstrfm, C. Regulation of the glucagon release from mouse pancreatic islets maintained in tissue culture at widely different glucose concentrations. Diab~te Metab. 2: 45-48; 1976. 11. Osteuson, C.-G.; Andersson, A.; Eriksson, U.; Hellerstr6m, C. Glucagon biosynthesis in isolated pancreatic islets of mice and guinea pigs. Diab6te Metab. 6: 141-149; 1980.

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12. Vance, J. E,; Buchanan, K. D.; Williams, R. H. Glucagon and insulin release. Influence of drugs affecting the autonomic nervous system. Diabetes 20: 78-82; 1971.

with collagenaseand in the isolated perlused pancreas of rats. Diabetes 24:961-970; 1975.

13. Buchanan, K. D.; Mawhinney, A. A. Insulin control of glucagon release from insulin-deficient rat islets. Diabetes 22: 801-803; 1973.

15. Turcot-Lemay, L.; Lemay, A.; Lacy, P.E. Somatostatin inhibition of insulin release from freshly isolated and organ cultured rat islets of Langerhans in vitro. Biochem. Biophys. Res. Commun. 63: 1130-1138; 1975.

14. Norfleet, W.T.; Pagliara, A.S.; Haymond, M. W.; Matschinsky, F. Comparison of alphaand beta-secretory responses in islets isolated

16. Ostenson, C.-G. Effects of insulin on the pancreatic A2-cell of the guinea pig. Diabetologia 17: 325-330; 1979.

The authors thank Margareta Engkvist and Astrid Nordin for valuable technical assistance. This work was supported by grants from the Swedish Medical Research Council (12X-109), the Nordic Insulin Fund, and the Swedish Diabetes Association.