ANALYSIS OF SECRETORY DYNAMICS AND DEVELOPMENT OF MEDIA FOR THE CONTROLLED SECRETION OF INSULIN-RELATED PEPTIDES FROM BETA-TC-3 INSULINOMA CELLS

Controlled secretion of proteins from endocrine-derived cell lines has been proposed as a means to produce some classes of post-translationa lly modified proteins in bioreactors. Under the right biological and e nvironmental conditions it may be possible to improve the product puri ty or quality relative to that obtained through steady (constitutive) secretion. The pancreatic-islet-derived cell line, beta TC-3, was sele cted as a model system to explore the secreton/dynamics of insulin und er various combinations of stimulatory or inhibitory environmental con ditions. The beta TC-3 cells exhibited a glucose-mediated stimulus-res ponse pattern which was saturated above 1 mM glucose and with an appar ent ''Kg'' of 0.1 mM glucose. However, the kinetics of insulin synthes is were closely coupled to those of secretion such that beta TC-3 cell s cycled between saturating and basal levels of glucose were never per turbed far from an intracellular synthesis-secretion equilibrium. When more powerful and selective agents were used to control secretion, th e system performance improved markedly. A combination of 1 mM isobytyl methylxanthine (IBMX) and 1 mu M carbachol (with saturating levels of glucose) could discharge 75% of stored insulin in 2 h. When this treat ment was followed by incubation in media adjusted to attenuate the inf lux of calcium into the cells, intracellular pools were efficiently re plenished within 24 h. Calcium attenuating treatments included hyperpo larization with reduced potassium (1 mM), calcium channel blockade wit h the dihydropyridine verapamil (1 mu M), and the direct mass-action e ffect of reduced environmental calcium (0.5 mM versus 1.8 mM). Other i nhibitory treatments were explored, but these tended to reduce both in sulin synthesis and secretion. The best recharging treatment found was a combination of verapamil (1 mu M) with reduced calcium level (0.5 m M). To demonstrate the feasibility of a controlled secretion process, beta TC-3 T-flask cultures were grown to confluence, then cycled throu gh two periods of discharging (2 h) and recharging (20 h) with the bes t combinations of secretagogues and calcium attenuators. The overall p rocess was quite efficient: Only 15% of the overall insulin secretion took place during the recharging episodes, and this residual secretion represented only 10% of the net insulin synthesis during these episod es. Discharging was very effective in the first episode (80% recovery of stored insulin), but slightly less efficient in subsequent discharg ing episodes, possibly due to a desensitization effect of the calcium attenuating media. Nevertheless, the regulated secretory pathway of be ta TC-3 cells could be successfully harnessed to a controlled secretio n process for the selective recovery of stored insulin. (C) 1997 John Wiley & Sons, Inc.