Analysis of secretory dynamics and development of media for the controlled secretion of insulin-related peptides from betaTC-3 insulinoma cells

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 muM 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 muM), 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 muM) 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. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 274-282, 1997.     

Characterization of the immature secretory granule, an intermediate in granule biogenesis

The events in the biogenesis of secretory granules after the budding of a dense-cored vesicle from the trans-Golgi network (TGN) were investigated in the neuroendocrine cell line PC12, using sulfate-labeled secretogranin II as a marker. The TGN-derived dense-cored vesicles, which we refer to as immature secretory granules, were found to be obligatory organellar intermediates in the biogenesis of the mature secretory granules which accumulate in the cell. Immature secretory granules were converted to mature secretory granules with a half-time of approximately 45 min. This conversion entailed an increase in their size, implying that the maturation of secretory granules includes a fusion event involving immature secretory granules. Pulse-chase labelling of PC12 cells followed by stimulation with high K+, which causes the release of secretogranin II, showed that not only mature, but also immature secretory granules were capable of undergoing regulated exocytosis. The kinetics of secretion of secretogranin II, as well as those of a constitutively secreted heparan sulfate proteoglycan, were reduced by treatment of PC12 cells with nocodazole, suggesting that both secretory granules and constitutive secretory vesicles are transported to the plasma membrane along microtubules. Our results imply that certain membrane proteins, e.g., those involved in the fusion of post-TGN vesicles with the plasma membrane, are sorted upon exit from the TGN, whereas other membrane proteins, e.g., those involved in the interaction of post-TGN vesicles with the cytoskeleton, may not be sorted.     

Imaging secretory vesicles by fluorescent protein insertion in propeptide rather than mature secreted peptide

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY^®TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.     

The trafficking of alpha 1-antitrypsin,a post-Golgi secretory pathway marker,in INS-1 pancreatic beta cells

A sulfated alpha1-antitrypsin (AAT), thought to be a default secretory pathway marker, is not stored in secretory granules when expressed in neuroendocrine PC12 cells. In search of a constitutive secretory pathway marker for pancreatic beta cells, we produced INS-1 cells stably expressing wild-type AAT. Because newly synthesized AAT arrives very rapidly in the Golgi complex, kinetics alone cannot resolve AAT release via distinct secretory pathways, although most AAT is secreted within a few hours and virtually none is stored in mature granules. Nevertheless, from pulse-chase analyses, a major fraction of newly synthesized AAT transiently exhibits secretogogue-stimulated exocytosis and localizes within immature secretory granules (ISGs). This trafficking occurs without detectable AAT polymerization or binding to lipid rafts. Remarkably, in a manner not requiring its glycans, all of the newly synthesized AAT is then removed from granules during their maturation, leading mostly to constitutive-like AAT secretion, whereas a smaller fraction (approximately 10%) goes on to lysosomes. Secretogogue-stimulated ISG exocytosis reroutes newly synthesized AAT directly into the medium and prevents its arrival in lysosomes. These data are most consistent with the idea that soluble AAT abundantly enters ISGs and then is efficiently relocated to the endosomal system, from which many molecules undergo constitutive-like secretion while a smaller fraction advances to lysosomes.     

Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.     

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.     

Constitutive protein secretion from the exocrine pancreas of fetal rats

Two general kinds of exocytotic secretion of proteins are known: that which is stimulated by secretagogues; and constitutive exocytosis, which is unable to be stimulated. The exocrine pancreas has often been cited as a model system for the first kind of secretion. However, the release of digestive enzymes from the exocrine pancreas of 1-day prenatal rats cannot be stimulated by secretagogues; therefore, its secretion is constitutive. To gain insight into the intracellular pathways which mediate secretion in the fetal gland, we examined the kinetics of release of newly synthesized proteins. We find that fetal pancreas in a steady state of secretion releases pulse-labeled secretory proteins in two kinetically distinct phases. The first phase occurring during 0-6.5 h of chase comprises approximately 12% of total incorporated radioactivity, the second phase beginning at greater than 7 h of chase comprises the remainder. Based on analysis by electron microscope autoradiography, radiolabel is localized during the first phase of secretion in immature granules/condensing vacuoles, Golgi compartments, and few mature granules. The second phase of secretion occurs when radiolabel is predominantly in mature granules. We propose that secretion occurs via (at least) 2 exocytotic routes, both of which are constitutive in fetal pancreatic tissue.     

Protein discharge from immature secretory granules displays both regulated and constitutive characteristics.

At physiological glucose concentrations, isolated pancreatic islets release a minor portion of their newly synthesized insulin and precursors in a phase of secretion which is largely complete by 4 h of chase. Discharge during this period can be amplified by secretagogues, yet is not abolished by conditions which fully suppress regulated release from dense core secretory granules. The ability to stimulate the secretion and the biochemical profile of released proinsulin-related peptides indicate that secretion during this period originates from immature granules. The stoichiometry of release of labeled C-peptide:insulin during this phase is 1:1 at high glucose concentrations. However, at physiologic or low concentrations, C-peptide is released in molar excess of insulin as if the exocytotic vesicles carrying this secretion were budding from a post-Golgi compartment in which the lumen was composed of condensing insulin and soluble C-peptide. These findings can be explained by a model for regulated secretory protein traffic in which direct exocytosis of young granules is stimulated by higher glucose concentrations and vesicle budding from immature granules occurs at lower concentrations. Thus, insulin targeting from immature granules exhibits both regulated and constitutive-like characteristics.     

Secretory protein trafficking

Acute insulin secretion from stimulated pancreatic β-cells is derived from the intracellular pool of insulin secretory granules wherein insulin is packaged in a highly concentrated (and in some species, crystalline) state. Here we review experimental work, principally from our laboratory, on the question of biogenesis of mature secretory granules within the broader context of intracellular protein trafficking. Events occurring in the lumen of organelles at various stages of intracellular transport within the secretory pathway and events at the limiting membrane of newly forming secretory granules each contribute to formation of the insulin storage compartment comprising the readily releasable pool.     

Protein sorting and secretion granule formation in regulated secretory cells

Formation of secretion granules in regulated secretory cells involves packaging a subject of proteins undergoing intracellular transport into specific vesicular carriers that function in stimulus-dependent exocytosis. Recent findings suggest that immature granules are a site of passive sorting, involving condensation of regulated secretory proteins. Proteins that are not condensed are stored to a lesser degree and are enriched in unstimulated, constitutive-like secretion. While these observations have helped to distinguish possible mechanisms of secretory protein sorting, there are only recent hints about the sorting processes that may be required to create the regulated secretory carrier membranes.     

Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach

Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.     

Extracellular ATP stimulates exocytosis via localized Ca(2+) release from acidic stores in rat pancreatic beta cells

Three different methods, membrane capacitance (C(m)) measurement, amperometry and FM dye labeling were used to investigate the role of extracellular ATP in insulin secretion from rat pancreatic beta cells. We found that extracellular application of ATP mobilized intracellular Ca(2+) stores and synchronously triggered vigorous exocytosis. No influence of ATP on the readily releasable pool of vesicles was observed, which argues against a direct modulation of the secretory machinery at a level downstream of Ca(2+) elevation. The stimulatory effects of ATP were greatly reduced by intracellular perfusion of BAPTA but not EGTA, suggesting a close spatial association of fusion sites with intracellular Ca(2+) releasing sites. ATP-induced Ca(2+) transients and exocytosis were not blocked by thapsigargin (TG), by a ryanodine receptor antagonist or by dissipation of pH in acidic stores by monensin alone, but they were greatly attenuated by IP(3) receptor inhibition as well as ionomycin plus monensin, suggesting involvement of IP(3)-sensitive acidic Ca(2+) stores. Taken together, our data suggest that extracellular ATP triggers exocytosis by mobilizing spatially limited acidic Ca(2+) stores through IP(3) receptors. This mechanism may explain how insulin secretion from the pancreas is coordinated through diffusible ATP that is co-released with insulin.     

Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment.

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.     

Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules

We have developed a method for separating purified parotid secretory granules according to their degree of maturation, and we have used this method to examine the relationship between granule formation and stimulus-independent (constitutive) protein secretion. Constitutive export of pulse-labeled secretory proteins occurs almost entirely after their appearance in newly formed granules, and this secretion can be resolved kinetically into two distinct components. Later-phase secretion is the more prominent component and, according to kinetic and compositional criteria, appears to result from basal exocytosis of mature granules. In contrast, early-phase secretion (1.5-15% of constitutive protein output) appears to originate from maturing granules but differs significantly from granule content in composition; that is, the early component exports individual protein species in different relative amounts. Maturing granules, which are labeled most highly before and during the appearance of early-phase secretion, possess numerous coated membrane evaginations suggestive of vesicular traffic. We propose that, in addition to basal exocytosis of relatively mature granules, constitutive exocrine secretion results from limited, selective removal of content proteins from forming and maturing granules. Thus protein sorting and packaging occur together in granule compartments. Exocrine secretory granules constitute an extension of the post-Golgi sorting system and are not merely terminal depots for proximally targeted polypeptides.     

Sorting ourselves out: seeking consensus on trafficking in the beta-cell

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans -Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.     

Incretin effect potentiates beta-cell responsivity to glucose as well as to its rate of change: OGTT and matched intravenous study

The aim of this study is to gain greater insight into the mechanism whereby "incretins" (greater insulinemia after oral than intravenous glucose) enhance insulin secretion. To do so, we use a model of C-peptide secretion to reanalyze data from a previously published study in which glycemic profiles observed following glucose ingestion were matched in the same 10 subjects by means of an intravenous glucose infusion. We report that incretins increase insulin secretion by enhancing both the dynamic (to the rate of increase of glucose) and static (to given glucose concentration) response with an increase of 58% for the static (Phi(s) = 16.4 +/- 1.8 vs. 24.6 +/- 2.0 10(-9) min(-1), P = 0.01) and 63% for the dynamic (Phi(d) = 278 +/- 32 vs. 463 +/- 86 10(-9), P = 0.02) indexes. Since increases in the dynamic response to glucose are believed to be due to an increase in the rate of docking, and exocytosis of insulin containing granules and increases in the static response to glucose are believed to be caused by a shift in the sensitivity of the beta-cell to glucose, these results suggest that incretins may modulate more than one step in the beta-cell insulin secretory cascade.     

The Rab-binding protein Noc2 is associated with insulin-containing secretory granules and is essential for pancreatic beta-cell exocytosis

The small GTPases Rab3 and Rab27 are associated with secretory granules of pancreatic beta-cells and regulate insulin exocytosis. In this study, we investigated the role of Noc2, a potential partner of these two GTPases, in insulin secretion. In the beta-cell line INS-1E wild-type Noc2, Noc265E, and Noc258A, a mutant capable of interacting with Rab27 but not Rab3, colocalized with insulin-containing vesicles. In contrast, two mutants (Noc2138S,141S and Noc2154A,155A,156A) that bind neither Rab3 nor Rab27 did not associate with secretory granules and were uniformly distributed throughout the cell cytoplasm. Overexpression of wild-type Noc2, Noc265E, or Noc258A inhibited hormone secretion elicited by insulin secretagogues. In contrast, overexpression of the mutants not targeted to secretory granules was without effect. Silencing of the Noc2 gene by RNA interference led to a strong impairment in the capacity of INS-1E cells to respond to insulin secretagogues, indicating that appropriate levels of Noc2 are essential for pancreatic beta-cell exocytosis. The defect was already detectable in the early secretory phase (0-10 min) but was particularly evident during the sustained release phase (10-45 min). Protein-protein binding studies revealed that Noc2 is a potential partner of Munc13, a component of the machinery that controls vesicle priming and insulin exocytosis. These data suggest that Noc2 is involved in the recruitment of secretory granules at the plasma membrane possibly via the interaction with Munc13.