title: Analysis of secretory dynamics and development of media for the controlled secretion of insulin-related peptides from betaTC-3 insulinoma cells.
abstract: Controlled secretion of proteins from endocrine-derived cell lines has been proposed as a means to produce some classes of post-translationally modified proteins in bioreactors. Under the right biological and environmental conditions it may be possible to improve the product purity or quality relative to that obtained through steady (constitutive) secretion. The pancreatic-islet-derived cell line, betaTC-3, was selected as a model system to explore the secretory dynamics of insulin under various combinations of stimulatory or inhibitory environmental conditions. The betaTC-3 cells exhibited a glucose-mediated stimulus-response pattern which was saturated above 1 mM glucose and with an apparent "Kg" of 0.1 mM glucose. However, the kinetics of insulin synthesis were closely coupled to those of secretion such that betaTC-3 cells cycled between saturating and basal levels of glucose were never perturbed far from an intracellular synthesis-secretion equilibrium. When more powerful and selective agents were used to control secretion, the system performance improved markedly. A combination of 1 mM isobytylmethylxanthine (IBMX) and 1 microM carbachol (with saturating levels of glucose) could discharge 75% of stored insulin in 2 h. When this treatment was followed by incubation in media adjusted to attenuate the influx of calcium into the cells, intracellular pools were efficiently replenished within 24 h. Calcium attenuating treatments included hyperpolarization with reduced potassium (1 mM), calcium channel blockade with the dihydropyridine verapamil (1 microM), and the direct mass-action effect of reduced environmental calcium (0.5 mM versus 1.8 mM). Other inhibitory treatments were explored, but these tended to reduce both insulin synthesis and secretion. The best recharging treatment found was a combination of verapamil (1 microM) with reduced calcium level (0.5 mM).To demonstrate the feasibility of a controlled secretion process, betaTC-3 T-flask cultures were grown to confluence, then cycled through two periods of discharging (2 h) and recharging (20 h) with the best combinations of secretagogues and calcium attenuators. The overall process 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 episodes. Discharging was very effective in the first episode (80% recovery of stored insulin), but slightly less efficient in subsequent discharging episodes, possibly due to a desensitization effect of the calcium attenuating media. Nevertheless, the regulated secretory pathway of betaTC-3 cells could be successfully harnessed to a controlled secretion process for the selective recovery of stored insulin.
authors.affiliation: Department of Chemical Engineering and Biotechnology Process Engineering Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. pat@cc.huji.ac.il
authors.forename: G E
authors.initials: GE
authors.lastname: Grampp
authors.forename: H F
authors.initials: HF
authors.lastname: Lodish
authors.forename: G
authors.initials: G
authors.lastname: Stephanopoulos