Newly Synthesized Proinsulin/Insulin and Stored Insulin Are Released from Pancreatic B Cells Predominantly via a Regulated, Rather Than a Constitutive, Pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15 % of the islet content of stored insulin was released at 16.7 mM glucose compared with 2 % at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to ll-fold by 16.7 mM glucose. At 180 min, 60 % of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that (a) glucose had no effect on intracellular proinsulin to insulin conversion; (b) release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; (c) 90% of the newly synthesized hormone was released as insulin; and (d) only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (<1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell. T HE general outline for the sequence of steps involved in the biosynthesis, processing, packaging, storage, and release of secretory products (including polypeptide hormones) has been known for many years (31, 33). The final step in this sequence is the extrusion of secretory granule contents by exocytosis. It has often been reported that there is preferential release of newly synthesized product over that which is stored intracellularly. This observation has been made for a variety of secretory products including insulin (6, 11, 22, 38), placental lactogen (43, 47), parathyroid hormone (26), alkaline phosphatase (4), pancreatic amylase (39), prolactin (35, 44), vasopressin (36), gonadotrophin (20), and also for some neurotransmitters, including acetylcholine (46), brain catecholamines (34), and serotonin (3). Within a given secretory cell type the total population of secretory granules will consist of a mixture of newly formed and older stored granules. Preferential release of a newly Dr. Rhodes' present address is Department of Clinical Biochemistry, Addenbrookes Hospital, Cambridge CB2 2QR, United Kingdom. Dr. Halban's present address is Unit6 Jeantet, Centre M&lical Universitaire, 1211 Geneva 4, Switzerland. synthesized product indicates that instead of random secretion of these two granule pools there is a defined subpopulation of recently formed granules destined for preferential release instead of storage. It has recently been suggested that there are two pathways for the release of a secretory product (24), the so-called regulated and constitutive pathways. For the regulated pathway, a product is concentrated into secretory granules which can accumulate in the cytoplasm and in which processing of any precursor can occur. The granules have a half-life of hours or days and the release of their products arises in response to a stimulus. For the constitutive pathway, newly synthesized products are packaged into membrane-limited vesicles which, unlike secretory granules, are destined for rapid release in a nonregulated fashion. There is only a short transit time between the Golgi complex and the plasma membrane, no cytoplasmic storage pool, and no processing of precursors. Since some cells display both pathways for the same product (27, 41, 44) it is tempting to speculate that preferential release of a newly synthesized hormone involves commitment of some of this material to the constitutive pathway. © The Rockefeller University Press, 0021-9525/87/07/145/9 $2.00 The Journal of Cell Biology, Volume 105, July 1987 145-153 145 on Jne 8, 2017 D ow nladed fom Published July 1, 1987