Role of Carboxypeptidase E in Processing of Pro-Islet Amyloid Polypeptide in ?-Cells
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内分泌学杂志 2005年第4期
Department of Pathology and Laboratory Medicine and 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@interchange.ubc.ca.
Abstract
Islet amyloid polypeptide (IAPP; amylin) is a peptide hormone that is cosecreted with insulin from ?-cells. Impaired processing of proIAPP, the IAPP precursor, has been implicated in islet amyloid formation in type 2 diabetes. We previously showed that proIAPP is processed to IAPP by the prohormone convertases PC1/3 and PC2 at its carboxyl (COOH) and amino (NH2) termini, respectively. In this study, we investigated the role of carboxypeptidase E (CPE) in the processing of proIAPP using mice lacking active CPE (Cpefat/Cpefat) and NIT-2 cells, a ?-cell line derived from their islets. Western blot analysis demonstrated that an approximately 6-kDa NH2-terminally unprocessed form of proIAPP was elevated approximately 86% in islets from Cpefat/Cpefat mice, compared with wild type. This increase was independent of the development of hyperglycemia (8 wk male) or obesity (18 wk female). Impaired proIAPP processing was associated with a decrease in PC2 (but not PC1/3) and both the 21- and 27-kDa forms of the PC2 chaperone protein 7B2, suggesting that PC2-mediated processing of proIAPP at its NH2 terminus was impaired in the absence of CPE. Formation of COOH-terminally amidated (pro)IAPP was reduced approximately 75% in NIT-2, compared with NIT-1 ?-cells, supporting a direct role for CPE in maturation of IAPP by removal of its COOH-terminal dibasic residues, the step essential for IAPP amidation. We conclude that lack of CPE in islet ?-cells results in a marked decrease in processing of proIAPP at its NH2 (but not COOH) terminus that is associated with attenuated levels of PC2 and (pro)7B2 and a great reduction in formation of mature amidated IAPP.
Introduction
ISLET AMYLOID POLYPEPTIDE (IAPP; amylin) (1, 2) is a neuroendocrine peptide hormone that is produced and cosecreted with insulin from pancreatic ?-cells (3, 4, 5). In type 2 diabetes, aggregation of this peptide results in the formation of islet amyloid deposits, which are toxic to ?-cells (6) and likely contribute to the progressive loss of ?-cells in this disease (5, 7, 8). Despite considerable study, it is still not understood why amyloid deposits form in type 2 diabetic patients. Elevated secretion of IAPP from ?-cells associated with an increased demand for insulin in this disease is likely an important contributor but not sufficient for amyloid formation (5, 7, 9, 10, 11, 12). It has been proposed that impaired processing of proIAPP, the IAPP precursor, in type 2 diabetes may lead to hypersecretion of unprocessed and/or partially processed form(s) of proIAPP that have a higher tendency for aggregation than mature IAPP and initiate amyloid formation (7, 13, 14, 15, 16, 17). Therefore, identifying the precise mechanism(s) by which proIAPP is processed in ?-cells in vivo is of considerable interest.
Pro-IAPP is processed by cleavage at basic residue pairs at both its amino (NH2) and carboxyl (COOH) termini (18) before its secretion as mature IAPP along with insulin (3, 4). We recently showed that normal processing of proIAPP in ?-cells is initiated by cleavage at its COOH terminus by the prohormone convertase PC1/3 to produce an NH2-terminally unprocessed intermediate proIAPP, which is subsequently cleaved in secretory granules by PC2 (19, 20). Previous studies have shown that processing of insulin is similarly initiated by cleavage at its B-chain/C-peptide junction, preferentially by PC1/3 followed by cleavage of the resulting conversion intermediate, des 31, 32 proinsulin, at the C-peptide/A-chain junction by PC2 (21, 22). It therefore appears that proinsulin and proIAPP processing may be similar in many ways, supporting the idea that like proinsulin (23, 24), processing of proIAPP might also be impaired in type 2 diabetes. Both subtilisin-like proprotein convertases PC1/3 and PC2 are colocalized with (pro)insulin and (pro)IAPP in ?-cell secretory granules (25, 26). After cleavage of proinsulin by PC1/3 or PC2, the remaining COOH-terminal basic residues are removed from the B-chain (Arg31-Arg32) and C-peptide (Lys64-Arg65) by carboxypeptidase E (CPE) resulting in the formation of mature insulin and C-peptide (27). CPE (EC 3.4.17.10), also known as carboxypeptidase H, is a metallocarboxypeptidase that is present in numerous neuroendocrine tissues including pancreatic ?-cells and is mainly localized in the secretory granules (27, 28, 29). As with proinsulin, after cleavage of proIAPP by PC1/3 the remaining COOH-terminal basic residues (Lys-Arg) are likely removed by a carboxypeptidase, a prerequisite for carboxyamidation of IAPP by the granule enzyme, peptidyl amidating monooxygenase complex (PAM) and formation of mature IAPP (30, 31).
Mice lacking active CPE (Cpefat/Cpefat) have been described previously (32) and shown to have a spontaneous point mutation (Ser202 to Pro) within the coding region of the CPE gene (Cpe), which leads to inactivation of the enzyme and degradation of the protein (32, 33). Cpefat/Cpefat mice are characterized by multiple endocrine disorders including maturity-onset obesity, infertility, and hyperproinsulinemia; diabetes develops only in males (32). Lack of active CPE in this animal model results in impaired processing of a number of neuroendocrine and endocrine prohormones including proinsulin (32), proglucagon, (34), pro-GnRH (35), procholecystokinin (36), pro-TRH (37), proopiomelanocortin, prodynorphin (38), proenkephalin, chromogranin A and B, secretogranin II (39), and progastrin (40). In the present study, we used islets from Cpefat/Cpefat mice and NIT-2 cells, a ?-cell line derived from their islets (41) to investigate the role of CPE in the processing of proIAPP in ?-cells.
Research Design and Methods
Materials
Collagenase (type XI); DNase; BSA; phenylmethylsulfonyl fluoride; dextran; dithizone; 2,2,2,tribromoethanol; 2-methyl-2-butanol; and aprotinin were obtained from Sigma-Aldrich (Oakville, Ontario, Canada); Hanks’ balanced salt solution (HBSS), F12-K medium, and fetal bovine serum from Invitrogen Canada Inc. (Burlington, Ontario, Canada), and protein G Sepharose beads from Amersham Biosciences (Baie d’Urfe, Québec, Canada). Rodent IAPP 1–37 was obtained from Bachem (Torrance, CA). [3H]leucine and [35S]cysteine were from American Radiolabeled Chemicals (St. Louis, MO). All electrophoresis chemicals were from Bio-Rad Laboratories (Hercules, CA).
Animals
Heterozygous Cpefat/+ mice (BKS.HRS-Cpefat/+/J) were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility of the BC Research Institute for Children’s and Women’s Health. Offspring were genotyped by PCR on genomic DNA prepared from tail biopsies according to the protocol supplied by the Jackson Laboratory. The animals were maintained on 9% fat diet (PMI Feeds Inc., Richmond, IN). Sex- and age-matched (8 and 18 wk old) mice were used in all experiments. The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.
Plasma measurements
Blood glucose levels were measured in blood sampled from the tail vein of fed mice between 0900 and 1000 h using a ONETOUCH glucometer from LifeScan (Milpitas, CA). Plasma insulin immunoreactivity (insulin plus proinsulin) was determined by a mouse insulin ELISA kit from ALPCO Diagnostics (Windham, NH).
Cell lines
NIT-1 cells, a ?-cell line derived from a transgenic nonobese diabetic (NOD/Lt) mouse (42), and NIT-2 cells, a ?-cell line derived from male Cpefat/Cpefat mice (41), were obtained from the American Type Culture Collection (Manassas, VA).
Antisera and recombinant adenoviruses
Antisera specific for the NH2-terminal and COOH-terminal regions of murine proIAPP were generated as previously described (19). IgG-purified antirodent IAPP antibody (RGG-7323) was obtained from Peninsula Laboratories (Belmont, CA). Rabbit antisera against the NH2-terminal region of PC1/3 and 7B2 were generous gifts from Dr. I. Lindberg (Louisiana State University, New Orleans, LA) and antiserum against COOH-terminal of PC2 from Dr. C. Rhodes (Pacific Northwest Research Institute, Seattle, WA). Adenovirus expressing PC2 (Ad-PC2) and 21-kDa 7B2 (Ad-7B2) were kindly provided by Dr. P. Halban (University of Geneva, Geneva, Switzerland) and Dr. I. Lindberg, respectively.
Islet isolation
Islets were isolated from mouse pancreas by collagenase digestion. Briefly, animals were anesthetized with Avertin (0.02 ml/g body weight, ip) consisting of 1.25% (wt/vol) 2,2,2,tribromoethanol, 2.5% (vol/vol) 2-methyl-2-butanol in distilled water, and killed by cervical dislocation. The abdominal cavity was opened, and 2.5 ml ice-cold collagenase (type XI) in HBSS (final concentration 525 U/ml) injected via the common bile duct. The pancreas was harvested and incubated with collagenase/HBSS (525 U/ml) for 9 min at 37 C (gentle shaking). 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 approximately 20 times and filtered through 800 μm mesh (Spectrum Laboratories, Rancho Dominguez, CA). After purification on a dextran gradient, islets were washed with HBSS/BSA and handpicked under a dissecting microscope. Purity of the islets as assessed by dithizone staining was greater than 95% in all experiments. For immunoblot experiments, approximately 300 freshly isolated islets pooled from two to three mice from each genotype were washed with PBS and lysed in 40 μl lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate for 25 min on ice. Samples were centrifuged (15,000 x g, 10 min, 4 C) and the supernatants frozen at –70 C until assayed. Total protein in the islet lysates was determined using the BCA assay (Pierce, Rockford, IL).
Cotransduction with Ad-PC2 and Ad-7B2 and metabolic labeling
NIT-2 cells were cultured in F12-K medium containing 7 mM glucose supplemented with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 μg/ml). Cells at approximately 70% confluency in 25-cm2 flasks were transduced with Ad-PC2 at multiplicity of infection 5.4 pfu/cell in 1 ml F12-K for 2 h at 37 C or cotransduced with Ad-PC2 plus Ad-7B2 (21 kDa). To assess transduction efficiency, NIT-2 cells were transduced with adenovirus expressing LacZ for 2 h at the same multiplicity of infection (5.4 pfu/cell), and after 24 h transduced cells were washed and fixed in 0.5% glutaraldehyde. The proportion of cells expressing ?-galactosidase was determined after incubation with the substrate X-gal (5-bromo-4-chloro-3-indole-?-D-galactoside). The transduction efficiency with adenovirus expressing LacZ was approximately 50% and was somewhat variable among different cells, with a few cells expressing large amounts of this marker, perhaps reflecting the nonclonal nature of the NIT-2 cell line.
Twenty-four hours after transduction with Ad-PC2 or Ad-PC2 plus Ad-7B2, cells were washed and preincubated with Krebs-Ringer bicarbonate (KRB) buffer containing 10 mM HEPES (pH 7.4), 16.7 mM glucose, 0.25% BSA (KRB-G16.7) for 15 min at 37 C. Cells were then pulse labeled (30 min) in 1 ml KRB-G16.7 containing 200 μCi/ml [3H]leucine (specific activity 110 Ci/mM) and 150 μCi/ml [35S]cysteine (1075 Ci/mM) for 30 min, washed two times with ice-cold PBS, and chased (60 min) in KRB buffer containing 1.67 mM glucose (KRB-G1.67) plus 1 mM cysteine and leucine. Cells were harvested with trypsin-EDTA and lysed with 150 μl lysis buffer as described. Cell extracts (500 μg) were precleared by incubation with 50 μl protein G-Sepharose beads (Amersham) for 45 min at 4 C. The supernatants were incubated with 5 μl of NH2-terminal proIAPP antiserum for 2 h followed by 1 h incubation with 50 μl of protein-G Sepharose beads at 4 C. The protein G-Sepharose immunocomplex was washed three times with lysis buffer and used for SDS-PAGE.
Electrophoresis and immunoblotting
Immunoprecipitated samples or aliquots of protein (10 or 15 μg) from islet or cell extracts were heated (100 C, 5 min) with Laemmli’s sample buffer and electrophoresed on 10% (PC1/3 and PC2) or 15% (7B2) polyacrylamide gels using Tris-tricine buffer for IAPP to enable separation of small proteins (43). Proteins were transferred to polyvinyl difluoride membrane using a Trans-Blot semidry transfer cell (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk for 1 h, washed, and incubated for 1 h at room temperature with appropriate antisera at the following dilutions: IgG-purified antirodent IAPP at 2 μg/ml, anti-PC2 and anti-7B2 at 1:1000; anti-PC1/3 at 1:2000; and proIAPP antisera specific for the COOH- and NH2-terminal regions at 1:100. Membranes were then washed and incubated (1 h) with horseradish peroxidase-conjugated antirabbit IgG (Amersham) diluted 1:5000 (or 1:2000 for anti-PC1/3) at room temperature. Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham). Protein bands on films (X-OMAT, Kodak, Rochester, NY) were analyzed by densitometry using Quantity One 4.2.1 (Bio-Rad Laboratories, Hercules, CA). Immunoprecipitated proteins from NIT-2 cell extracts were electrophoresed on a polyacrylamide gel using Tris-tricine buffer as described and the separated proteins detected by fluorography using EN3HANCE (Perkin-Elmer Life Sciences, Woodbridge, Ontario, Canada) followed by exposure to Kodak X-OMAT film at –70 C for 3 d.
Double immunostain for insulin and somatostatin
Pancreas from 17- to 18-wk-old female Cpefat/Cpefat mice were fixed in 4% paraformaldehyde in 0.1 mol/liter PBS (pH 7.5) for 1 h and washed in 70% ethanol. Paraffin-embedded sections (5 μm) were blocked in 0.05 mol/liter PBS plus 0.25% Triton X-100 containing 2% normal goat serum (Vector Laboratories, Burlingame, CA), washed, and incubated with mouse antisomatostatin (a gift from Dr. C. McIntosh, Vancouver, British Columbia, Canada) at a 1:1000 dilution in the same buffer containing 1% BSA for 1 h at room temperature and then washed and incubated (1 h) with goat antimouse antibody conjugated to the green fluorophore, Alexa Fluor 488 (Molecular Probes, Eugene, OR). Sections were then similarly incubated with guinea pig antiinsulin antibody (Dako, Carpinteria, CA) and goat antiguinea pig antibody conjugated to Texas Red (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h (1:100). The slides were viewed using an Axioplan 2 microscope (Carl Zeiss, New York, NY) equipped for epifluorescence with a Sensys high-performance charge-coupled device camera (Photometrics, Tucson, AZ) and Quips Pathvysion imaging software (Applied Imaging, Santa Clara, CA). Images were captured with green and red filters and merged to give the final image.
ELISA for detection of amidated IAPP
NIT-1 and NIT-2 cells were boiled in 1 M acetic acid plus 0.1% BSA for 10 min and the lysates centrifuged (15,000 x g, 10 min, 4 C) to remove the cell debris. The cell content of amidated IAPP was measured in the diluted supernatants (1:200) by an amylin ELISA kit (EZHA-52K) from Linco Research Inc. (St. Charles, MO). The capture antibody (F002) used in this assay binds to all molecular forms of (pro)IAPP, whereas the detection antibody (F025) recognizes only the amidated form of IAPP (44). Total protein levels were matched in the samples.
Statistical analysis
Values are expressed as the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a Newman-Keuls test or by Student’s t test as appropriate. P < 0.05 was taken as the level of significance.
Results
Metabolic characteristics of Cpefat/Cpefat and wild-type mice
Body weight, blood glucose, and plasma insulin levels of animals are summarized in Table 1. All parameters were measured in nonfasted animals (0900–1000 h) on the termination day. As shown previously (32), there was a significant elevation in body weight in 8-wk-old male Cpefat/Cpefat, compared with wild-type mice. Although Cpefat/Cpefat female mice were already heavier than their wild-type littermates by 8 wk of age, this difference was more profound in 18-wk-old animals. As expected, Cpefat/Cpefat mice of both sexes had markedly high levels of plasma insulin immunoreactivity, whereas only male Cpefat/Cpefat mice developed hyperglycemia.
TABLE 1. Metabolic characteristics of Cpefat/Cpefat and wild-type mice at termination (n = 7–9/group)
Pro-IAPP processing is impaired in islets from Cpefat/Cpefat mice lacking CPE
To investigate the role of CPE in proIAPP processing, Western blot analysis was performed on islet extracts from hyperglycemic male and normoglycemic female Cpefat/Cpefat mice as well as their heterozygous and wild-type littermates. Animals were studied before (8 wk old) and after (18 wk old) the development of the fat phenotype. The antibody used for these studies recognizes both unprocessed and processed forms of proIAPP. Consistent with our previous studies (19, 20), the major form of IAPP immunoreactivity in the islets from wild-type mice was fully processed IAPP (4 kDa) (Fig. 1A). Approximately 72, 22, and 6% of total IAPP immunoreactivity was composed of mature IAPP, partially processed, and unprocessed proIAPP, respectively (Fig. 1B). Interestingly, although islets from Cpefat/Cpefat mice were able to process proIAPP to mature IAPP, a partially processed intermediate form of proIAPP with a molecular mass of approximately 6 kDa was elevated by 86% in Cpefat/Cpefat mouse islets, compared with wild-type mice (Fig. 1). Therefore, in Cpefat/Cpefat mouse islets, approximately 53, 40, and 7% of total IAPP immunoreactivity was composed of IAPP, partially processed, and unprocessed proIAPP, respectively (Fig. 1B). This increase in partially processed proIAPP was observed to a similar extent in islets from both hyperglycemic male Cpefat/Cpefat mice and normoglycemic females before and after the development of overt obesity.
FIG. 1. Pro-IAPP processing is impaired in the absence of active CPE in islet ?-cells. A, Islets were isolated from age-matched homozygous Cpefat/Cpefat (–/–), heterozygous (+/–), and wild-type (+/+) mice. Western blot analysis was performed on islet extracts (10 μg) from hyperglycemic male (8 wk old) and normoglycemic female (8 and 18 wk old) mice using antirodent IAPP antibody (Peninsula). A partially processed intermediate form(s) of (pro)IAPP (6 kDa) is elevated in islets from Cpefat/Cpefat mice, compared with heterozygous and wild-type mice. B, Densitometric analysis of immunoblots from three independent experiments. Results are presented as the percentage of each IAPP-immunoreactive molecular form, with total IAPP immunoreactivity taken as 100%. Data are expressed as mean ± SEM *, Significantly different from corresponding molecular form in wild-type islets (P < 0.05, ANOVA).
Lack of CPE results in impaired processing at the NH2-terminal cleavage site of proIAPP in islet ?-cells
To identify the partially processed intermediate form of proIAPP elevated in Cpefat/Cpefat mouse islets, we used antisera raised against the NH2- and COOH-terminal flanking regions of proIAPP (19). Western blot analysis using these antisera revealed that an NH2-terminally unprocessed intermediate form of proIAPP (6 kDa) was markedly increased in islets from both normoglycemic (female) and hyperglycemic (male) Cpefat/Cpefat mice (Fig. 2A). By contrast, the COOH-terminal proIAPP antiserum detected only low levels of a COOH-terminally unprocessed intermediate form (6 kDa) in both Cpefat/Cpefat and wild-type mouse islets (Fig. 2B). Therefore, in islets from Cpefat/Cpefat mice, processing of proIAPP is impaired at its NH2-terminal cleavage site but appears to be normal at its COOH terminus. The COOH-terminal antiserum also detected small amounts of unprocessed (8 kDa) proIAPP in islets from both mutant and wild-type mice. Low levels of proIAPP could also be detected by the NH2-terminal antiserum after a longer exposure of the film (data not shown).
FIG. 2. Lack of active CPE results in impaired processing at the NH2-terminal cleavage site of proIAPP. Western blot analysis was performed on islet extracts (15 μg) from Cpefat/Cpefat mice and their heterozygous and wild-type littermates using antisera specific for the COOH or NH2 termini of mouse proIAPP. A representative blot from four independent experiments is shown. Note the elevated levels of an NH2-terminally unprocessed proIAPP intermediate (6 kDa) detected in Cpefat/Cpefat mouse islets, compared with the almost undetectable levels of this intermediate in islets from heterozygous and wild-type mice (A). The COOH-terminal antiserum detected similar levels of the COOH-terminally unprocessed intermediate form in islets from all three genotypes studied (B). Small amounts of unprocessed (8 kDa) proIAPP were detected by the COOH-terminal proIAPP antiserum.
It is worth noting that in mice, (pro)IAPP is also expressed along with somatostatin in islet -cells (45), although these cells normally form a small proportion of total islet cells. To determine whether an increased proportion of - relative to ?-cells may be present in Cpefat/Cpefat mouse islets, thus possibly contributing to the (pro)IAPP immunoreactivity that we observed in this study, pancreatic sections from female Cpefat/Cpefat mice were double immunostained for insulin and somatostatin (Fig. 3). Despite the larger size and greater number of both ?-cells and -cells in islets from Cpefat/Cpefat mice, compared with islets from their heterozygous and wild-type littermates, there did not appear to be any noticeable difference between the proportion of ?-cells relative to -cells in islets from the three different genotypes.
FIG. 3. Immunofluorescent staining of pancreatic sections from female Cpefat/Cpefat mice for insulin and somatostatin. Paraffin-embedded pancreatic sections from female Cpefat/Cpefat mice (17–18 wk old) and their heterozygous and wild-type littermates were double immunostained for the presence of insulin (red) and somatostatin (green) immunoreactivity. Despite the larger size and greater number of both ?- and -cells in islets from Cpefat/Cpefat mice, compared with those from their heterozygous and wild-type littermates, the proportion of somatostatin-producing -cells to insulin-producing ?-cells appears to be approximately the same in islets from mice of all three genotypes. Figures are representative of immunostains performed on pancreatic sections from three animals of each genotype.
Impaired proIAPP processing in the absence of CPE is associated with reduced levels of 7B2 and PC2 but not PC1/3 in ?-cells
We recently showed that proIAPP is processed by sequential cleavage at its COOH- and NH2-termini by PC1/3 and PC2, respectively (19, 20). To determine whether impaired processing of proIAPP in ?-cells that lack CPE is associated with changes in either one or both of these prohormone convertases, we examined the protein levels of PC1/3, PC2 and its binding protein 7B2 by Western blot. Because relatively high levels of PC2 are also expressed in -cells, the level of PC2 observed in immunoblots of islet extracts may not reflect that in ?-cells. We therefore looked at PC2 protein expression in a transformed ?-cell line (NIT-2) that is derived from Cpefat/Cpefat mice and lacks CPE. Unlike NIT-1 ?-cells, which express CPE and in which mature PC2 was the major form of PC2-immunoreactivity detected, NIT-2 cells contained mainly proPC2 and very low levels of mature PC2, suggesting that processing of proPC2 is impaired in NIT-2 cells (Fig. 4A). This decrease in mature PC2 level was associated with a decrease in both pro7B2 and the 21-kDa NH2-terminal 7B2 protein fragment, and a small but statistically insignificant rise in the ratio of pro7B2 to 7B2 (Fig. 4B). Therefore, total (pro)7B2 immunoreactivity was lower in NIT-2 cells, compared with NIT-1 cells. By contrast, PC1/3 levels in NIT-2 cells were comparable with those in control NIT-1 cells (Fig. 5A). We also examined PC1/3 levels in islets of Cpefat/Cpefat mice because unlike PC2, PC1/3 is expressed mainly in ?-cells, and therefore, results of immunoblots performed on islets will reflect PC1/3 protein levels in ?-cells. PC1/3 levels were decreased only in the male Cpefat/Cpefat islets but were comparable in islets from normoglycemic female Cpefat/Cpefat and wild-type mice, although a slight but significant increase (n = 4, P < 0.05) in the levels of PC1/3 precursor, proPC1/3, was detected in the islets from 18-wk-old (but not 8 wk) females (Fig. 5B).
FIG. 4. Mature forms of PC2 and 7B2 are reduced in NIT-2 ?-cells that lack CPE. Western blot analysis was performed on NIT-1 and NIT-2 ?-cell extracts for PC2 (A) and 7B2 (B) as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading marker. The right panels show the densitometric analyses of immunoblots from four independent experiments. Levels of mature PC2 and both NH2-terminal 7B2 (21 kDa) and its precursor molecule, pro7B2, were significantly lower in NIT-2 cells that lack active CPE, compared with the control (NIT-1) cells, whereas levels of pro-PC2 were approximately the same in the two cell lines. Total (pro)PC2 and (pro)7B2 immunoreactivity in NIT-2 cells were thus decreased, compared with NIT-1 cells. Results are presented as the percentage of each PC2 (or 7B2) immunoreactive molecular form, with total PC2 (or 7B2) immunoreactivity taken as 100%. Data are expressed as mean ± SEM. *, Significantly different from corresponding molecular form in NIT-1 cells (P < 0.05, Student’s t test).
FIG. 5. PC1/3 levels are reduced in islets from hyperglycemic male but not normoglycemic female Cpefat/Cpefat mice. Western blot analysis of cell extracts from NIT-1 and NIT-2 ?-cells (A) or islet extracts from Cpefat/Cpefat mice and their heterozygous and wild-type littermates (B) is shown. PC1/3 protein levels were comparable in (control) NIT-1 and NIT-2 cells that lack CPE and islets from normoglycemic female Cpefat/Cpefat and wild-type mice but were reduced in hyperglycemic male Cpefat/Cpefat mice, suggesting that the decrease in islet PC1/3 content is likely secondary to hyperglycemia and not the absence of CPE per se. Blots are representative of four independent experiments.
To determine whether overexpression of proPC2 or its chaperone protein 7B2 could rescue proIAPP processing in the absence of CPE, NIT-2 cells were transduced with Ad-PC2 alone (46) or cotransduced with Ad-PC2 and Ad-7B2 (21 kDa), pulse labeled with [3H]leucine/[35S]cysteine (30 min) and chased for 60 min to allow processing of the newly synthesized proIAPP to mature IAPP (Marzban, L., and C. B. Verchere, unpublished data), followed by immunoprecipitation with an antiserum specific for the NH2-terminal flanking region of proIAPP. Although NIT-2 cells transduced with Ad-PC2 produced markedly more proPC2, they were unable to process the precursor to generate more mature PC2 (Fig. 6A). Furthermore, adenoviral-mediated expression of 21-kDa 7B2 (Fig 6, A and B) did not improve impaired proPC2 processing. Accordingly, the adenoviral overexpression of proPC2 alone or along with 21-kDa 7B2 in NIT-2 cells did not enhance proIAPP processing, having no effect on the elevated levels of NH2-terminally unprocessed proIAPP in those cells (Fig. 6C).
FIG. 6. Processing of the NH2-terminal intermediate form of proIAPP in NIT-2 cells overexpressing pro-PC2 and 21-kDa 7B2. NIT-2 cells were transduced with recombinant adenovirus expressing pro-PC2 (Ad-PC2; 5.4 pfu/cell) or cotransduced with Ad-PC2 and Ad-7B2 (21 kDa). (Pro)PC2 (A) and (pro)7B2 (B) levels in transduced NIT-2 cells were detected by Western blot. Expression of (pro)PC2 in Ad-PC2 transduced NIT-1 cells is shown on the right side for comparison. C, Transduced cells were pulse labeled with [3H]leucine and [35S]cysteine for 30 min and chased for 60 min, by which time very little newly synthesized proIAPP and small amounts of NH2-terminal intermediate are normally observed. Pro-IAPP and its NH2-terminally unprocessed intermediate form were immunoprecipitated from cell lysates using an NH2-terminal antiserum followed by SDS-PAGE and fluorography. Pro-PC2 expressed in transduced NIT-2 cells remained mainly unprocessed in the presence or absence of 7B2 and was thus unable to restore impaired NH2-terminal processing of proIAPP. Experiments were repeated twice with identical results.
Formation of amidated IAPP is markedly decreased in the absence of CPE in ?-cells
Removal of the COOH-terminal dibasic residues (Lys-Arg) is essential for subsequent amidation of IAPP and formation of mature peptide (30). Therefore, IAPP that is correctly processed at its COOH terminus will be rapidly amidated in ?-cell granules, whereas IAPP that has not had its COOH-terminal extended basic residues removed will remain nonamidated. To investigate whether CPE is the major carboxypeptidase responsible for removal of the dibasic residues from the COOH terminus of IAPP in ?-cells, we measured the concentration of amidated IAPP in NIT-1 and NIT-2 ?-cell extracts using an ELISA that detects COOH-terminally amidated forms of IAPP immunoreactivity (44, 47). The capture antibody used in this assay is predicted to bind to all molecular forms of (pro)IAPP, regardless of modifications of its termini, whereas the detection antibody recognizes only the amidated form of IAPP (44) (Fig. 7A). The level of amidated IAPP was reduced approximately 75% in NIT-2 cells, compared with NIT-1 cells (Fig. 7B), suggesting that lack of CPE results in marked impairment in removal of the COOH-terminal dibasic residues in IAPP. Based on Western blot analysis, expression of IAPP was comparable in NIT-1 and NIT-2 ?-cells (data not shown), indicating that the attenuated levels of IAPP immunoreactivity observed in NIT-2 cells using this ELISA reflected a reduction in amidation and not synthesis.
FIG. 7. COOH-terminally amidated IAPP is significantly reduced in ?-cells lacking CPE. Cellular content of amidated IAPP was measured by ELISA in extracts of NIT-1 and NIT-2 ?-cells as described in Research Design and Methods. The capture antibody used in this assay is predicted to bind both unprocessed and partially processed forms of proIAPP as well as mature IAPP, whereas the detection antibody binds specifically to amidated (pro)IAPP and does not recognize nonamidated IAPP forms with the COOH-terminal glycine and basic residues intact (A). Note the marked decrease in amidated forms of IAPP immunoreactivity in NIT-2 ?-cells that lack CPE, compared with control (NIT-1) ?-cells (B). Values are expressed as the mean ± SEM of three independent experiments performed in triplicate. *, Significantly different from control NIT-1 cells (P < 0.05, Student’s t test). AP, Alkaline phosphatase; Ab, antibody.
Discussion
In the present study, we demonstrate that CPE is an important enzyme for processing and maturation of proIAPP in islet ?-cells. Western blot analyses of islets from Cpefat/Cpefat mice using specific antisera for unprocessed forms of proIAPP showed that processing of proIAPP is markedly impaired at its NH2 terminus in the absence of CPE, whereas cleavage at its COOH terminus is normal. The finding that this NH2-terminally unprocessed intermediate form was elevated to a similar extent in both hyperglycemic male (8 wk old) and normoglycemic female mice before (8 wk old) and after (18 wk old) the development of obesity suggests that impairment in the NH2-terminal processing of proIAPP observed in those animals is primarily due to the absence of ?-cell CPE per se and not secondary to the development of hyperglycemia or the fat phenotype. Because the proportion of IAPP immunoreactive species was not significantly different in islets from heterozygous and wild-type mice, half of normal islet CPE activity appears to be sufficient for mediating the normal processing of proIAPP.
The accumulation of the NH2-terminally extended intermediate form of proIAPP in Cpefat/Cpefat islets suggests that the absence of CPE may be associated with an alteration in the level and/or activity of a specific endopeptidase responsible for proIAPP processing at its NH2 terminus. We recently showed that in ?-cells proIAPP is processed by cleavage at its COOH and NH2 termini by the sequential action of PC1/3 and PC2, respectively (19, 20), the two ?-cell prohormone convertases that also mediate sequential processing of proinsulin (21, 22). Interestingly, the pattern of impairment in proIAPP processing observed in Cpefat/Cpefat mice resembles that seen in mice lacking PC2, although less severe, because in PC2 null mice NH2-terminal processing of proIAPP is completely blocked (19). Considering that PC2 is essential for NH2-terminal processing of proIAPP (19), these findings suggest that the impaired proIAPP processing observed in the absence of CPE might arise from reduced expression and/or activity of PC2. In agreement with this idea, processing of proglucagon to glucagon, which is normally mediated by PC2, has similarly been shown to be impaired in islets from Cpefat/Cpefat mice (34). Impaired processing of proIAPP in NIT-2 cells was accompanied by a significant decrease in the mature form of PC2 and normal or slightly elevated levels of proPC2. Adenoviral expression of proPC2 in NIT-2 cells was unable to restore proIAPP processing at its NH2-terminal cleavage site, and most of the pro-PC2 expressed by Ad-PC2 remained unprocessed, supporting the idea that loss of CPE is associated with a marked impairment in pro-PC2 processing to active PC2. On the other hand, PC1/3 protein levels were decreased only in hyperglycemic male Cpefat/Cpefat mice, whereas they were comparable in the islets from female Cpefat/Cpefat mice and their wild-type littermates. Furthermore, the reduced level of PC1/3 detected in islets from hyperglycemic Cpefat/Cpefat mice was not observed in NIT-2 ?-cells, suggesting that in islets, PC1/3 content is not affected by the absence of CPE per se, although it is decreased after prolonged hyperglycemia, as previously reported (23). Taken together, these data suggest that lack of CPE interferes with PC2-mediated processing of proIAPP associated with an impairment in the processing of pro-PC2 to active PC2, whereas PC1/3 cleavage of proIAPP appears to be normal.
PC2 is first synthesized as a 75-kDa zymogen that undergoes proteolysis to produce mature enzyme (48). Maturation of PC2 is regulated by the neuroendocrine protein 7B2, which binds to proPC2 in the endoplasmic reticulum and facilitates its transport to the trans-Golgi network (TGN), in which its autocatalytic activation occurs due to the low pH (49, 50). 7B2 is a bifunctional peptide: its 21-kDa NH2-terminal domain facilitates the maturation of PC2 and is the main form of the protein stored in the secretory granules, whereas its COOH-terminal peptide is a potent inhibitor of PC2 (51, 52, 53). The inhibitory activity of the 7B2 COOH-terminal peptide is terminated by cleavage at its internal basic sites (Lys17–Lys18), most likely by PC2, followed by the removal of the COOH-terminal dibasic residues by CPE (54, 55). Absence of CPE may lead to elevated levels of Lys-Lys-extended 7B2 COOH-terminal peptide in ?-cell secretory granules and may further contribute to accumulation of NH2-terminally unprocessed proIAPP and proinsulin intermediates by blocking the action of PC2. In agreement with this idea, 7B2 COOH-terminal peptide has been shown to inhibit cleavage of des 31, 32 proinsulin by PC2 in vitro. Interestingly, this inhibitory effect was considerably decreased in the presence of CPE (54). Alternatively, reduced levels of the 21-kDa NH2-terminal 7B2 fragment, which has been proposed to play a role in regulation of the intracellular activity of PC2 (52), may contribute to decreased PC2 activity in ?-cells that lack CPE. However, this possibility seems unlikely, given that adenoviral expression of 21-kDa 7B2 was unable to restore pro-PC2 processing in NIT-2 cells. The lower levels of 7B2 in NIT-2 cells appears not to be due to a significant defect in processing of pro7B2 because the ratio of pro7B2 to 7B2 was only slightly elevated in these cells. Determining the cause of the lower levels of 7B2 in NIT-2 cells will require further study but may be due to decreased synthesis or loss through increased secretion.
Our findings further suggest that CPE is the major carboxypeptidase responsible for removal of the remaining COOH-terminal basic residues of (pro)IAPP after cleavage by PC1/3 in ?-cells. Using an IAPP ELISA, which detects only COOH-terminally amidated forms of pro(IAPP) immunoreactivity, we found that the cellular content of amidated IAPP was markedly reduced (75%) in NIT-2 cells, compared with control (NIT-1) cells. This decrease could not be explained by differences in the expression of either IAPP or the granule-amidating enzyme PAM (32), which are unchanged in the absence of CPE. Because removal of COOH-terminal basic residues is essential for amidation of IAPP at this site, this profound decrease in amidated IAPP in NIT-2 cells must reflect the presence of significant amounts of IAPP and/or NH2-terminally extended proIAPP with intact COOH-terminal basic residues, providing evidence that CPE plays an important role in maturation of IAPP by removal of these basic residues. Similarly, it has been shown that lack of CPE results in reduced processing and accumulation of B-chain extended (31, 32) diarginyl insulins and proinsulin in both Cpefat/Cpefat mouse islets and NIT-2 ?-cells (32, 41). By contrast, in -cells CPE does not appear to be important in removal of COOH-terminal dibasic residues in proglucagon processing (34).
The finding that some fully processed mature insulin and COOH-terminally amidated IAPP (25%) are produced in the absence of CPE suggests that another carboxypeptidase is able to compensate for the CPE deficiency. Carboxypeptidase D (CPD) is a potential candidate because it has similar enzyme activities to those of CPE, is present in most cells containing CPE, and like CPE removes COOH-terminal basic residues from peptides (28, 56). However, unlike CPE that is primarily present in the secretory granules of neuroendocrine cells, CPD is mainly localized in the TGN (29). It is worth noting that cleavage of proIAPP at its COOH terminus by PC1/3 likely can occur in the TGN (Marzban, L., and C. B. Verchere, unpublished data) in which CPD is primarily localized, raising the possibility that CPD may contribute to removal of the COOH-terminal dibasic residues in proIAPP shortly after cleavage by PC1/3 and before its entry into secretory granules.
Although the true biological role of IAPP is not known, physiological concentrations of amidated IAPP have been shown to suppress food intake (57) and gastric emptying (58). The amidation of IAPP is conserved across species and is thought to be important for its biological activity. It is possible therefore that loss of COOH-terminal processing and amidation of IAPP in the absence of CPE could contribute to the obese phenotype of Cpefat/Cpefat mice. Finally, a rare missense mutation in the CPE gene, which is associated with an earlier onset of hyperglycemia, has been reported in a subpopulation of Ashkenazi type 2 diabetic families (59). It will be of interest to determine whether this mutation is also associated with prohormone processing impairments consistent with a decrease in PC2 activity.
In summary, these data demonstrate that CPE has a dual role in processing and maturation of proIAPP in islet ?-cells. First, CPE mediates its effects on proIAPP processing by regulation of PC2 maturation, the enzyme essential for NH2-terminal processing of proIAPP. Thus, CPE is required for efficient processing of proIAPP by PC2 in ?-cells. Second, CPE is the major enzyme responsible for removal of COOH-terminal dibasic residues of IAPP after cleavage by PC1/3, which is required for subsequent amidation at this site and formation of mature IAPP. Lack of CPE results in accumulation of NH2-terminally unprocessed proIAPP as well as a marked decrease in the formation of mature (amidated) IAPP in ?-cells. These findings, taken together with our previous studies with PC2 and PC1/3 null mice, further support our contention that proIAPP processing closely resembles the pathway for normal processing of proinsulin in ?-cells (Fig. 8).
FIG. 8. Proposed pathway for processing of proIAPP in ?-cells. Pro-IAPP processing is initiated by cleavage at its COOH terminus preferentially by PC1/3 (20 ) likely in the TGN (Marzban, L., and C. B. Verchere, unpublished data) followed by cleavage of the NH2-terminally unprocessed proIAPP intermediate by PC2 (19 ) in the immature and/or mature secretory granules. After cleavage by PC1/3, the COOH-terminal dibasic residues (KR) are removed by the action of CPE. This step is essential for removal of Gly49 and amidation of IAPP at the COOH terminus by the PAM complex (30 ). G, Gly; K, Lys; R, Arg.
Acknowledgments
We thank Dr. Iris Lindberg (Louisiana State University) for 7B2 and PC1/3 antibodies and adenovirus expressing 7B2, Dr. Christopher J. Rhodes (University of Washington) for PC2 antibody, Dr. Christopher McIntosh (University of British Columbia) for somatostatin antibody, and Dr. Philippe A. Halban (University of Geneva, Switzerland) for adenovirus expressing PC2.
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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@interchange.ubc.ca.
Abstract
Islet amyloid polypeptide (IAPP; amylin) is a peptide hormone that is cosecreted with insulin from ?-cells. Impaired processing of proIAPP, the IAPP precursor, has been implicated in islet amyloid formation in type 2 diabetes. We previously showed that proIAPP is processed to IAPP by the prohormone convertases PC1/3 and PC2 at its carboxyl (COOH) and amino (NH2) termini, respectively. In this study, we investigated the role of carboxypeptidase E (CPE) in the processing of proIAPP using mice lacking active CPE (Cpefat/Cpefat) and NIT-2 cells, a ?-cell line derived from their islets. Western blot analysis demonstrated that an approximately 6-kDa NH2-terminally unprocessed form of proIAPP was elevated approximately 86% in islets from Cpefat/Cpefat mice, compared with wild type. This increase was independent of the development of hyperglycemia (8 wk male) or obesity (18 wk female). Impaired proIAPP processing was associated with a decrease in PC2 (but not PC1/3) and both the 21- and 27-kDa forms of the PC2 chaperone protein 7B2, suggesting that PC2-mediated processing of proIAPP at its NH2 terminus was impaired in the absence of CPE. Formation of COOH-terminally amidated (pro)IAPP was reduced approximately 75% in NIT-2, compared with NIT-1 ?-cells, supporting a direct role for CPE in maturation of IAPP by removal of its COOH-terminal dibasic residues, the step essential for IAPP amidation. We conclude that lack of CPE in islet ?-cells results in a marked decrease in processing of proIAPP at its NH2 (but not COOH) terminus that is associated with attenuated levels of PC2 and (pro)7B2 and a great reduction in formation of mature amidated IAPP.
Introduction
ISLET AMYLOID POLYPEPTIDE (IAPP; amylin) (1, 2) is a neuroendocrine peptide hormone that is produced and cosecreted with insulin from pancreatic ?-cells (3, 4, 5). In type 2 diabetes, aggregation of this peptide results in the formation of islet amyloid deposits, which are toxic to ?-cells (6) and likely contribute to the progressive loss of ?-cells in this disease (5, 7, 8). Despite considerable study, it is still not understood why amyloid deposits form in type 2 diabetic patients. Elevated secretion of IAPP from ?-cells associated with an increased demand for insulin in this disease is likely an important contributor but not sufficient for amyloid formation (5, 7, 9, 10, 11, 12). It has been proposed that impaired processing of proIAPP, the IAPP precursor, in type 2 diabetes may lead to hypersecretion of unprocessed and/or partially processed form(s) of proIAPP that have a higher tendency for aggregation than mature IAPP and initiate amyloid formation (7, 13, 14, 15, 16, 17). Therefore, identifying the precise mechanism(s) by which proIAPP is processed in ?-cells in vivo is of considerable interest.
Pro-IAPP is processed by cleavage at basic residue pairs at both its amino (NH2) and carboxyl (COOH) termini (18) before its secretion as mature IAPP along with insulin (3, 4). We recently showed that normal processing of proIAPP in ?-cells is initiated by cleavage at its COOH terminus by the prohormone convertase PC1/3 to produce an NH2-terminally unprocessed intermediate proIAPP, which is subsequently cleaved in secretory granules by PC2 (19, 20). Previous studies have shown that processing of insulin is similarly initiated by cleavage at its B-chain/C-peptide junction, preferentially by PC1/3 followed by cleavage of the resulting conversion intermediate, des 31, 32 proinsulin, at the C-peptide/A-chain junction by PC2 (21, 22). It therefore appears that proinsulin and proIAPP processing may be similar in many ways, supporting the idea that like proinsulin (23, 24), processing of proIAPP might also be impaired in type 2 diabetes. Both subtilisin-like proprotein convertases PC1/3 and PC2 are colocalized with (pro)insulin and (pro)IAPP in ?-cell secretory granules (25, 26). After cleavage of proinsulin by PC1/3 or PC2, the remaining COOH-terminal basic residues are removed from the B-chain (Arg31-Arg32) and C-peptide (Lys64-Arg65) by carboxypeptidase E (CPE) resulting in the formation of mature insulin and C-peptide (27). CPE (EC 3.4.17.10), also known as carboxypeptidase H, is a metallocarboxypeptidase that is present in numerous neuroendocrine tissues including pancreatic ?-cells and is mainly localized in the secretory granules (27, 28, 29). As with proinsulin, after cleavage of proIAPP by PC1/3 the remaining COOH-terminal basic residues (Lys-Arg) are likely removed by a carboxypeptidase, a prerequisite for carboxyamidation of IAPP by the granule enzyme, peptidyl amidating monooxygenase complex (PAM) and formation of mature IAPP (30, 31).
Mice lacking active CPE (Cpefat/Cpefat) have been described previously (32) and shown to have a spontaneous point mutation (Ser202 to Pro) within the coding region of the CPE gene (Cpe), which leads to inactivation of the enzyme and degradation of the protein (32, 33). Cpefat/Cpefat mice are characterized by multiple endocrine disorders including maturity-onset obesity, infertility, and hyperproinsulinemia; diabetes develops only in males (32). Lack of active CPE in this animal model results in impaired processing of a number of neuroendocrine and endocrine prohormones including proinsulin (32), proglucagon, (34), pro-GnRH (35), procholecystokinin (36), pro-TRH (37), proopiomelanocortin, prodynorphin (38), proenkephalin, chromogranin A and B, secretogranin II (39), and progastrin (40). In the present study, we used islets from Cpefat/Cpefat mice and NIT-2 cells, a ?-cell line derived from their islets (41) to investigate the role of CPE in the processing of proIAPP in ?-cells.
Research Design and Methods
Materials
Collagenase (type XI); DNase; BSA; phenylmethylsulfonyl fluoride; dextran; dithizone; 2,2,2,tribromoethanol; 2-methyl-2-butanol; and aprotinin were obtained from Sigma-Aldrich (Oakville, Ontario, Canada); Hanks’ balanced salt solution (HBSS), F12-K medium, and fetal bovine serum from Invitrogen Canada Inc. (Burlington, Ontario, Canada), and protein G Sepharose beads from Amersham Biosciences (Baie d’Urfe, Québec, Canada). Rodent IAPP 1–37 was obtained from Bachem (Torrance, CA). [3H]leucine and [35S]cysteine were from American Radiolabeled Chemicals (St. Louis, MO). All electrophoresis chemicals were from Bio-Rad Laboratories (Hercules, CA).
Animals
Heterozygous Cpefat/+ mice (BKS.HRS-Cpefat/+/J) were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in the animal facility of the BC Research Institute for Children’s and Women’s Health. Offspring were genotyped by PCR on genomic DNA prepared from tail biopsies according to the protocol supplied by the Jackson Laboratory. The animals were maintained on 9% fat diet (PMI Feeds Inc., Richmond, IN). Sex- and age-matched (8 and 18 wk old) mice were used in all experiments. The animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care.
Plasma measurements
Blood glucose levels were measured in blood sampled from the tail vein of fed mice between 0900 and 1000 h using a ONETOUCH glucometer from LifeScan (Milpitas, CA). Plasma insulin immunoreactivity (insulin plus proinsulin) was determined by a mouse insulin ELISA kit from ALPCO Diagnostics (Windham, NH).
Cell lines
NIT-1 cells, a ?-cell line derived from a transgenic nonobese diabetic (NOD/Lt) mouse (42), and NIT-2 cells, a ?-cell line derived from male Cpefat/Cpefat mice (41), were obtained from the American Type Culture Collection (Manassas, VA).
Antisera and recombinant adenoviruses
Antisera specific for the NH2-terminal and COOH-terminal regions of murine proIAPP were generated as previously described (19). IgG-purified antirodent IAPP antibody (RGG-7323) was obtained from Peninsula Laboratories (Belmont, CA). Rabbit antisera against the NH2-terminal region of PC1/3 and 7B2 were generous gifts from Dr. I. Lindberg (Louisiana State University, New Orleans, LA) and antiserum against COOH-terminal of PC2 from Dr. C. Rhodes (Pacific Northwest Research Institute, Seattle, WA). Adenovirus expressing PC2 (Ad-PC2) and 21-kDa 7B2 (Ad-7B2) were kindly provided by Dr. P. Halban (University of Geneva, Geneva, Switzerland) and Dr. I. Lindberg, respectively.
Islet isolation
Islets were isolated from mouse pancreas by collagenase digestion. Briefly, animals were anesthetized with Avertin (0.02 ml/g body weight, ip) consisting of 1.25% (wt/vol) 2,2,2,tribromoethanol, 2.5% (vol/vol) 2-methyl-2-butanol in distilled water, and killed by cervical dislocation. The abdominal cavity was opened, and 2.5 ml ice-cold collagenase (type XI) in HBSS (final concentration 525 U/ml) injected via the common bile duct. The pancreas was harvested and incubated with collagenase/HBSS (525 U/ml) for 9 min at 37 C (gentle shaking). 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 approximately 20 times and filtered through 800 μm mesh (Spectrum Laboratories, Rancho Dominguez, CA). After purification on a dextran gradient, islets were washed with HBSS/BSA and handpicked under a dissecting microscope. Purity of the islets as assessed by dithizone staining was greater than 95% in all experiments. For immunoblot experiments, approximately 300 freshly isolated islets pooled from two to three mice from each genotype were washed with PBS and lysed in 40 μl lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 1% Nonidet P-40, and 0.5% sodium deoxycholate for 25 min on ice. Samples were centrifuged (15,000 x g, 10 min, 4 C) and the supernatants frozen at –70 C until assayed. Total protein in the islet lysates was determined using the BCA assay (Pierce, Rockford, IL).
Cotransduction with Ad-PC2 and Ad-7B2 and metabolic labeling
NIT-2 cells were cultured in F12-K medium containing 7 mM glucose supplemented with 10% fetal bovine serum, penicillin (50 U/ml), and streptomycin (50 μg/ml). Cells at approximately 70% confluency in 25-cm2 flasks were transduced with Ad-PC2 at multiplicity of infection 5.4 pfu/cell in 1 ml F12-K for 2 h at 37 C or cotransduced with Ad-PC2 plus Ad-7B2 (21 kDa). To assess transduction efficiency, NIT-2 cells were transduced with adenovirus expressing LacZ for 2 h at the same multiplicity of infection (5.4 pfu/cell), and after 24 h transduced cells were washed and fixed in 0.5% glutaraldehyde. The proportion of cells expressing ?-galactosidase was determined after incubation with the substrate X-gal (5-bromo-4-chloro-3-indole-?-D-galactoside). The transduction efficiency with adenovirus expressing LacZ was approximately 50% and was somewhat variable among different cells, with a few cells expressing large amounts of this marker, perhaps reflecting the nonclonal nature of the NIT-2 cell line.
Twenty-four hours after transduction with Ad-PC2 or Ad-PC2 plus Ad-7B2, cells were washed and preincubated with Krebs-Ringer bicarbonate (KRB) buffer containing 10 mM HEPES (pH 7.4), 16.7 mM glucose, 0.25% BSA (KRB-G16.7) for 15 min at 37 C. Cells were then pulse labeled (30 min) in 1 ml KRB-G16.7 containing 200 μCi/ml [3H]leucine (specific activity 110 Ci/mM) and 150 μCi/ml [35S]cysteine (1075 Ci/mM) for 30 min, washed two times with ice-cold PBS, and chased (60 min) in KRB buffer containing 1.67 mM glucose (KRB-G1.67) plus 1 mM cysteine and leucine. Cells were harvested with trypsin-EDTA and lysed with 150 μl lysis buffer as described. Cell extracts (500 μg) were precleared by incubation with 50 μl protein G-Sepharose beads (Amersham) for 45 min at 4 C. The supernatants were incubated with 5 μl of NH2-terminal proIAPP antiserum for 2 h followed by 1 h incubation with 50 μl of protein-G Sepharose beads at 4 C. The protein G-Sepharose immunocomplex was washed three times with lysis buffer and used for SDS-PAGE.
Electrophoresis and immunoblotting
Immunoprecipitated samples or aliquots of protein (10 or 15 μg) from islet or cell extracts were heated (100 C, 5 min) with Laemmli’s sample buffer and electrophoresed on 10% (PC1/3 and PC2) or 15% (7B2) polyacrylamide gels using Tris-tricine buffer for IAPP to enable separation of small proteins (43). Proteins were transferred to polyvinyl difluoride membrane using a Trans-Blot semidry transfer cell (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk for 1 h, washed, and incubated for 1 h at room temperature with appropriate antisera at the following dilutions: IgG-purified antirodent IAPP at 2 μg/ml, anti-PC2 and anti-7B2 at 1:1000; anti-PC1/3 at 1:2000; and proIAPP antisera specific for the COOH- and NH2-terminal regions at 1:100. Membranes were then washed and incubated (1 h) with horseradish peroxidase-conjugated antirabbit IgG (Amersham) diluted 1:5000 (or 1:2000 for anti-PC1/3) at room temperature. Immunodetection was performed using an enhanced chemiluminescence detection kit (Amersham). Protein bands on films (X-OMAT, Kodak, Rochester, NY) were analyzed by densitometry using Quantity One 4.2.1 (Bio-Rad Laboratories, Hercules, CA). Immunoprecipitated proteins from NIT-2 cell extracts were electrophoresed on a polyacrylamide gel using Tris-tricine buffer as described and the separated proteins detected by fluorography using EN3HANCE (Perkin-Elmer Life Sciences, Woodbridge, Ontario, Canada) followed by exposure to Kodak X-OMAT film at –70 C for 3 d.
Double immunostain for insulin and somatostatin
Pancreas from 17- to 18-wk-old female Cpefat/Cpefat mice were fixed in 4% paraformaldehyde in 0.1 mol/liter PBS (pH 7.5) for 1 h and washed in 70% ethanol. Paraffin-embedded sections (5 μm) were blocked in 0.05 mol/liter PBS plus 0.25% Triton X-100 containing 2% normal goat serum (Vector Laboratories, Burlingame, CA), washed, and incubated with mouse antisomatostatin (a gift from Dr. C. McIntosh, Vancouver, British Columbia, Canada) at a 1:1000 dilution in the same buffer containing 1% BSA for 1 h at room temperature and then washed and incubated (1 h) with goat antimouse antibody conjugated to the green fluorophore, Alexa Fluor 488 (Molecular Probes, Eugene, OR). Sections were then similarly incubated with guinea pig antiinsulin antibody (Dako, Carpinteria, CA) and goat antiguinea pig antibody conjugated to Texas Red (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h (1:100). The slides were viewed using an Axioplan 2 microscope (Carl Zeiss, New York, NY) equipped for epifluorescence with a Sensys high-performance charge-coupled device camera (Photometrics, Tucson, AZ) and Quips Pathvysion imaging software (Applied Imaging, Santa Clara, CA). Images were captured with green and red filters and merged to give the final image.
ELISA for detection of amidated IAPP
NIT-1 and NIT-2 cells were boiled in 1 M acetic acid plus 0.1% BSA for 10 min and the lysates centrifuged (15,000 x g, 10 min, 4 C) to remove the cell debris. The cell content of amidated IAPP was measured in the diluted supernatants (1:200) by an amylin ELISA kit (EZHA-52K) from Linco Research Inc. (St. Charles, MO). The capture antibody (F002) used in this assay binds to all molecular forms of (pro)IAPP, whereas the detection antibody (F025) recognizes only the amidated form of IAPP (44). Total protein levels were matched in the samples.
Statistical analysis
Values are expressed as the mean ± SEM. Statistical analyses were performed using one-way ANOVA followed by a Newman-Keuls test or by Student’s t test as appropriate. P < 0.05 was taken as the level of significance.
Results
Metabolic characteristics of Cpefat/Cpefat and wild-type mice
Body weight, blood glucose, and plasma insulin levels of animals are summarized in Table 1. All parameters were measured in nonfasted animals (0900–1000 h) on the termination day. As shown previously (32), there was a significant elevation in body weight in 8-wk-old male Cpefat/Cpefat, compared with wild-type mice. Although Cpefat/Cpefat female mice were already heavier than their wild-type littermates by 8 wk of age, this difference was more profound in 18-wk-old animals. As expected, Cpefat/Cpefat mice of both sexes had markedly high levels of plasma insulin immunoreactivity, whereas only male Cpefat/Cpefat mice developed hyperglycemia.
TABLE 1. Metabolic characteristics of Cpefat/Cpefat and wild-type mice at termination (n = 7–9/group)
Pro-IAPP processing is impaired in islets from Cpefat/Cpefat mice lacking CPE
To investigate the role of CPE in proIAPP processing, Western blot analysis was performed on islet extracts from hyperglycemic male and normoglycemic female Cpefat/Cpefat mice as well as their heterozygous and wild-type littermates. Animals were studied before (8 wk old) and after (18 wk old) the development of the fat phenotype. The antibody used for these studies recognizes both unprocessed and processed forms of proIAPP. Consistent with our previous studies (19, 20), the major form of IAPP immunoreactivity in the islets from wild-type mice was fully processed IAPP (4 kDa) (Fig. 1A). Approximately 72, 22, and 6% of total IAPP immunoreactivity was composed of mature IAPP, partially processed, and unprocessed proIAPP, respectively (Fig. 1B). Interestingly, although islets from Cpefat/Cpefat mice were able to process proIAPP to mature IAPP, a partially processed intermediate form of proIAPP with a molecular mass of approximately 6 kDa was elevated by 86% in Cpefat/Cpefat mouse islets, compared with wild-type mice (Fig. 1). Therefore, in Cpefat/Cpefat mouse islets, approximately 53, 40, and 7% of total IAPP immunoreactivity was composed of IAPP, partially processed, and unprocessed proIAPP, respectively (Fig. 1B). This increase in partially processed proIAPP was observed to a similar extent in islets from both hyperglycemic male Cpefat/Cpefat mice and normoglycemic females before and after the development of overt obesity.
FIG. 1. Pro-IAPP processing is impaired in the absence of active CPE in islet ?-cells. A, Islets were isolated from age-matched homozygous Cpefat/Cpefat (–/–), heterozygous (+/–), and wild-type (+/+) mice. Western blot analysis was performed on islet extracts (10 μg) from hyperglycemic male (8 wk old) and normoglycemic female (8 and 18 wk old) mice using antirodent IAPP antibody (Peninsula). A partially processed intermediate form(s) of (pro)IAPP (6 kDa) is elevated in islets from Cpefat/Cpefat mice, compared with heterozygous and wild-type mice. B, Densitometric analysis of immunoblots from three independent experiments. Results are presented as the percentage of each IAPP-immunoreactive molecular form, with total IAPP immunoreactivity taken as 100%. Data are expressed as mean ± SEM *, Significantly different from corresponding molecular form in wild-type islets (P < 0.05, ANOVA).
Lack of CPE results in impaired processing at the NH2-terminal cleavage site of proIAPP in islet ?-cells
To identify the partially processed intermediate form of proIAPP elevated in Cpefat/Cpefat mouse islets, we used antisera raised against the NH2- and COOH-terminal flanking regions of proIAPP (19). Western blot analysis using these antisera revealed that an NH2-terminally unprocessed intermediate form of proIAPP (6 kDa) was markedly increased in islets from both normoglycemic (female) and hyperglycemic (male) Cpefat/Cpefat mice (Fig. 2A). By contrast, the COOH-terminal proIAPP antiserum detected only low levels of a COOH-terminally unprocessed intermediate form (6 kDa) in both Cpefat/Cpefat and wild-type mouse islets (Fig. 2B). Therefore, in islets from Cpefat/Cpefat mice, processing of proIAPP is impaired at its NH2-terminal cleavage site but appears to be normal at its COOH terminus. The COOH-terminal antiserum also detected small amounts of unprocessed (8 kDa) proIAPP in islets from both mutant and wild-type mice. Low levels of proIAPP could also be detected by the NH2-terminal antiserum after a longer exposure of the film (data not shown).
FIG. 2. Lack of active CPE results in impaired processing at the NH2-terminal cleavage site of proIAPP. Western blot analysis was performed on islet extracts (15 μg) from Cpefat/Cpefat mice and their heterozygous and wild-type littermates using antisera specific for the COOH or NH2 termini of mouse proIAPP. A representative blot from four independent experiments is shown. Note the elevated levels of an NH2-terminally unprocessed proIAPP intermediate (6 kDa) detected in Cpefat/Cpefat mouse islets, compared with the almost undetectable levels of this intermediate in islets from heterozygous and wild-type mice (A). The COOH-terminal antiserum detected similar levels of the COOH-terminally unprocessed intermediate form in islets from all three genotypes studied (B). Small amounts of unprocessed (8 kDa) proIAPP were detected by the COOH-terminal proIAPP antiserum.
It is worth noting that in mice, (pro)IAPP is also expressed along with somatostatin in islet -cells (45), although these cells normally form a small proportion of total islet cells. To determine whether an increased proportion of - relative to ?-cells may be present in Cpefat/Cpefat mouse islets, thus possibly contributing to the (pro)IAPP immunoreactivity that we observed in this study, pancreatic sections from female Cpefat/Cpefat mice were double immunostained for insulin and somatostatin (Fig. 3). Despite the larger size and greater number of both ?-cells and -cells in islets from Cpefat/Cpefat mice, compared with islets from their heterozygous and wild-type littermates, there did not appear to be any noticeable difference between the proportion of ?-cells relative to -cells in islets from the three different genotypes.
FIG. 3. Immunofluorescent staining of pancreatic sections from female Cpefat/Cpefat mice for insulin and somatostatin. Paraffin-embedded pancreatic sections from female Cpefat/Cpefat mice (17–18 wk old) and their heterozygous and wild-type littermates were double immunostained for the presence of insulin (red) and somatostatin (green) immunoreactivity. Despite the larger size and greater number of both ?- and -cells in islets from Cpefat/Cpefat mice, compared with those from their heterozygous and wild-type littermates, the proportion of somatostatin-producing -cells to insulin-producing ?-cells appears to be approximately the same in islets from mice of all three genotypes. Figures are representative of immunostains performed on pancreatic sections from three animals of each genotype.
Impaired proIAPP processing in the absence of CPE is associated with reduced levels of 7B2 and PC2 but not PC1/3 in ?-cells
We recently showed that proIAPP is processed by sequential cleavage at its COOH- and NH2-termini by PC1/3 and PC2, respectively (19, 20). To determine whether impaired processing of proIAPP in ?-cells that lack CPE is associated with changes in either one or both of these prohormone convertases, we examined the protein levels of PC1/3, PC2 and its binding protein 7B2 by Western blot. Because relatively high levels of PC2 are also expressed in -cells, the level of PC2 observed in immunoblots of islet extracts may not reflect that in ?-cells. We therefore looked at PC2 protein expression in a transformed ?-cell line (NIT-2) that is derived from Cpefat/Cpefat mice and lacks CPE. Unlike NIT-1 ?-cells, which express CPE and in which mature PC2 was the major form of PC2-immunoreactivity detected, NIT-2 cells contained mainly proPC2 and very low levels of mature PC2, suggesting that processing of proPC2 is impaired in NIT-2 cells (Fig. 4A). This decrease in mature PC2 level was associated with a decrease in both pro7B2 and the 21-kDa NH2-terminal 7B2 protein fragment, and a small but statistically insignificant rise in the ratio of pro7B2 to 7B2 (Fig. 4B). Therefore, total (pro)7B2 immunoreactivity was lower in NIT-2 cells, compared with NIT-1 cells. By contrast, PC1/3 levels in NIT-2 cells were comparable with those in control NIT-1 cells (Fig. 5A). We also examined PC1/3 levels in islets of Cpefat/Cpefat mice because unlike PC2, PC1/3 is expressed mainly in ?-cells, and therefore, results of immunoblots performed on islets will reflect PC1/3 protein levels in ?-cells. PC1/3 levels were decreased only in the male Cpefat/Cpefat islets but were comparable in islets from normoglycemic female Cpefat/Cpefat and wild-type mice, although a slight but significant increase (n = 4, P < 0.05) in the levels of PC1/3 precursor, proPC1/3, was detected in the islets from 18-wk-old (but not 8 wk) females (Fig. 5B).
FIG. 4. Mature forms of PC2 and 7B2 are reduced in NIT-2 ?-cells that lack CPE. Western blot analysis was performed on NIT-1 and NIT-2 ?-cell extracts for PC2 (A) and 7B2 (B) as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading marker. The right panels show the densitometric analyses of immunoblots from four independent experiments. Levels of mature PC2 and both NH2-terminal 7B2 (21 kDa) and its precursor molecule, pro7B2, were significantly lower in NIT-2 cells that lack active CPE, compared with the control (NIT-1) cells, whereas levels of pro-PC2 were approximately the same in the two cell lines. Total (pro)PC2 and (pro)7B2 immunoreactivity in NIT-2 cells were thus decreased, compared with NIT-1 cells. Results are presented as the percentage of each PC2 (or 7B2) immunoreactive molecular form, with total PC2 (or 7B2) immunoreactivity taken as 100%. Data are expressed as mean ± SEM. *, Significantly different from corresponding molecular form in NIT-1 cells (P < 0.05, Student’s t test).
FIG. 5. PC1/3 levels are reduced in islets from hyperglycemic male but not normoglycemic female Cpefat/Cpefat mice. Western blot analysis of cell extracts from NIT-1 and NIT-2 ?-cells (A) or islet extracts from Cpefat/Cpefat mice and their heterozygous and wild-type littermates (B) is shown. PC1/3 protein levels were comparable in (control) NIT-1 and NIT-2 cells that lack CPE and islets from normoglycemic female Cpefat/Cpefat and wild-type mice but were reduced in hyperglycemic male Cpefat/Cpefat mice, suggesting that the decrease in islet PC1/3 content is likely secondary to hyperglycemia and not the absence of CPE per se. Blots are representative of four independent experiments.
To determine whether overexpression of proPC2 or its chaperone protein 7B2 could rescue proIAPP processing in the absence of CPE, NIT-2 cells were transduced with Ad-PC2 alone (46) or cotransduced with Ad-PC2 and Ad-7B2 (21 kDa), pulse labeled with [3H]leucine/[35S]cysteine (30 min) and chased for 60 min to allow processing of the newly synthesized proIAPP to mature IAPP (Marzban, L., and C. B. Verchere, unpublished data), followed by immunoprecipitation with an antiserum specific for the NH2-terminal flanking region of proIAPP. Although NIT-2 cells transduced with Ad-PC2 produced markedly more proPC2, they were unable to process the precursor to generate more mature PC2 (Fig. 6A). Furthermore, adenoviral-mediated expression of 21-kDa 7B2 (Fig 6, A and B) did not improve impaired proPC2 processing. Accordingly, the adenoviral overexpression of proPC2 alone or along with 21-kDa 7B2 in NIT-2 cells did not enhance proIAPP processing, having no effect on the elevated levels of NH2-terminally unprocessed proIAPP in those cells (Fig. 6C).
FIG. 6. Processing of the NH2-terminal intermediate form of proIAPP in NIT-2 cells overexpressing pro-PC2 and 21-kDa 7B2. NIT-2 cells were transduced with recombinant adenovirus expressing pro-PC2 (Ad-PC2; 5.4 pfu/cell) or cotransduced with Ad-PC2 and Ad-7B2 (21 kDa). (Pro)PC2 (A) and (pro)7B2 (B) levels in transduced NIT-2 cells were detected by Western blot. Expression of (pro)PC2 in Ad-PC2 transduced NIT-1 cells is shown on the right side for comparison. C, Transduced cells were pulse labeled with [3H]leucine and [35S]cysteine for 30 min and chased for 60 min, by which time very little newly synthesized proIAPP and small amounts of NH2-terminal intermediate are normally observed. Pro-IAPP and its NH2-terminally unprocessed intermediate form were immunoprecipitated from cell lysates using an NH2-terminal antiserum followed by SDS-PAGE and fluorography. Pro-PC2 expressed in transduced NIT-2 cells remained mainly unprocessed in the presence or absence of 7B2 and was thus unable to restore impaired NH2-terminal processing of proIAPP. Experiments were repeated twice with identical results.
Formation of amidated IAPP is markedly decreased in the absence of CPE in ?-cells
Removal of the COOH-terminal dibasic residues (Lys-Arg) is essential for subsequent amidation of IAPP and formation of mature peptide (30). Therefore, IAPP that is correctly processed at its COOH terminus will be rapidly amidated in ?-cell granules, whereas IAPP that has not had its COOH-terminal extended basic residues removed will remain nonamidated. To investigate whether CPE is the major carboxypeptidase responsible for removal of the dibasic residues from the COOH terminus of IAPP in ?-cells, we measured the concentration of amidated IAPP in NIT-1 and NIT-2 ?-cell extracts using an ELISA that detects COOH-terminally amidated forms of IAPP immunoreactivity (44, 47). The capture antibody used in this assay is predicted to bind to all molecular forms of (pro)IAPP, regardless of modifications of its termini, whereas the detection antibody recognizes only the amidated form of IAPP (44) (Fig. 7A). The level of amidated IAPP was reduced approximately 75% in NIT-2 cells, compared with NIT-1 cells (Fig. 7B), suggesting that lack of CPE results in marked impairment in removal of the COOH-terminal dibasic residues in IAPP. Based on Western blot analysis, expression of IAPP was comparable in NIT-1 and NIT-2 ?-cells (data not shown), indicating that the attenuated levels of IAPP immunoreactivity observed in NIT-2 cells using this ELISA reflected a reduction in amidation and not synthesis.
FIG. 7. COOH-terminally amidated IAPP is significantly reduced in ?-cells lacking CPE. Cellular content of amidated IAPP was measured by ELISA in extracts of NIT-1 and NIT-2 ?-cells as described in Research Design and Methods. The capture antibody used in this assay is predicted to bind both unprocessed and partially processed forms of proIAPP as well as mature IAPP, whereas the detection antibody binds specifically to amidated (pro)IAPP and does not recognize nonamidated IAPP forms with the COOH-terminal glycine and basic residues intact (A). Note the marked decrease in amidated forms of IAPP immunoreactivity in NIT-2 ?-cells that lack CPE, compared with control (NIT-1) ?-cells (B). Values are expressed as the mean ± SEM of three independent experiments performed in triplicate. *, Significantly different from control NIT-1 cells (P < 0.05, Student’s t test). AP, Alkaline phosphatase; Ab, antibody.
Discussion
In the present study, we demonstrate that CPE is an important enzyme for processing and maturation of proIAPP in islet ?-cells. Western blot analyses of islets from Cpefat/Cpefat mice using specific antisera for unprocessed forms of proIAPP showed that processing of proIAPP is markedly impaired at its NH2 terminus in the absence of CPE, whereas cleavage at its COOH terminus is normal. The finding that this NH2-terminally unprocessed intermediate form was elevated to a similar extent in both hyperglycemic male (8 wk old) and normoglycemic female mice before (8 wk old) and after (18 wk old) the development of obesity suggests that impairment in the NH2-terminal processing of proIAPP observed in those animals is primarily due to the absence of ?-cell CPE per se and not secondary to the development of hyperglycemia or the fat phenotype. Because the proportion of IAPP immunoreactive species was not significantly different in islets from heterozygous and wild-type mice, half of normal islet CPE activity appears to be sufficient for mediating the normal processing of proIAPP.
The accumulation of the NH2-terminally extended intermediate form of proIAPP in Cpefat/Cpefat islets suggests that the absence of CPE may be associated with an alteration in the level and/or activity of a specific endopeptidase responsible for proIAPP processing at its NH2 terminus. We recently showed that in ?-cells proIAPP is processed by cleavage at its COOH and NH2 termini by the sequential action of PC1/3 and PC2, respectively (19, 20), the two ?-cell prohormone convertases that also mediate sequential processing of proinsulin (21, 22). Interestingly, the pattern of impairment in proIAPP processing observed in Cpefat/Cpefat mice resembles that seen in mice lacking PC2, although less severe, because in PC2 null mice NH2-terminal processing of proIAPP is completely blocked (19). Considering that PC2 is essential for NH2-terminal processing of proIAPP (19), these findings suggest that the impaired proIAPP processing observed in the absence of CPE might arise from reduced expression and/or activity of PC2. In agreement with this idea, processing of proglucagon to glucagon, which is normally mediated by PC2, has similarly been shown to be impaired in islets from Cpefat/Cpefat mice (34). Impaired processing of proIAPP in NIT-2 cells was accompanied by a significant decrease in the mature form of PC2 and normal or slightly elevated levels of proPC2. Adenoviral expression of proPC2 in NIT-2 cells was unable to restore proIAPP processing at its NH2-terminal cleavage site, and most of the pro-PC2 expressed by Ad-PC2 remained unprocessed, supporting the idea that loss of CPE is associated with a marked impairment in pro-PC2 processing to active PC2. On the other hand, PC1/3 protein levels were decreased only in hyperglycemic male Cpefat/Cpefat mice, whereas they were comparable in the islets from female Cpefat/Cpefat mice and their wild-type littermates. Furthermore, the reduced level of PC1/3 detected in islets from hyperglycemic Cpefat/Cpefat mice was not observed in NIT-2 ?-cells, suggesting that in islets, PC1/3 content is not affected by the absence of CPE per se, although it is decreased after prolonged hyperglycemia, as previously reported (23). Taken together, these data suggest that lack of CPE interferes with PC2-mediated processing of proIAPP associated with an impairment in the processing of pro-PC2 to active PC2, whereas PC1/3 cleavage of proIAPP appears to be normal.
PC2 is first synthesized as a 75-kDa zymogen that undergoes proteolysis to produce mature enzyme (48). Maturation of PC2 is regulated by the neuroendocrine protein 7B2, which binds to proPC2 in the endoplasmic reticulum and facilitates its transport to the trans-Golgi network (TGN), in which its autocatalytic activation occurs due to the low pH (49, 50). 7B2 is a bifunctional peptide: its 21-kDa NH2-terminal domain facilitates the maturation of PC2 and is the main form of the protein stored in the secretory granules, whereas its COOH-terminal peptide is a potent inhibitor of PC2 (51, 52, 53). The inhibitory activity of the 7B2 COOH-terminal peptide is terminated by cleavage at its internal basic sites (Lys17–Lys18), most likely by PC2, followed by the removal of the COOH-terminal dibasic residues by CPE (54, 55). Absence of CPE may lead to elevated levels of Lys-Lys-extended 7B2 COOH-terminal peptide in ?-cell secretory granules and may further contribute to accumulation of NH2-terminally unprocessed proIAPP and proinsulin intermediates by blocking the action of PC2. In agreement with this idea, 7B2 COOH-terminal peptide has been shown to inhibit cleavage of des 31, 32 proinsulin by PC2 in vitro. Interestingly, this inhibitory effect was considerably decreased in the presence of CPE (54). Alternatively, reduced levels of the 21-kDa NH2-terminal 7B2 fragment, which has been proposed to play a role in regulation of the intracellular activity of PC2 (52), may contribute to decreased PC2 activity in ?-cells that lack CPE. However, this possibility seems unlikely, given that adenoviral expression of 21-kDa 7B2 was unable to restore pro-PC2 processing in NIT-2 cells. The lower levels of 7B2 in NIT-2 cells appears not to be due to a significant defect in processing of pro7B2 because the ratio of pro7B2 to 7B2 was only slightly elevated in these cells. Determining the cause of the lower levels of 7B2 in NIT-2 cells will require further study but may be due to decreased synthesis or loss through increased secretion.
Our findings further suggest that CPE is the major carboxypeptidase responsible for removal of the remaining COOH-terminal basic residues of (pro)IAPP after cleavage by PC1/3 in ?-cells. Using an IAPP ELISA, which detects only COOH-terminally amidated forms of pro(IAPP) immunoreactivity, we found that the cellular content of amidated IAPP was markedly reduced (75%) in NIT-2 cells, compared with control (NIT-1) cells. This decrease could not be explained by differences in the expression of either IAPP or the granule-amidating enzyme PAM (32), which are unchanged in the absence of CPE. Because removal of COOH-terminal basic residues is essential for amidation of IAPP at this site, this profound decrease in amidated IAPP in NIT-2 cells must reflect the presence of significant amounts of IAPP and/or NH2-terminally extended proIAPP with intact COOH-terminal basic residues, providing evidence that CPE plays an important role in maturation of IAPP by removal of these basic residues. Similarly, it has been shown that lack of CPE results in reduced processing and accumulation of B-chain extended (31, 32) diarginyl insulins and proinsulin in both Cpefat/Cpefat mouse islets and NIT-2 ?-cells (32, 41). By contrast, in -cells CPE does not appear to be important in removal of COOH-terminal dibasic residues in proglucagon processing (34).
The finding that some fully processed mature insulin and COOH-terminally amidated IAPP (25%) are produced in the absence of CPE suggests that another carboxypeptidase is able to compensate for the CPE deficiency. Carboxypeptidase D (CPD) is a potential candidate because it has similar enzyme activities to those of CPE, is present in most cells containing CPE, and like CPE removes COOH-terminal basic residues from peptides (28, 56). However, unlike CPE that is primarily present in the secretory granules of neuroendocrine cells, CPD is mainly localized in the TGN (29). It is worth noting that cleavage of proIAPP at its COOH terminus by PC1/3 likely can occur in the TGN (Marzban, L., and C. B. Verchere, unpublished data) in which CPD is primarily localized, raising the possibility that CPD may contribute to removal of the COOH-terminal dibasic residues in proIAPP shortly after cleavage by PC1/3 and before its entry into secretory granules.
Although the true biological role of IAPP is not known, physiological concentrations of amidated IAPP have been shown to suppress food intake (57) and gastric emptying (58). The amidation of IAPP is conserved across species and is thought to be important for its biological activity. It is possible therefore that loss of COOH-terminal processing and amidation of IAPP in the absence of CPE could contribute to the obese phenotype of Cpefat/Cpefat mice. Finally, a rare missense mutation in the CPE gene, which is associated with an earlier onset of hyperglycemia, has been reported in a subpopulation of Ashkenazi type 2 diabetic families (59). It will be of interest to determine whether this mutation is also associated with prohormone processing impairments consistent with a decrease in PC2 activity.
In summary, these data demonstrate that CPE has a dual role in processing and maturation of proIAPP in islet ?-cells. First, CPE mediates its effects on proIAPP processing by regulation of PC2 maturation, the enzyme essential for NH2-terminal processing of proIAPP. Thus, CPE is required for efficient processing of proIAPP by PC2 in ?-cells. Second, CPE is the major enzyme responsible for removal of COOH-terminal dibasic residues of IAPP after cleavage by PC1/3, which is required for subsequent amidation at this site and formation of mature IAPP. Lack of CPE results in accumulation of NH2-terminally unprocessed proIAPP as well as a marked decrease in the formation of mature (amidated) IAPP in ?-cells. These findings, taken together with our previous studies with PC2 and PC1/3 null mice, further support our contention that proIAPP processing closely resembles the pathway for normal processing of proinsulin in ?-cells (Fig. 8).
FIG. 8. Proposed pathway for processing of proIAPP in ?-cells. Pro-IAPP processing is initiated by cleavage at its COOH terminus preferentially by PC1/3 (20 ) likely in the TGN (Marzban, L., and C. B. Verchere, unpublished data) followed by cleavage of the NH2-terminally unprocessed proIAPP intermediate by PC2 (19 ) in the immature and/or mature secretory granules. After cleavage by PC1/3, the COOH-terminal dibasic residues (KR) are removed by the action of CPE. This step is essential for removal of Gly49 and amidation of IAPP at the COOH terminus by the PAM complex (30 ). G, Gly; K, Lys; R, Arg.
Acknowledgments
We thank Dr. Iris Lindberg (Louisiana State University) for 7B2 and PC1/3 antibodies and adenovirus expressing 7B2, Dr. Christopher J. Rhodes (University of Washington) for PC2 antibody, Dr. Christopher McIntosh (University of British Columbia) for somatostatin antibody, and Dr. Philippe A. Halban (University of Geneva, Switzerland) for adenovirus expressing PC2.
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