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.     

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.     

Heterogeneity of expression and secretion of native and mutant [AspB10]insulin in AtT20 cells

AtT20 (pituitary corticotroph) cells were transfected with either the native or a mutant [AspB10]rat insulin II gene, using a plasmid containing the insulin gene and a neomycin resistance gene under the control of independent constitutive promoters. The cellular immunoreactive insulin (IRI) content ranged from 0.8-440 ng/10(6) cells, with the highest value similar to that found for a rat insulinoma cell line (RIN) and corresponding to approximately 1% that of native pancreatic B-cells. There was a direct correlation between insulin mRNA levels and IRI content and no correlation between mRNA levels and rat insulin II gene copy number. Furthermore, in some lines the insulin II transgene was lost even though the gene encoding neomycin resistance was retained. IRI release was stimulated up to 4-fold by isobutylmethylxanthine in all lines transfected with the native rat insulin II gene, and HPLC analysis showed most IRI as fully processed insulin, with less than 5% as proinsulin. These cells, thus, directed most proinsulin to secretory granules for conversion and regulated release regardless of the absolute amount of IRI expressed. One of the lines transfected with the AspB10 mutant gene (line AA9) released nearly 50% of IRI as proinsulin under basal conditions, with stimulation of insulin, but not proinsulin, release by isobutylmethylxanthine. This confirmed our previous finding of partial diversion of this mutant proinsulin from the regulated to the constitutive pathway. A second line (IC6) expressing the same mutant gene at much higher levels appeared to direct all mutant proinsulin to the regulated pathway, suggesting that for this particular mutant proinsulin, the secretory pathway employed by the transfected cells can be affected by the amount of proinsulin synthesized.     

Distinct molecular mechanisms for protein sorting within immature secretory granules of pancreatic beta-cells

In the beta-cells of pancreatic islets, insulin is stored as the predominant protein within storage granules that undergo regulated exocytosis in response to glucose. By pulse-chase analysis of radiolabeled protein condensation in beta-cells, the formation of insoluble aggregates of regulated secretory protein lags behind the conversion of proinsulin to insulin. Condensation occurs within immature granules (IGs), accounting for passive protein sorting as demonstrated by constitutive-like secretion of newly synthesized C- peptide in stoichiometric excess of insulin (Kuliawat, R., and P. Arvan. J. Cell Biol. 1992. 118:521-529). Experimental manipulation of condensation conditions in vivo reveals a direct relationship between sorting of regulated secretory protein and polymer assembly within IGs. By contrast, entry from the trans-Golgi network into IGs does not appear especially selective for regulated secretory proteins. Specifically, in normal islets, lysosomal enzyme precursors enter the stimulus-dependent secretory pathway with comparable efficiency to that of proinsulin. However, within 2 h after synthesis (the same period during which proinsulin processing occurs), newly synthesized hydrolases are fairly efficiently relocated out of the stimulus- dependent pathway. In tunicamycin-treated islets, while entry of new lysosomal enzymes into the regulated secretory pathway continues unperturbed, exit of nonglycosylated hydrolases from this pathway does not occur. Consequently, the ultimate targeting of nonglycosylated hydrolases in beta-cells is to storage granules rather than lysosomes. These results implicate a post-Golgi mechanism for the active removal of lysosomal hydrolases away from condensed granule contents during the storage process for regulated secretory proteins.     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

Deletion of a highly conserved tetrapeptide sequence of the proinsulin connecting peptide (C-peptide) inhibits proinsulin to insulin conversion by transfected pituitary corticotroph (AtT20) cells

The biological function of the connecting peptide (C-peptide) of proinsulin is unknown. Comparison of all known C-peptide sequences reveals the presence of a highly conserved peptide sequence, Glu/Asp-X-Glu/Asp (X being a hydrophobic amino acid), adjacent to the Arg-Arg doublet at the B chain/C-peptide junction. Furthermore, the next amino acid in the C-peptide sequence is also acidic in many animal species. To test the possible involvement of this hydrophilic domain in insulin biosynthesis, we constructed a mutant of the rat proinsulin II gene lacking the first four amino acids of the C-peptide and expressed either the normal (INS) on the mutated (INSDEL) genes in the AtT20 pituitary corticotroph cell line. In both cases immunoreactive insulin (IRI) was stored by the cells and released upon stimulation by cAMP. In the INS expressing cells, the majority of IRI, whether stored or released in response to a secretagogue, was mature insulin. By contrast, most of the stored and releasable IRI in the INSDEL expressing cells appeared to be (mutant) proinsulin or conversion intermediate with little detectable native insulin. Release of the mutant proinsulin and/or conversion intermediates was stimulated by cAMP. These results suggest that the mutant proinsulin was appropriately targeted to secretory granules and released predominantly via the regulated pathway, but that the C-peptide deletion prevented its conversion to native insulin.     

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.     

Proinsulin modified by analogues of arginine and lysine is degraded rapidly in pancreatic B-cells.

Modified cytosolic proteins are known to be degraded more rapidly than their native counterparts. In order to determine whether the same applies to a modified protein within the potentially protective environment of secretory granules, rat islets were labelled [( 3H]leucine) in the presence or absence (controls) of 3 mM-canavanine and 3 mM-thialysine (analogues of arginine and lysine respectively), followed by a 24h 'chase' period without analogues. The results showed the following. (1) Incorporation of the analogues into newly synthesized labelled proinsulin inhibited its conversion into insulin during the chase period. (2) Despite this block in conversion, the modified proinsulin was released from islets at the same rate as native proinsulin and insulin from control islets. (3) Morphometric analysis of high-resolution autoradiographs showed that products labelled in the presence of analogues were sequestered into secretory granules at the same rate as native products in control B-cells. (4) Only 7% of prelabelled proinsulin had been degraded within islet cells during the chase period in control islets, compared with 36% for proinsulin prelabelled in the presence of analogues. (5) Control experiments showed that the analogues had no effect on the release or intracellular degradation of unmodified stored insulin (present in islets before exposure to the analogues). (6) Despite sequestration into secretory granules, modified proinsulin, if not released from B-cells, is thus degraded more rapidly than native products.     

Constitutive and basal secretion from the endocrine cell line, AtT-20

A variant of the ACTH-secreting pituitary cell line, AtT-20, has been isolated that does not make ACTH, sulfated proteins characteristic of the regulated secretory pathway, or dense-core secretory granules but retains constitutive secretion. Unlike wild type AtT-20 cells, the variant cannot store or release on stimulation, free glycosaminoglycan (GAG) chains. In addition, the variant cells cannot store trypsinogen or proinsulin, proteins that are targeted to dense core secretory granules in wild type cells. The regulated pathway could not be restored by transfecting with DNA encoding trypsinogen, a soluble regulated secretory protein targeted to secretory granules. A comparison of secretion from variant and wild type cells allows a distinction to be made between constitutive secretion and basal secretion, the spontaneous release of regulated proteins that occurs in the absence of stimulation.     

Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.     

Dibasic cleavage site is required for sorting to the regulated secretory pathway for both pro- and neuropeptide Y

To investigate the signals governing routing of biologically active peptides to the regulated secretory pathway, we have expressed mutated and non-mutated proneuropeptide Y (ProNPY) in pituitary-derived AtT20 cells. The mutations were carried out on dibasic cleavage site and or ProNPY C-terminal sequence. Targeting to the regulated secretory pathway was studied using protein kinase A (8-BrcAMP), protein kinase C (phorbol myristate acetate) specific activators and protein synthesis inhibitor cycloheximide, and by pulse chase. The analysis of expressed peptides in cells and culture media indicated that: neuropeptide Y (NPY) and ProNPY were differently secreted, whilst NPY was exclusively secreted via regulatory pathway; ProNPY was secreted via regulated and constitutive-like secretory pathways. ProNPY secretion behaviour was not Proteolytic cleavage efficiency-dependent. The dibasic cleavage was essential for ProNPY and NPY cAMP-dependent regulated secretion and may have function as a retention signal.     

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.     

Processing of proinsulin by transfected hepatoma (FAO) cells.

Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.     

Novel regulation by Rac1 of glucose- and forskolin-induced insulin secretion in INS-1 beta-cells

Stimulation of insulin secretion by glucose and other secretagogues from pancreatic islet beta-cells is mediated by multiple signaling pathways. Rac1 is a member of Rho family GTPases regulating cytoskeletal organization, and recent evidence also implicates Rac1 in exocytotic processes. Herein, we report that exposure of insulin-secreting (INS) cells to stimulatory glucose concentrations caused translocation of Rac1 from cytosol to the membrane fraction (including the plasmalemma), an indication of Rac1 activation. Furthermore, glucose stimulation increased Rac1 GTPase activity. Time course study indicates that such an effect is demonstrable only after 15 min stimulation with glucose. Expression of a dominant-negative Rac1 mutant (N17Rac1) abolished glucose-induced translocation of Rac1 and significantly inhibited insulin secretion stimulated by glucose and forskolin. This inhibitory effect on glucose-stimulated insulin secretion was more apparent in the late phase of secretion. However, N17Rac1 expression did not significantly affect insulin secretion induced by high K+. INS-1 cells expressing N17Rac1 also displayed significant morphological changes and disappearance of F-actin structures. Expression of wild-type Rac1 or a constitutively active Rac1 mutant (V12Rac1) did not significantly affect either the stimulated insulin secretion or basal release, suggesting that Rac1 activation is essential, but not sufficient, for evoking secretory process. These data suggest, for the first time, that Rac1 may be involved in glucose- and forskolin-stimulated insulin secretion, possibly at the level of recruitment of secretory granules through actin cytoskeletal network reorganization.     

Evidence for defects in membrane traffic in Paramecium secretory mutants unable to produce functional storage granules

The ciliated protozoan Paramecium has a regulated secretory system amenable to genetic analysis. The secretory storage granules, known as trichocysts, enclose a crystalline matrix with a genetically determined shape whose biogenesis involves proteolytic maturation of a family of precursor molecules into a heterogeneous set of small acidic polypeptides that crystallize within the maturing vesicles. We have developed an original pulse-chase protocol for monoxenic Paramecium cultures using radiolabeled bacteria to study the processing of trichocyst matrix proteins in wild-type and mutant cells. In wild-type cells, proteolytic processing is blocked in the presence of monensin and otherwise rapidly completed after approximately 20 min of chase, suggesting that the conversion occurs in the trans-Golgi and/or in small vesicles soon after sorting to the regulated pathway, probably before crystallization begins. In trichless mutant cells, which contain no visible trichocysts, secretory proteins are synthesized but not processed and we report constitutive secretion of the uncleaved precursor molecules. The mutation thus appears to affect sorting to the regulated pathway and should prove useful for analysis of the sorting machinery and of the relationship between sorting and proteolytic processing of secretory proteins. In mutants bearing misshapen trichocysts with poorly crystallized contents (tam33, tam38, stubbyA), the proteolytic processing of the trichocyst matrix proteins appears to be normal, while both pulse-chase and morphological data indicate that intracellular transport is perturbed, probably between ER and Golgi. Precursor molecules are present in the mutant trichocysts but not in wild-type trichocysts and may account for the defective crystallization. Our analysis of these mutants suggests that the temporal coordination of intracellular traffic plays a regulatory role in granule maturation.     

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line,CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain — C-peptide — A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.     

Protein traffic from the secretory pathway to the endosomal system in pancreatic beta-cells

Constitutive-like secretion involves vesicular trafficking corresponding kinetically and biochemically with a post-trans-Golgi network (TGN) origin. In pancreatic beta-cells, the budding of AP-1/clathrin-coated vesicles, a portion of which is derived from immature secretory granules, has been hypothesized to initiate constitutive-like trafficking. However, approximately 30 min after release of a 20 degrees C intracellular transport block in pancreatic beta-cells (to synchronize protein egress from the TGN), addition of brefeldin A (BFA) (which inhibits AP-1 recruitment) was reported not to block subsequent constitutive-like secretion. To further explore post-TGN trafficking in pancreatic beta-cell lines, we have followed the fate of pulse-labeled procathepsin B (ProB, a lysosomal proenyzme) after postpulse wortmannin treatment or the BFA treatment described above. We find that continuous wortmannin treatment allows ProB to reach immature secretory granules but inhibits its egress from maturing granules. Remarkably, BFA treatment causes augmented unstimulated secretion of newly synthesized ProB that is not paralleled by insulin. This effect requires a delay of 25-35 min after release from the 20 degrees C block. Further, when ProB delivery to endosomes is inhibited, its BFA-augmented secretion is eliminated. We hypothesize that the constitutive-like pathway involves an endosomal intermediate.     

Ectopic expression of syncollin in INS-1 beta-cells sorts it into granules and impairs regulated secretion

Syncollin was first demonstrated to be a protein capable of affecting granule fusion in a cell-free system, but later studies revealed its luminal localization in zymogen granules. To determine its possible role in exocytosis in the intact cell, syncollin and a truncated form of the protein (lacking the N-terminal hydrophobic domain) were stably transfected in insulin-secreting INS-1 cells since these well-studied exocytotic cells appear not to express the protein per se. Studies by subcellular fractionation analysis, double immunofluorescence staining, and electron microscopy examination revealed that transfection of syncollin produced strong signals in the insulin secretory granules, whereas the product from transfecting the truncated syncollin was predominantly associated with the Golgi apparatus and to a lesser degree with the endoplasmic reticulum. The expressed products were associated with membranes and not the soluble fractions in either cytoplasm or the lumens of organelles. Importantly, insulin release stimulated by various secretagogues was severely impaired in cells expressing syncollin, but not affected by expressing truncated syncollin. Transfection of syncollin appeared not to impede insulin biosynthesis and processing, since cellular contents of proinsulin and insulin and the number of secretory granules were not altered. In addition, the early signals (membrane depolarization and Ca(2+) responses) for regulated insulin secretion were unaffected. These findings indicate that syncollin may be targeted to insulin secretory granules specifically and impair regulated secretion at a distal stage.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

The Granular Chloride Channel ClC-3 Is Permissive for Insulin Secretion

Insulin secretion from pancreatic β cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated β cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of β cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3^−/− mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3^−/− β cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3^−/− mice, while in Clcn3^+/+ cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic β cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.