Processing of Pro-Islet Amyloid Polypeptide in the Constitutive and Regulated Secretory Pathways of ß Cells

Lucy Marzban, Genny Trigo-Gonzalez and C. Bruce Verchere

Department of Pathology and Laboratory Medicine & British Columbia Research Institute for Children’s and Women’s Health, University of British Columbia, Vancouver, British Columbia, Canada V5Z 4H4

Address all correspondence and requests for reprints to: Dr. C. Bruce Verchere, British Columbia Research Institute for Children’s and Women’s Health, 3084-950 West 28th Avenue, Vancouver, British Columbia, Canada V5Z 4H4. E-mail: verchere{at}interchange.ubc.ca.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Islet amyloid is a pathologic characteristic of the pancreas in type 2 diabetes comprised mainly of the ß-cell peptide islet amyloid polypeptide (IAPP; amylin). We used a pulse-chase approach to investigate the kinetics of processing and secretion of the IAPP precursor, proIAPP, in ß cells. By only 20 min after synthesis, a COOH-terminally processed proIAPP intermediate (~6 kDa) was already present in ß cells. Formation of this NH2-terminally extended intermediate was not prevented by arresting secretory pathway transport at the trans-Golgi network (TGN) by either brefeldin A or temperature blockade, suggesting that this initial cleavage step occurs in the TGN before entry of (pro)IAPP into granules. Mature IAPP (~4 kDa) was not detected until 60 min of chase, suggesting that NH2-terminal cleavage occurs in granules. Cells chased in low glucose without Ca2+ or with diazoxide, to block regulated release, secreted both proIAPP (~8 kDa) and a partially processed form (~6 kDa) via the constitutive secretory pathway. Stimulation of regulated secretion resulted in secretion primarily of mature IAPP as well as low levels of both unprocessed (~8 kDa) and partially processed (~6 kDa) proIAPP. We conclude that normal processing of proIAPP is a two-step process initiated by cleavage at its COOH terminus (likely by prohormone convertase 1/3 in the TGN) followed by cleavage at its NH2 terminus (by prohormone convertase 2 in granules) to form IAPP. Both proIAPP and its NH2-terminally extended intermediate appear to be normal secretory products of the ß cell that can be released via either the regulated or constitutive secretory pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
ISLET AMYLOID DEPOSITS are a pathologic characteristic of the pancreas in type 2 diabetes (1, 2) and may play an important role in the progressive loss of ß cell mass in this disease (1, 2, 3, 4). Islet amyloid is comprised mainly of islet amyloid polypeptide (IAPP; amylin) (5, 6), a 37-amino acid peptide that is cosecreted from ß cells along with insulin (7, 8). In humans, IAPP is first synthesized in ß cells as a 67-amino acid precursor molecule, proIAPP (9, 10).

Despite intensive studies during the past decade, it is still not clear why soluble IAPP molecules aggregate to from toxic amyloid deposits in type 2 diabetes. It has been proposed that defects in the processing, sorting, and/or secretion of (pro)IAPP by ß cells may initiate amyloid formation (2, 11, 12, 13). The findings that proinsulin processing is impaired in type 2 diabetes (14, 15, 16) and that proIAPP appears to be processed and secreted in parallel with proinsulin (8, 17, 18, 19) indirectly support this idea. Impaired processing of proIAPP could lead to hypersecretion of unprocessed or partially processed form(s) of the IAPP precursor that may initiate or contribute to amyloid formation (2, 11, 12, 20). Understanding the precise mechanisms mediating proIAPP processing and secretion from ß cells may lead to the identification of new targets to inhibit islet amyloid formation.

Endocrine cells, such as islet ß cells, are equipped with both regulated and constitutive pathways of secretion. After their synthesis in the rough endoplasmic reticulum, prohormones are transported to the cis- and through the trans-Golgi network (TGN), the latter being the final compartment common to both secretory pathways (21, 22). In the regulated secretory pathway, (pro)proteins are sorted into secretory granules for processing and storage before their release by exocytosis. In contrast, the constitutive secretory pathway is thought to be the default pathway by which newly synthesized (pro)proteins exit the TGN in small vesicles and are rapidly released without storage (21, 22, 23). Unlike the regulated secretory pathway in which exocytosis of stored granules occurs in response to secretagogues, secretion via the constitutive pathway is only regulated at the level of biosynthesis and is not subject to regulation by secretagogues (21, 23). The major endoproteolytic enzymes mediating processing of secreted proteins are the subtilisin-like proprotein convertases, among which the two prohormone convertases PC1/3 and PC2 are localized in the regulated secretory pathway of ß cells, whereas furin is the major processing enzyme of the constitutive secretory pathway (21, 24).

Although IAPP is colocalized with insulin in ß cell secretory granules and is cosecreted along with insulin in response to ß cell secretagogues (8, 17, 25), evidence from in vitro studies suggests that the release of IAPP and insulin is not always proportionate (17, 26, 27). Unlike proinsulin, which is efficiently (>99%) sorted to the regulated secretory pathway in ß cells and is released primarily from secretory granules (17, 28), IAPP immunoreactive forms have been shown to be secreted not only via the regulated but also the constitutive secretory pathway (29), at least in immature (neonatal) rat ß cells (17, 26), as well as human islets cultured in high glucose (27). These findings suggest that under certain conditions, a significant proportion of IAPP secretion, unlike insulin, may occur via the constitutive secretory pathway, although the predominant molecular forms of (pro)IAPP released from the constitutive secretory pathway of ß cells have yet to be determined.

ProIAPP is processed to mature IAPP by cleavage at two pairs of basic residues (Lys-Arg) located at its NH2 and COOH termini (9, 10, 30), by the prohormone convertases PC1/3 and PC2 (18, 19), which also mediate processing of proinsulin to insulin (31, 32). In previous studies, using islets from mice lacking either PC1/3 or PC2, we demonstrated that proIAPP is cleaved preferentially by PC1/3 at its COOH terminus and by PC2 at its NH2 terminus (18, 19). After cleavage by either PC1/3 or PC2, the remaining dibasic residues are removed by the action of carboxypeptidase E (CPE) (24, 33, 34). It has been shown that normal processing of proinsulin is initiated by cleavage at the B-chain/C-peptide junction on the COOH-terminal side of a pair of basic residues (Arg-Arg), preferentially by PC1/3. Further cleavage of the resulting intermediate form, des 31,32 proinsulin, at its C-peptide/A-chain junction on the COOH-terminal side of dibasic residues (Lys-Arg) preferentially by PC2, followed by removal of these dibasic residues from the B-chain and C-peptide by CPE then results in the formation of mature insulin and C-peptide (24, 31, 32, 33, 35, 36). ProIAPP processing might similarly be mediated by the sequential action of PC1/3 and PC2. The precise sequence of events in proIAPP processing have not, however, been elucidated. In the present study, we have investigated the kinetics of proIAPP processing and sought to identify the predominant molecular forms of (pro)IAPP released from both the constitutive and regulated secretory pathways of ß cells.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Kinetics of proIAPP Processing in ß Cells
To investigate the kinetics of proIAPP processing, INS-1 ß cells were pulse-labeled with [3H]Leu and chased for different time periods. Newly synthesized proIAPP (~8 kDa) was, as expected, the only form detected in cell extracts after a 10-min pulse-label. By 20 min, significant levels of a partially processed intermediate form of proIAPP (~6 kDa) were already present in cells, suggesting that the initial cleavage step in proIAPP processing occurs early in the secretory pathway (Fig. 1AGo). Mature IAPP (~4 kDa) was not detected until 60 min of chase (Fig. 1BGo), indicating that the second cleavage step in proIAPP processing occurs later in the secretory pathway. Processing of newly synthesized proIAPP was complete by 120 min of chase (Fig. 1BGo). In primary islets, as in INS-1 ß cells, initial cleavage of proIAPP occurred rapidly, within 20 min after synthesis (Fig. 1AGo). Mature IAPP was the only detectable form in islets by 120 min of chase (Fig. 1BGo).



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Fig. 1. Time Course of proIAPP Processing in ß Cells

A, INS-1 cells were pulse-labeled with [3H]Leu (300 µCi/ml) for 10, 20, or 30 min in KRB-G16.7. Batches of 300–350 mouse islets were labeled with [35S]Cys (230 µCi/100 µl) for 20 or 30 min. B, INS-1 cells were labeled with [3H]Leu (300 µCi/ml) for 30 min and chased for 60 or 120 min in KRB-G16.7 with 1 mM leucine. Mouse islets were labeled with [35S]Cys (230 µCi/100 µl) for 30 min and chased for 120 min in KRB-G16.7 with 1 mM cysteine. (Pro)IAPP forms were immunoprecipitated from the cell lysates with IgG-purified IAPP antibody (RGG-7323; Peninsula) as described in Materials and Methods. The immunoprecipitates were subjected to SDS-PAGE followed by fluorography. Partially processed intermediate form(s) of proIAPP were detectable as early as 20 min after labeling and mature IAPP was formed in less than 90 min. ProIAPP processing was complete by 120 min of chase.

 
Processing of proIAPP Is Initiated in the TGN
The finding that significant levels of a partially processed proIAPP intermediate were detectable by only 20 min of pulse-label, raised the possibility that the initial cleavage of proIAPP might occur in the TGN, before its entry into immature secretory granules. To test this hypothesis, we blocked transport of newly synthesized proteins from the TGN into secretory granules using either brefeldin A (BFA) or temperature blockade (20 C) (37, 38). After a short pulse (15 min) at 37 C in high glucose (16.7 mM) to stimulate proIAPP synthesis and allow its entry into the Golgi, cells were lysed or chased for a further 45 min at either 37 C (as control) or 20 C. The efficiency of temperature blockade in arresting protein transport at the TGN was confirmed by the absence of any radiolabeled (pro)IAPP immunoreactivity in the chase media (Fig. 2AGo). Cells chased for 45 min at 20 C still contained significant levels of proIAPP but also had increasing amounts of the partially processed (~6 kDa) proIAPP intermediate, supporting the idea that initiation of proIAPP processing may occur before its exit from the TGN. In a second approach, after a short pulse (15 min), cells were chased (45 min) in the presence or absence of BFA (10 µg/ml) (Fig. 2BGo). BFA treatment blocked transport of newly synthesized proteins from the TGN, manifest as increased accumulation of radiolabeled (pro)IAPP in cells and the absence of (pro)IAPP forms in the chase media (Fig. 2BGo) but did not block continued formation of the proIAPP intermediate form.



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Fig. 2. The Initial Cleavage of proIAPP Can Occur Early in the Secretory Pathway, Likely in the TGN

A, INS-1 cells were labeled for 15 min with [3H]Leu and [35S]Cys (each 200 µCi/ml) in KRB-G16.7 and chased for 45 min in KRB-G1.67 (containing 1 mM leucine and cysteine) at either 37 C or 20 C, the latter used to block secretory vesicle exit from the TGN. B, INS-1 cells were pulse-chased with the same conditions detailed in panel A in the absence or presence of BFA (10 µg/ml) to arrest exit of proIAPP and its partially processed intermediate from the TGN. Cell extracts or chase media were immunoprecipitated with IAPP antibody, followed by SDS-PAGE and fluorography. Note the presence of an approximately 6-kDa band of radiolabeled (pro)IAPP immunoreactivity even when cells were chased at 20 C (A) or in the presence of BFA (B). Initial cleavage of proIAPP therefore likely occurs in the TGN before its entry into secretory granules.

 
ProIAPP Processing Is Initiated by Cleavage at Its COOH Terminus in ß Cells
To determine the site at which proIAPP is initially cleaved, we identified the intermediate form that first appears in cell lysates. Mouse proIAPP has a methionine residue that is unique to its NH2-terminal flanking region and is lost during NH2-terminal processing of proIAPP (Fig. 3AGo). We therefore radiolabeled NIT-1 mouse ß cells with [35S]Met to identify this initial intermediate form. NIT-1 cells were first pulse-labeled with [3H]Leu using the same conditions as for INS-1 cells in Fig. 1Go, confirming that the kinetics of initial proIAPP cleavage are similar in NIT-1 and INS-1 ß cells (Fig. 3BGo). NIT-1 cells were then pulse-labeled with [35S]Met (20 min) and chased for 0, 30, or 120 min, to determine whether the initial intermediate form (~6 kDa) detected in cell lysates contains the NH2-terminal methionine residue. By 20 min of radiolabel, a band of [35S]Met-labeled proIAPP immunoreactivity appeared at (~6 kDa), indicating that this initial intermediate form was indeed NH2-terminally unprocessed proIAPP and that proIAPP is first processed at its COOH-terminal cleavage site (Fig. 3CGo). Cleavage of this partially processed form results in loss of detectable [35S]Met-labeled IAPP immunoreactivity in cells chased for 120 min (Fig. 3CGo) because mature IAPP does not have the NH2-terminal methionine residue. The rapid COOH-terminal cleavage observed in NIT-1 cells was subsequently confirmed using primary mouse islets. After only 20 min label with [35S]Met, NH2-terminal intermediate form of proIAPP (~6 kDa) was already present in islet lysates (Fig. 3CGo).



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Fig. 3. ProIAPP Processing Is Initiated by Cleavage at its COOH Terminus in NIT-1 ß Cells

A, Amino acid sequences of rat (P12969) and mouse (P12968) proIAPP are shown in single letter code along with approximate molecular weights of unprocessed, partially processed and mature IAPP. Dashes indicate identical amino acids in mouse and rat proIAPP. After cleavage by PC1/3 or PC2, the COOH-terminal dibasic residues are removed by CPE (34 ) and glycine is lost during COOH-terminal amidation. A methionine residue (bold) unique to the NH2 terminus of mouse proIAPP was used to identify the presence of the NH2-terminally unprocessed proIAPP intermediate form in cells or incubation media. B, NIT-1 mouse ß cells were pulse-labeled with [3H]Leu (300 µCi/ml) for 20 or 30 min in KRB-G16.7 with or without chase in KRB-G1.67 containing 1 mM leucine. C, NIT-1 cells were labeled with [35S]Met (300 µCi/ml) for 20 min and chased for 0, 30, or 120 min, in KRB-G1.67 containing 1 mM methionine, to identify the intermediate form (~6 kDa) detected in panel B. Mouse islets labeled with [35S]Met (pulse 20 min, chase: 0) are shown on the left side for comparison. Cell extracts were immunoprecipitated with anti-IAPP antibody, followed by SDS-PAGE and fluorography. In NIT-1 cells, as in INS-1 cells, partially processed proIAPP (~6 kDa) was detected after a 20-min pulse label (B). This intermediate form contained the labeled methionine residue and therefore was not processed at its NH2 terminus (C).

 
Processing of proIAPP at Its COOH Terminus Is Not Mediated by Furin
Because furin is the major enzyme present in its active form in the early secretory pathway, we investigated whether processing of proIAPP at its COOH terminus in the TGN might be mediated by furin. NIH/3T3 cells, a nonendocrine (fibroblast) cell line that expresses furin but not IAPP, PC1/3 or PC2 (39) were infected with a retrovirus expressing rat proIAPP and (pro)IAPP immunoreactive forms were detected by Western blot analysis of cell lysates. ProIAPP (~8 kDa) was the only IAPP-immunoreactive form detected in NIH/3T3 cells infected with retrovirus (Fig. 4Go), suggesting that initiation of proIAPP processing in the TGN at its COOH terminus is not mediated by furin.



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Fig. 4. Furin Is Unable to Process proIAPP at Its Either NH2- or COOH-Terminal Cleavage Sites

NIH/3T3 cells were transfected with retrovirus coding for the expression of rat proIAPP as described in Materials and Methods. (Pro)IAPP immunoreactive forms were detected in cell lysates by Western blot. A lysate of INS-1 ß cells, which are able to completely process proIAPP to mature IAPP but have detectable amounts of proIAPP, is shown for comparison.

 
ProIAPP and Its Intermediate Form Are Secreted by Both the Constitutive and Regulated Secretory Pathways
To determine whether proIAPP and/or the NH2-terminal proIAPP intermediate are secreted from ß cells, INS-1 cells were pulse-labeled for 30 min and chased for 3 h, with the addition of a ß-cell secretagogue cocktail in the final hour of chase to stimulate granule exocytosis (as detailed in Materials and Methods). Both proIAPP (~8 kDa) and its partially processed form(s) (~6 kDa) were present in the basal chase medium (0–60 min), suggesting that both proIAPP and its intermediate are released via the constitutive secretory pathway. This finding was confirmed in primary islets (Fig. 5AGo). Stimulation (60 min) of granule exocytosis after a 120-min chase resulted in secretion primarily of mature IAPP as well as low levels of unprocessed and partially processed proIAPP, indicating that not only IAPP but also unprocessed and partially processed proIAPP can be released via the regulated secretory pathway. Because small amounts of mature IAPP were observed in the 0- to 60-min basal chase medium (and more in the 60- to 120-min chase medium; Fig. 5AGo), we could not exclude the possibility that some of the newly synthesized (pro)IAPP secreted in the first 60 min of chase was due to basal release of secretory granules. Therefore, to confirm that the proIAPP intermediate (~6-kDa) was indeed released via the constitutive secretory pathway, we performed a shorter pulse-label (15 min) and chased cells for only 45 min in the presence of EGTA (5 mM) or diazoxide (0.5 mM), to inhibit regulated release of granules. Removal of extracellular Ca2+ by EGTA inhibits granule exocytosis and therefore blocks regulated but not constitutive secretion, whereas diazoxide inhibits granule secretion via opening of ATP-dependent K+-channels and hyperpolarization of the ß-cell plasma membrane. Cells chased in low glucose (1.67 mM) either in the absence of Ca2+ or presence of diazoxide, continued to secrete both the 8- and 6-kDa but not 4-kDa form of (pro)IAPP (Fig. 5BGo), confirming that both proIAPP and a partially processed proIAPP intermediate but not mature IAPP are secreted via the constitutive pathway. It therefore appears that secretion of not only unprocessed proIAPP but also its intermediate form can occur via both the regulated and constitutive secretory pathways in INS-1 ß cells.



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Fig. 5. ProIAPP and Its Incompletely Processed Form(s) Are Normal Secretory Products of ß Cells

A, INS-1 ß cells were pulse-labeled with [3H]Leu and [35S]Cys each (200 µCi/ml) in KRB-G16.7 for 30 min and chased in KRB-G1.67 for 3 h. The chase buffer was collected after 60 min (Ch 1), replaced with fresh buffer, and collected after a further 60 min (Ch 2). After 120 min of chase, granule exocytosis was stimulated for 60 min with a cocktail of ß-cell secretagogues as detailed in Materials and Methods. Islets were pulse-labeled with [35S]Cys (230 µCi/100 µl) in KRB-G16.7 for 30 min and chased in KRB-G1.67 for 1 h. B, INS-1 cells were pulse-labeled for 15 min with the same conditions as above and chased for 45 min in low glucose (1.67 mM) with diazoxide (0.5 mM), EGTA (5 mM) or vehicle (control). (Pro)IAPP forms were immunoprecipitated from the chase media and analyzed by SDS-PAGE followed by fluorography.

 
The proIAPP Intermediate Secreted by the Constitutive and Regulated Secretory Pathways Is an NH2 Terminally Unprocessed Form
To determine whether the intermediate form of proIAPP secreted constitutively is NH2-terminally unprocessed proIAPP, we performed additional pulse-chase experiments using [35S]Met as radiolabel. NIT-1 ß cells were pulse-labeled (20 min) followed by chase for different periods for up to 165 min. Both proIAPP and its NH2-terminally extended form were detected in the initial nonstimulated chase medium (0–45 min). To investigate whether the NH2-terminally extended intermediate is also secreted from the regulated secretory pathway, in a parallel experiment, after 120 min of chase, secretory granule release was stimulated by a mixture of ß-cell secretagogues for 60 min. Both proIAPP and its NH2-terminally extended form were present in the stimulated chase media (105–165 min; Fig. 6Go).



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Fig. 6. The proIAPP Intermediate Secreted by the Constitutive and Regulated Secretory Pathways Is an NH2 Terminally Unprocessed Form

NIT-1 cells were pulse-labeled with [35S]-Met (300 µCi/ml) in KRB-G16.7 for 20 min and chased in KRB-G1.67 with 1 mM methionine for 165 min. Media were collected after 45 min and then every 60 min for 2 h (Ch 1, 0–45 min; Ch 2, 45–105 min; Ch 3, 105–165 min). In a parallel study, after 105 min of chase, granule exocytosis was stimulated for 60 min with a cocktail of ß-cell secretagogues (Ch 3+, 105–165 min).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Impaired processing and secretion of proIAPP, the IAPP precursor, have been implicated in islet amyloid formation in type 2 diabetes and insulinomas (2, 11, 12). We therefore sought to understand the kinetics of proIAPP processing and to determine whether incompletely processed forms of proIAPP are secreted from ß cells. Studies from our laboratory and others have shown that, like primary ß cells, INS-1 and NIT-1 ß cells express (pro)IAPP, are able to process both proIAPP and proinsulin to mature peptides, and secrete insulin and IAPP in a glucose-sensitive manner (19, 40, 41, 42). Using a pulse-chase approach, we found that processing of newly synthesized proIAPP in both INS-1 and NIT-1 ß cells is initiated as early as 20 min after synthesis, suggesting that the first cleavage of proIAPP occurs very early in the secretory pathway. This finding was confirmed by pulse-label studies performed on isolated mouse islets, indicating that it is not a peculiarity of transformed cells. We further found that the initial cleavage of proIAPP produces an NH2-terminally unprocessed proIAPP intermediate, indicating that proIAPP is processed first at its COOH terminus. It is worth noting that although our results suggest that the intermediate form detected in cell lysates is primarily NH2-terminally extended proIAPP, we cannot rule out the possibility that low levels of a COOH-terminally extended proIAPP intermediate might also be present. These data are in keeping with Western blot data indicating the presence of an NH2- (but not COOH-) terminally extended form of proIAPP in rodent ß cells (19, 43). Mature IAPP was not detectable until 60 min of chase and the processing of newly synthesized proIAPP appeared to be complete by 120 min, suggesting that the second step in proIAPP processing is slower and occurs later in the secretory pathway, likely in secretory granules. Taken with previous findings (18, 19, 29), these data suggest that production of mature IAPP from proIAPP is a two-step process, initiated predominantly by cleavage at its COOH terminus by the prohormone convertase PC1/3, followed by cleavage of the resulting NH2-terminally extended intermediate by PC2 (Fig. 7Go). Our findings support a model in which the pathway for processing of proIAPP in ß cells resembles that for proinsulin in that both are sequential processes in which PC1/3 first cleaves the intact propeptide to produce an intermediate (des 31,32 proinsulin or NH2-terminally extended proIAPP) followed by PC2 cleavage to form the mature peptide (insulin or IAPP).



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Fig. 7. Proposed Model for proIAPP Processing in the Regulated and Constitutive Secretory Pathways of ß Cells

Normal processing of proIAPP is a two-step process that is initiated by cleavage at its COOH terminus by PC1/3, resulting in the formation of an NH2-terminally unprocessed intermediate form. Cleavage at the COOH terminus of proIAPP may occur before its exit from the TGN resulting in secretion of both intact proIAPP and an NH2-terminally extended intermediate form via the constitutive pathway. Alternatively, the COOH-terminally processed intermediate form may be sorted to granules where it is processed at its NH2 terminus by PC2 to form mature IAPP, which is then released via the regulated secretory pathway along with small amounts of proIAPP and its NH2-terminally unprocessed form.

 
The minimum transit time for newly synthesized proinsulin from the endoplasmic reticulum into immature granules is thought to be about 30 min (44). Therefore, our finding that the NH2-terminally unprocessed proIAPP intermediate was detectable in ß cells only 20 min after synthesis suggests that the initial cleavage of proIAPP must occur before its entry into immature secretory granules, likely in the TGN. Prevention of transport of newly synthesized proIAPP from the TGN into secretory granules, using either BFA or temperature blockade during the chase period, did not inhibit the continued formation of the proIAPP intermediate form, further supporting the idea that COOH-terminal processing of proIAPP can be initiated in the TGN. Although the enzyme furin is active in the TGN, it is unlikely to be involved in initiation of proIAPP processing in the TGN, because expression of proIAPP in cells that express furin but lack PC1/3 and PC2 resulted in formation of only unprocessed proIAPP. Furthermore, proIAPP does not have a consensus furin cleavage sequence (RXK/RR) (45) at either its NH2- or COOH-terminal cleavage sites (13), and furin was unable to cleave proIAPP in an in vitro translation assay (30).

We previously have shown that cleavage at the COOH terminus of proIAPP is preferentially mediated by PC1/3, although PC2 is able to cleave at this site as well (19). Activation of PC1/3 is initiated by autocatalytic cleavage of proPC1/3 at its NH2 terminus in the endoplasmic reticulum, followed by cleavage at its COOH terminus in secretory granules (46, 47). Thus, PC1/3 is present in a partially active state before entry into granules where it is processed to its fully active state (47) under conditions of slightly acidic pH and micromolar concentrations of calcium (48). Such conditions are found early in the secretory pathway (48, 49), suggesting that PC1/3 might be present in an active form in the TGN where it could conceivably initiate processing of proIAPP at its COOH terminus. In fact, the PC1/3-mediated processing of proopiomelanocortin has been shown to occur in the TGN in AtT-20 neuroendocrine cells (38, 46, 50). Therefore, PC1/3 is the most likely candidate for the enzymatic activity initiating proIAPP processing at its COOH terminus in the TGN. When considered with previous findings that proinsulin is cleaved primarily in secretory granules with limited levels of conversion occurring in the TGN and constitutive secretory pathway (21, 51, 52), these data raise the possibility that the initial cleavage of proIAPP may occur earlier than proinsulin in the secretory pathway.

In contrast to PC1/3, activation of PC2 is achieved in a post-Golgi compartment. Maturation of proPC2 is regulated by the neuroendocrine protein 7B2, which binds to proPC2 and facilitates its transport from the endoplasmic reticulum to the TGN. ProPC2 is then activated by autocleavage in low pH and millimolar concentrations of calcium (48, 53, 54, 55), two conditions known to be present in the secretory granules. Unlike PC1/3, the PC2 precursor is enzymatically inert (21). The late activation of this enzyme in the secretory pathway (38, 48) is consistent with our findings because processing of the NH2-terminally extended intermediate, which is dependent on the action of PC2 (18), was not seen until at least 60 min after proIAPP synthesis, and therefore likely occurs in granules.

In this study, we identified the major forms of IAPP secreted from ß cells via the constitutive pathway as proIAPP and its NH2-terminally extended intermediate form. We propose that as a result of COOH-terminal cleavage of proIAPP in the TGN, not only proIAPP but also the NH2-terminally extended proIAPP intermediate could exit the TGN and may either be sorted into granules for further processing before secretion, or released via the constitutive pathway. No mature IAPP is released constitutively when regulated secretion is blocked (Fig. 5BGo) in keeping with the idea that no NH2-terminal processing of proIAPP by PC2 can occur in either the TGN or constitutive vesicles. Previous studies have suggested that under certain conditions, (pro)IAPP may be less efficiently sorted to secretory granules and disproportionately released via the constitutive secretory pathway (17, 26, 27, 29). Thus, in both immature rat ß cells (17, 26) and human islets cultured in high glucose concentrations (27), the selective secretion of immunoreactive IAPP forms via the constitutive secretory pathway may be favored. Missorting and increased secretion of proIAPP and its NH2-terminally extended proIAPP via the constitutive secretory pathway thus remains a plausible mechanism to explain islet amyloid formation in type 2 diabetes and insulinomas (11). Interestingly, NH2-terminally extended proIAPP has been proposed to be a component of islet amyloid (20). We cannot rule out the possibility that proIAPP and/or its NH2-terminally extended form may also be secreted in part from ß cells via the constitutive-like pathway in which small vesicles are thought to release contents primarily from the halo of immature secretory granules (56, 57, 58), where IAPP is thought to reside (59). However, a previous finding that BFA, which inhibits secretion from the constitutive (but not the constitutive-like) pathway (37), prevents calcium-independent release of immunoreactive IAPP from neonatal rat islet cells (17), suggests that the constitutive pathway is the major contributor to secretion of newly synthesized (pro)IAPP under basal conditions.

Stimulation of granule exocytosis resulted in secretion primarily of mature IAPP but also low levels of proIAPP and the NH2-terminally extended intermediate, indicating that these precursors may also be released via the regulated secretory pathway. Because IAPP is processed by the same prohormone convertases (PC1/3 and PC2) (18, 19) that mediate proinsulin processing in ß cell granules (31, 32), any granular defect in proinsulin processing is also likely to affect proIAPP processing. Indeed, secretion of both proinsulin and des 31,32 proinsulin is disproportionately elevated (relative to insulin) in patients with type 2 diabetes (16). Because des 31,32 proinsulin is produced primarily by the action of PC1/3, it is possible that in type 2 diabetes this intermediate may be released from immature granules before PC2 is activated and can complete its processing to insulin and C peptide (15). Because our findings suggest that proIAPP is processed by sequential cleavage at its COOH and NH2 termini by PC1/3 and PC2, respectively, it follows that immature granule secretion before PC2 activation would lead to disproportionate secretion of not only proIAPP but also its NH2-terminally extended intermediate form from granules. We predict, based on these findings, that disproportionate secretion of this proIAPP conversion intermediate will occur in type 2 diabetes and in fact may be more profound than that of des 31,32 proinsulin because PC2 is essential for the NH2-terminal processing of proIAPP (18), whereas PC1/3 can substitute for PC2 in the processing of des 31,32 proinsulin (31). We previously demonstrated that the NH2-terminal cleavage site of proIAPP contains a heparin/heparan sulfate binding domain that may mediate binding of proIAPP (or the NH2-terminally extended intermediate) to glycosaminoglycan side chains of the heparan sulfate proteoglycan perlecan (60), a major component of islet amyloid and cell basement membranes (2). This heparin binding domain is lost during normal NH2-terminal processing of proIAPP to IAPP by PC2 (60). If indeed there is disproportionate secretion of the NH2-terminally extended proIAPP intermediate in type 2 diabetes and insulinomas, binding of this intermediate to perlecan in the basement membrane of islet ß cells or endothelial cells may create a local nidus for amyloid formation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Avertin, collagenase (type XI), deoxyribonuclease, BSA, dextran, dithizone, phenylmethylsulfonyl fluoride, aprotinin, pepstatin A, leupeptin, diazoxide, BFA, forskolin, phorbol 12-myristate 13-acetate, and 3-isobutyl-1-methylxanthine were obtained from Sigma-Aldrich (Oakville, Ontario, Canada); RPMI-1640, Ham’s-F10, F12-K medium, DMEM, trypsin-EDTA, Hanks’ balanced salt solution (HBSS), fetal bovine serum from Invitrogen Canada Inc. (Burlington, Ontario, Canada), and protein G Sepharose beads from Amersham Biosciences (Baie d’Urfe, Quebec, Canada). Radiolabeled amino acids ([3H]leucine, [35S]cysteine, and [35S]methionine) were from American Radiolabeled Chemicals (St. Louis, MO) and autoradiography enhancer was from PerkinElmer Life Sciences (Woodbridge, Ontario, Canada). All electrophoresis chemicals were from Bio-Rad Laboratories (Mississauga, Ontario, Canada). Antirodent IAPP antibody (RGG-7323) was obtained from Peninsula Laboratories (Belmont, CA).

Cell Cultures
INS-1 (832/13) cells, a transformed rat ß-cell line, were a generous gift of Dr. Christopher Newgard (Duke University Medical Center, Durham, NC). NIT-1 cells, a ß cell line derived from an islet tumor in a transgenic NOD-Lt mouse (40) and NIH/3T3 cells, a mouse fibroblast cell line established from mouse embryo, were obtained from the American Type Culture Collection (ATCC; Manassas, VA). INS-1 cells were grown in RPMI-1640 medium and NIT-1 cells in F12-K medium containing 11.1 and 7 mmol/l glucose, respectively, supplemented with 10% fetal bovine serum, ß-mercaptoethanol (50 µM; for INS-1 only), penicillin (50 U/ml) and streptomycin (50 µg/ml) at 37 C in a humidified atmosphere of 95% air 5% CO2. NIH/3T3 cells were grown in DMEM containing 10% calf serum.

Islet Isolation
C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Islets were isolated by collagenase digestion. Briefly, animals (8–10 wk old) were anesthetized with Avertin as described previously (34) and killed by cervical dislocation. Then 2.5 ml ice-cold collagenase (type XI) in HBSS (525 U/ml) was injected via the common bile duct. The pancreas was harvested and incubated with collagenase/HBSS (525 U/ml) for 9 min at 37 C. The digestion was stopped by addition of ice-cold HBSS containing 0.1% BSA. Digested pancreatic tissue was homogenized by passing through a glass Pasteur pipette and filtered through 800-µm mesh. After partial purification on a dextran gradient, islets were washed with HBSS and hand-picked under a dissecting microscope. Purity of the islets as assessed by dithizone staining was greater than 95% in all experiments. Islets were cultured in Ham’s-F10 containing 0.5% BSA and 10 mM glucose overnight to recover before radiolabel studies.

Pulse-Chase Experiments
INS-1 or NIT-1 ß cells (equal number of cells cultured in 25-cm2 flasks) were preincubated in Krebs-Ringer bicarbonate (KRB) buffer containing 10 mM HEPES (pH: 7.4), 0.25% BSA, and 16.7 mM glucose (KRB-G16.7) for 15 min at 37 C. Cells were then pulse-labeled in KRB-G16.7 containing [35S]Met (specific activity: 1175 Ci/mM) or [3H]Leu (110 Ci/mM) alone or with [35S]Cys (1075 Ci/mM) for different time periods (10–30 min). After pulse-label, cells were washed two times with ice-cold PBS and then chased (0–180 min) in KRB buffer containing 1.67 mM glucose (KRB-G1.67) plus 1 mM methionine, cysteine, and/or leucine for various time periods and conditions as detailed in each figure legend. Radiolabeling was performed at 16.7 mM glucose to stimulate proIAPP biosynthesis, whereas chase experiments (nonstimulated) were performed at basal (1.67 mM) glucose to minimize regulated secretion of (pro)IAPP. To stimulate release of newly synthesized (pro)IAPP via the regulated pathway, cells were incubated in KRB-G16.7 with a secretagogue mixture containing 1 mM 3-isobutyl-1-methylxanthine, 10 µM forskolin, 0.1 µM phorbol 12-myristate 13-acetate, and 1 mM leucine and arginine. The chase or stimulated incubation media were then collected and centrifuged to remove cellular debris. Cells were harvested with trypsin-EDTA and lysed in 160 µl lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 20 µg/ml leupeptin, for 25 min on ice, vortexed every 5 min. Samples were centrifuged (15,000 x g, 10 min, 4 C) and the supernatants (or chase media) frozen at –70 C until analysis.

For experiments on primary ß cells, batches of 300–350 isolated mouse islets were preincubated in 0.5 ml of KRB-G16.7 for 30 min at 37 C and then pulse-labeled (20 or 30 min) in 100 µl of the same buffer containing [35S]Cys or [35S]Met and chased for 0 or 120 min. Islets were then washed two times with ice-cold KRB buffer and lysed in 50 µl of lysis buffer as described earlier. Protein concentration in the islet or cell lysates was measured using the BCA protein assay (Pierce, Rockford, IL). All experiments were repeated at least three times.

Immunoprecipitation and Electrophoresis
Cell extracts containing equal amounts of total protein (650–850 µg) or islet extracts (100–110 µg) and chase media (1 ml) were incubated with 4 µg of IgG purified antirodent IAPP antibody (RGG-7323, Peninsula) for 2.5 h (4 h for media) followed by 1.5 h incubation with 70 µl protein-G Sepharose beads (50% slurry in lysis buffer) at 4 C. The protein G-Sepharose immunocomplex was washed three times with lysis buffer and the immunoprecipitated samples from cell lysates or chase media were heated (100 C) in Laemmli’s sample buffer for 5 min. Samples were electrophoresed on a polyacrylamide gel using Tris-tricine buffer for separation of small proteins (61). Gels were fixed in 10% glacial acetic acid and 30% methanol (vol/vol) for 1 h, soaked in a fluorographic enhancing solution (EN3HANCE; PerkinElmer) for 1 h, and then in ice-cold water (0.5 h) to precipitate the fluorescent material inside the gel. Dried gels were exposed to Kodak (Rochester, NY) X-OMAT film at –70 C for 4–6 d.

Transduction with Retrovirus Overexpressing Rat proIAPP
A cDNA construct encoding rat proIAPP (NM012586) was subcloned into the retroviral vector MSCVpac (62) and a high-titer virus was generated as described previously (63). Briefly, the virus packaging BOSC23 cells were transfected with the retroviral vector MSCVpac containing the coding region for proIAPP. The supernatant containing retrovirus-proIAPP particles was used to infect NIH/3T3 cells (8 h). The NIH/3T3 cells constitutively expressing rat proIAPP were selected by culture in the presence of puromycin (2 µg/ml) for 3 d. ProIAPP immunoreactive forms were detected in cell lysates from control and transfected NIH/3T3 cells by Western blot.


    ACKNOWLEDGMENTS
 
We thank Dr. Christopher Newgard (Duke University Medical Center, Durham, NC) for INS-1 (832/13) cells, Dr. Robert Kay (Terry Fox Laboratories, University of British Columbia, Vancouver, British Columbia, Canada) for providing NIH/3T3 cells and his expert advice on preparation of retrovirus. Technical support of Ms. Ghazaleh Tazmini (Terry Fox Laboratories) is gratefully acknowledged. We also would like to thank Dr. Philippe Halban (University of Geneva, Geneva, Switzerland) and Dr. Christopher Rhodes (Pacific Northwest Research Institute, Seattle, WA) for their helpful suggestions in the design of these studies.


    FOOTNOTES
 
This work was supported by the Canadian Institutes of Health Research Grant MT-14682 (to C.B.V.). L.M. is supported by a Postdoctoral Fellowship Award from the Canadian Diabetes Association.

First Published Online March 31, 2005

Abbreviations: BFA, Brefeldin A; CPE, carboxypeptidase E; HBSS, Hanks’ balanced salt solution; IAPP, islet amyloid polypeptide; KRB, Krebs-Ringer bicarbonate; PC, prohormone convertase; TGN, trans-Golgi network.

Received for publication October 12, 2004. Accepted for publication March 25, 2005.


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