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Processing and secretion of insulin‐related peptides in an insulinoma cell line
Author(s) -
Grampp Gustavo E.,
Lodish Harvey F.,
Stephanopoulos Gregory
Publication year - 1997
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19970205)53:3<283::aid-bit6>3.0.co;2-e
Subject(s) - exocytosis , insulin , medicine , endocrinology , secretion , enteroendocrine cell , proinsulin , secretagogue , chromogranin a , biology , intracellular , insulinoma , calmodulin , cell culture , islet , calcium in biology , radioimmunoassay , calcium , biochemistry , endocrine system , hormone , genetics , immunohistochemistry
Abstract Certain classes of prohormones and other neuroendocrine or endocrine‐derived secretory proteins are post‐translationally modified in the secretory storage granules. If such molecules were to be biosynthesized to acceptable quantity and yield using endocrine‐derived cell lines, it would be important to understand the relationship between the secretory dynamics and the conversion and release of the immature and mature forms of the molecule. We studied aspects of such a relationship using the endocrine‐derived cell line βTC‐3, which synthesizes murine proinsulin, sequesters it into secretory granules, and converts it into mature insulin. In T‐flask experiments with confluent cultures of βTC‐3 cells, intracellular and secreted (pro)insulin was sampled before and after episodes of stimulated exocytosis and recharging and quantified by radioimmunoassay and reversed‐phase high‐performance liquid chromatography (HPLC). Under conditions of steady‐state secretion in glucose‐rich growth medium the cells turned over their (pro)insulin inventory (90 ± 5% mature insulin) at 2–3% per hour through secretion of (pro)insulin which was less than 70% mature. During an episode of hyperstimulated exocytosis induced by the combined secretagogues carbachol (1 μ M ) and isobutylmethylxanthine (1 m M ), ∼80% of the intracellular (pro)insulin stores were depleted within 2 h and 84 ± 4% of the secreted (pro)insulin was in the mature form. Following the discharging episode, exocytosis was suppressed to 10% of its steady‐state rate with a treatment which attenuated calcium influx (20 μ M verapamil with reduced levels of calcium in the medium). Under this condition the secreted protein was only ∼50% converted to mature insulin, but 85 ± 10% of the net (pro)insulin accumulating within the intracellular stores was converted to the mature form. The inverse relationship between rate of secretion and degree of conversion of secreted (pro)insulin is consistent with a previously observed phenomenon of preferential basal secretion from immature secretory granules. This tends to enrich the secreted peptides in immature forms relative to the total intracellular pool. Preferential early secretion can best be overcome by rapid discharging of the long‐term and predominantly mature stores. Thus, a cyclic controlled secretion process wherein product is collected during intermittent discharging episodes would provide a better yield of mature product than would steady‐state secretion. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 283–289, 1997.