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Department of Molecular Pharmacology, Albert Einstein College of Medicine (O.V., L.D.F.), Bronx, New York 10461; the Department of Biochemistry and Molecular Biology (H.F., D.F.S.) and Howard Hughes Medical Institute (D.F.S.), University of Chicago, Chicago, Illinois 60637; and The Jackson Laboratory (S.H.L., E.H.L.), Bar Harbor, Maine 04609
Address all correspondence and requests for reprints to: Lloyd D. Fricker, Ph.D., Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461.
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Introduction |
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A missense mutation in the CPE gene (Cpe) is the molecular basis for the recessive obesity-producing mutation on mouse chromosome 8 originally named fat (6) and now designated Cpefat. Although a full-length messenger RNA transcript is produced, and CPE protein is translated, the protein is inactive due to a 729TCT to 729CCT point mutation in the gene effecting replacement of serine 202 by proline 202 in a highly conserved region of the enzyme (7). No CPE activity can be detected in any of the Cpefat/Cpefat mouse endocrine/neuroendocrine tissues examined, including pancreatic islets, pituitary, brain, and intestine (8, 9). Pulse-chase analysis shows that the mutant protein is extremely unstable, being degraded intracellularly within hours after synthesis (10). The absence of functional CPE in ß-granules leads to an incomplete conversion of proinsulin to fully mature, active insulin, producing the phenotype of hyperproinsulinemia (7). Approximately 80% of the insulin measured in the serum of homozygous Cpefat mice is comprised of proinsulins I and II and their C-terminally extended (diarginyl) forms. This material has low biological potency compared with fully processed insulins and explains the insulin responsiveness of the hyperglycemic syndrome that develops in C57BLKS/J-Cpefat/Cpefat male mice.
The finding that some mature insulin is generated in Cpefat/Cpefat mice indicates that an additional enzyme is able to compensate for the deficiency of CPE. Recently, a second carboxypeptidase, designated metallocarboxypeptidase D (CPD), has been identified in the secretory pathway (11). CPD has similar enzymatic properties as CPE, but different physical properties (11). CPD is predominantly a 180-kDa protein that is attached to membranes by a conventional transmembrane-spanning domain (11, 12), whereas CPE is a 50- to 56-kDa protein found in soluble and membrane-associated forms (13, 14). CPD has a broader tissue distribution than CPE (12, 15). Whereas CPE is enriched in secretory vesicles, CPD is enriched in the trans-Golgi network (Varlamov, O., and L. D. Fricker, submitted).
The mechanism by which the defect in CPE reduces the processing of proinsulin by the prohormone convertases in the Cpefat/Cpefat mice is not well understood. Cultured ß-cells provide a model to study the effect of the CPE mutation on the processing and secretion of insulin in a system without the pressure to compensate for reduced levels of insulin. One purpose of the present study was to investigate whether the CPE defect is sufficient to affect proinsulin processing in a cell culture system. In the present study, we have produced two immortalized ß-cell lines homozygously expressing the Cpefat mutation. The CPE defect alters proinsulin processing in these cultured cells, indicating that it is the mutation itself that causes the proinsulin-processing defect and not a feedback mechanism resulting from the reduction in mature insulin in the Cpefat/Cpefat mice. Another purpose of the present study was to test the proposal that CPE is a sorting receptor required for the targeting of proinsulin into the regulated pathway (16). The finding that insulin/proinsulin is secreted from NIT-2 and -3 cells via the regulated pathway indicates that CPE is not essential for the targeting of proinsulin. NIT-2 and -3 cells provide an important research tool for further analysis of the role of the CPE deficiency in the aberrant processing of proinsulin.
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Materials and Methods |
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The NIT-2 cell line was derived from dissociated adenomas from a 10-week-old F2 male; NIT-3 was established from cells from a 12-week-old female sibling. After death, pancreata were inflated by bile duct injection of 4 mg/ml collagenase P (Boehringer Mannheim, Mannheim, Germany). Adenomatous islets were isolated using the method of Gotoh et al. (19). The largest islets were transferred by micropipette through three rinses in sterile Hanks’ solution and then dispersed into a cell suspension by a 10-min digestion at 37 C in 0.025% trypsin-0.1 mM EDTA in calcium-magnesium free (CMF)-Hanks’ solution. Cells were pelleted at 500 x g for 2 min, and the pellet was resuspended and repelleted in DMEM supplemented with glucose to a final concentration of 16.5 mM, with HEPES to 15 mM, and with gentamicin sulfate to 50 µg/ml and further supplemented with Eagle’s MEM nonessential amino acid formulation. Heat-inactivated and dialyzed FBS was added at 10% (vol/vol). All culture reagents were obtained from Life Technologies (Grand Island, NY). Washed pellets were resuspended in 1 ml of the above medium and inoculated into 25-cm2 plastic tissue culture flasks (Corning Plastics, Corning, NY). After a 30-min incubation at 37 C in a CO2 incubator, nonadherent cells were decanted into a single well of a plastic six-well cluster dish and maintained in a humidified CO2 incubator. Cultures were fed twice weekly, with one change of medium lacking serum to eliminate any contaminating fibroblasts. NIT-2 cells were first subcultured into 25-cm2 flasks after 2 weeks of growth in the primary culture, whereas the primary culture of NIT-3 expanded more rapidly, such that first passage was performed within a week, and second passage was performed 3 weeks thereafter. Cells were subcultured by 10-min incubation at 37 C in nonenzymatic cell dissociation solution (C-5789, Sigma, St. Louis, MO; diluted 1:10 in CMF-Hanks’ solution), followed by mechanical agitation and trituration with CMF-Hanks’ solution. After the 12th passage, cell growth was rapid, such that cell harvests from a single 25-cm2 flask were split at 1:3 or 1:4 ratios. The NIT-1, NIT-2, and NIT-3 lines have been deposited with the American Type Culture Collection (Rockville, MD).
Ultrastructural characterization
Cells detached from the substratum as described above were
rinsed in serum-free culture medium and then fixed for 1 h in ice
in 2% paraformaldehyde-1% glutaraldehyde buffered in 0.1
M sodium cacodylate, pH 7.4. Fixed pellets were Epon
embedded, thin sectioned, and examined in a JEOL transmission electron
microscope (model JEOL 1000 CXII, Peabody, MA). NIT-1 cells (passage
24) were used as a CPE-positive control for comparison.
Western blot analysis of CPE and CPD
NIT cells were cultured on 10-cm plates in high (i.e.
25 mM) glucose DMEM containing 10% heat-inactivated FBS
(Life Technologies). The cells were washed three times with PBS and
then incubated at 37 C in 2 ml serum-free medium. After 4 h of
incubation, media were removed, the cells were resuspended in 1 ml 10
mM NaAc, pH 5.5, and aliquots of both fractions (30 µl
cells and 100 µl media) were analyzed by a Western blot using the
antiserum to the C-terminal region of CPE (10) and antiserum to the
180-kDa form of CPD (15).
Affinity purification and enzymatic assays of CPE
Media were diluted to 5 ml with 200 mM NaAc, pH 5.5,
and cells were extracted with 5 ml 50 mM NaAc buffer, pH
5.5, containing 1 M NaCl and 1% Triton X-100. The media or
cell extracts were then subjected to purification on
p-aminobenzoylarginine Sepharose 6B affinity columns as
previously described (11, 13). CPE was eluted with 2 ml 50
mM Tris-HCl, pH 8.0, containing 100 mM NaCl and
0.01% Triton X-100. The CPE-containing aliquots of affinity-purified
fraction were analyzed by Western blot, as described above.
Carboxypeptidase activity was measured using 200 µM
dansyl-Phe-Ala-Arg in 100 mM NaAc, pH 5.5, buffer as
previously described (20).
Pulse-chase analysis of CPE
NIT-1 and NIT-3 cells cultured on six-well plates were labeled
with [35S]Met (180 µCi/ml) in high glucose DMEM lacking
unlabeled Met for 30 min (pulse) and then washed twice with DMEM
containing nonradioactive Met. Some of the plates were further
incubated in high glucose DMEM with nonradioactive Met for different
time periods. Media were removed, and the cells were frozen in 100 µl
20 mM Tris-HCl, pH 7.4, in the presence of 1 mM
phenylmethylsulfonylfluoride. The cells and media were then subjected
to immunoprecipitation as previously described (21) using antiserum to
the N-terminal region of CPE (13).
Immunocytochemistry
NIT-1 and NIT-3 cells were cultured for 24 h on 18-mm
coverslips precoated with 1 mg/ml polylysine (Sigma). Cells were washed
with DMEM, fixed in 4% paraformaldehyde for 10 min, and then
permeabilized for 15 min in 0.1% Triton X-100 in PBS. After 1 h
of blocking in 5% BSA, the cells were immunostained for 1 h with
the primary antisera (1:2500 dilution): rabbit polyclonal antisera
directed against the C-terminal region of CPE, the pro region of CPE,
the 180-kDa form of CPD, or calnexin (gift from Ari Helenius, Yale
University). The cells were washed three times with 0.2% Tween-20 in
PBS and then incubated with fluorescein-labeled antirabbit secondary
antibody and rhodamine-labeled wheat-germ agglutinin (Vector
Laboratories, Burlingame, CA; 1:200 dilution) for 1 h, followed by
extensive PBS washing. Immunofluorescence staining was examined using a
Bio-Rad confocal microscope (Richmond, CA).
Insulin biosynthesis
Cells grown to near confluence in 60-mm plastic disks in DMEM
with 10% FBS and 2 mg/ml (11 mM) glucose were rinsed with
Hanks’ Balanced Salt solution and then labeled for periods of 2 or
8 h by incubation at 37 C in HEPES-buffered Krebs-Ringer
bicarbonate (KRB) buffer containing 2.0 mg/ml BSA, 3.0 mg/ml glucose
(16.7 mM), and 100 µCi/ml [3H]Arg and
[35S]Met (Amersham, Arlington Heights, IL) and
supplemented with 48 µg/ml Cys, 584 µg/ml Gln, 146 µg/ml Lys, 72
µg/ml Tyr, 105 µg/ml Leu, 105 µg/ml Ile, 94 µg/ml Val, 16
µg/ml Trp, 66 µg/ml Phe, 95 µg/ml Thr, and 42 µg/ml His. After
incubation, the medium was removed and saved for analysis. The cells
were rinsed three times with cold KRB buffer and then extracted with
acid-ethanol, essentially as previously described (22). The protein
pellet after alcohol-ether precipitation was dissolved in a small
volume of 3 M acetic acid and applied to a 1 x 50-cm
column of Bio-Gel P30 eluted with 3 M acetic acid
containing 50 µg/ml BSA (22). Radioactivity in small aliquots of each
fraction was measured in a liquid scintillation counter. The
proinsulin- and insulin-containing fractions were then pooled
separately, divided into aliquots, and evaporated to dryness. Aliquots
of the proinsulin- and insulin-containing fractions were redissolved in
RIA buffer (0.13 M sodium borate, pH 8.0; 0.003
M NaN3; and 5 µg/ml BSA) and assayed for
insulin immunoreactivity (23). Aliquots of the insulin peak (which also
contains C peptide) were reserved for analysis by HPLC (23), whereas
others were treated with carboxypeptidase B (CPB) to determine the
portion of C-terminally extended material. For treatment of the insulin
peak fractions with CPB, dried aliquots were dissolved in 0.1
M Tris-HCl, pH 8.4, and treated for 5 min at 37 C with 14.5
µg/ml CPB (Worthington Biochemical Corp., Freehold, NJ). After
incubation, the reaction mixture was quenched with 50% acetic acid and
applied to Bio-Gel P30 columns for rechromatography as described above.
Fractions were counted in a liquid scintillation spectrometer.
Secretion of insulin/proinsulin from NIT cells
Cells were cultured in six-well plates. Medium was removed, and
cells were washed three times with PBS and then incubated in KRB buffer
containing 0.1% BSA for 1 h. This medium was discarded and
replaced with 1 ml KRB-0.1% BSA containing no addition, 16.7
mM glucose, and/or 45 mM KCl. After 2-h
incubation, the medium was removed and assayed for insulin/proinsulin
by RIA. This assay has been previously described (24) and detects both
insulin and proinsulin. Cells were extracted with 2 M
acetic acid, and an aliquot was assayed for insulin/proinsulin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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shows
the typical morphology of NIT-1 cells; most of the ß-granules were
typically dense core, mature granules found at the periphery of the
cells, indicating a state of active secretion. Cell surface shedding of
an endogenously encoded type C ecotropic retrovirus is also
characteristic of this cell line (17). Figure 1B, depicting NIT-2
cells, shows the enlarged and more electron-lucent (immature)
appearance of many of the ß-granules. A similar result was observed
in NIT-3 cells (data not shown). Neither NIT-2 nor NIT-3 cells
exhibited budding type C retroviral particles at their cell surfaces,
indicating that they may carry C57BLKS/J rather than the NOD genome on
proximal chromosome 11, the location of the Emv30 locus
encoding the retrovirus expressed in NIT-1 cells (25). The cell lines
were H2 typed using a polymorphism in a simple sequence repeat in the
H2-Ab gene (D17Mit21). NIT-1 cells derive directly from
NOD/Lt mice and, thus, are homozygous for NOD alleles comprising the
H2g7 haplotype. The NIT-2 and NIT-3 cells, which
were derived from F2 hybrids between NOD and C57BLKS/J
(H2d haplotype), are
H2g7/H2d heterozygous.
Thus, transplantation of these cells in vivo will require
either a (NOD x C57BLKS/J) F1 host or an immunocompromised
NOD/LtSz-scid/scid (severe combined immunodeficiency)
host.
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, left). This size corresponds to that of the mature form of
CPE found in various bovine and rat tissues (13). In contrast, the CPE
in NIT-2 and NIT-3 cells was approximately 56 kDa, which corresponds to
the size of pro-CPE (i.e. the precursor form of CPE that
contains 14 additional N-terminal amino acids). Although the levels of
immunoreactive CPE/pro-CPE in NIT-1, -2, and -3 cells were generally
similar, CPE was only detected in the medium from NIT-1 cells (Fig. 2).
Immunoreactive CPD was also detected in NIT cells, with a major band of
approximately 180 kDa (Fig. 2, right). This size corresponds
to the predominant form of CPD in bovine pituitary and other tissues
(11, 15). The levels of immunoreactive CPD in NIT-1, -2, and -3 cells
were comparable. No immunoreactive CPD was detected in the medium,
consistent with the previous finding that the major form of CPD is a
membrane-bound protein (11, 15).
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). The affinity column eluate from
NIT-2 and -3 cells had no detectable CPE activity, whereas the material
from NIT-1 cells showed a relatively large amount of enzyme activity
(Fig. 3B). In contrast, all three cell lines contained CPD activity
(data not shown), which eluted from the affinity column in a later
fraction than CPE (11). The medium from NIT-1 cells, but not the medium
from NIT-2 or -3 cells, contained detectable levels of CPE-like enzyme
activity (Fig. 3B). The CPE activity secreted from NIT-1 cells over
4 h was 11-fold lower than the cellular activity, indicating that
these cells secrete approximately 2% of the cellular CPE per h in high
glucose medium.
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). After 30 min of chase, a second
band of approximately 53 kDa was present at levels comparable to the
56-kDa band, and then by 90 min of chase, the 53-kDa band was the
predominant form (Fig. 4). In contrast to the results with NIT-1 cells,
NIT-3 cells showed only the 56-kDa form of pro-CPE, with no detectable
conversion to a smaller form (Fig. 4). After 6 h of chase, there
was virtually no labeled pro-CPE remaining in NIT-3 cells (Fig. 4). As
CPE is not found in the medium of NIT-3 cells (Fig. 2
), the
disappearance of labeled pro-CPE presumably reflects degradation.
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, top left).
The antiserum to the pro region of CPE (i.e. raised against
a peptide corresponding to the 14-residue N-terminal extension on
pro-CPE that was not present on the mature form of CPE) only showed
perinuclear staining and did not stain the tips of NIT-1 cells (Fig. 5, top middle). This observation was similar to what was found
in the AtT-20 cell line and fits with the proposal that the processing
of pro-CPE into CPE occurs in the trans-Golgi network (TGN)
or immature secretory vesicles (26). In contrast, NIT-3 cells showed
diffuse staining throughout the cell with either of these two antisera
(Fig. 5, bottom). This diffuse staining pattern was similar
to that observed with calnexin (Fig. 5, right), a resident
protein of the endoplasmic reticulum (ER) (27). The defect in NIT-3
cells was not a general defect in ER to Golgi transport, as
immunoreactive CPD was detected in a perinuclear compartment in both
NIT-1 and NIT-3 cells (Fig. 6, left). This perinuclear staining was similar to that
observed with wheat-germ agglutinin (Fig. 6, right), a Golgi
marker (28).
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show the relative proportions of
[3H]Arg-labeled proinsulin in the three cell lines after
an 8-h labeling period. The NIT-2 and NIT-3 cells showed an increased
level of proinsulin (55.1% and 44.1%, respectively) compared with
NIT-1 cells (27.2%). Similar results were obtained for the
35S-labeled material (Fig. 7B), confirming that proinsulin
is present in increased amounts within NIT-2 and NIT-3 cells. When
total immunoassayable insulin and proinsulin were measured in cell
extracts fractionated by Bio-Gel P30 chromatography, a similar pattern
of elevated proinsulin was demonstrated in the two CPE-deficient
cell lines (Table 1). These results
confirm the existence of a block in proinsulin conversion and correlate
with the observed immature morphology of the secretory granules in
NIT-2 and NIT-3 cell lines (Fig. 1).
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), confirming the
presence of some B chain-extended diarginyl insulin and C
peptide-Lys-Arg in the insulin fractions. From these results the
relative amounts of extended material in the CPE-deficient cell lines
could be calculated (Table 2). The
presence of only about 4–15% of extended material in NIT-2 and NIT-3
cells, compared with about 40% found in pancreas extracts of
Cpefat/Cpefat mice (7),
may reflect the presence of increased levels of CPD or other
carboxypeptidases in the transformed NIT cell lines compared to those
in the nondividing ß-cells in vivo.
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). High
glucose (16.7 mM) stimulates the secretion of
immunoreactive insulin/proinsulin approximately 4-fold from NIT-1 cells
and 1-fold (i.e. a 100% increase) from NIT-2 and NIT-3
cells (Fig. 9). KCl (45 mM) causes a 14-fold increase in
immunoreactive insulin/proinsulin secretion from NIT-1 cells and a 5-
to 6-fold increase from NIT-2 and -3 cells (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The size of the immunoreactive CPE produced in NIT-2 and -3 cells is consistent with the size of pro-CPE, and not with the mature form of CPE that predominates in NIT-1 cells and in many tissues (13, 30). Previous studies on the AtT-20 mouse corticotropic cell line have found that pro-CPE is converted to CPE in the TGN or in a post-Golgi compartment (26). The present results from pulse-chase analysis and immunocytochemistry suggest that the wild-type pro-CPE in NIT-1 cells is also processed to CPE in a late Golgi or post-Golgi compartment. The size change in CPE observed during the pulse-chase analysis of NIT-1 cells is presumably due to removal of the pro region and processing within the C-terminal region, as previously found for CPE in the AtT-20 cell line (26). In contrast, the mutant form of pro-CPE in NIT-3 cells is not converted into CPE, based on the size of the immunoreactive protein and on the pulse-chase analysis. Furthermore, the mutant CPE in NIT-3 cells does not appear in a perinuclear compartment typical of Golgi proteins. These findings are similar to those obtained when the Pro202 mutant form of pro-CPE was expressed in AtT-20 cells (10). The substitution of Pro for Ser202 presumably affects the folding of pro-CPE; this Ser is predicted to be present in a ß-sheet, and a Pro would significantly alter the structure. Misfolded proteins are thought to be degraded either in the ER or by proteosomes after transit out of the ER (31).
The lack of transport and processing of the mutant pro-CPE is not due to a general transport defect in NIT-3 cells, as CPD appears in a perinuclear compartment in both NIT-1 and NIT-3 cells. CPD is enriched in the TGN in AtT-20 cells (Varlamov, O., and L. D. Fricker, submitted). As found for other enzymes that are present in the TGN, CPD cycles to the surface of the AtT-20 cells and returns to a TGN-like perinuclear compartment (Varlamov, O., and L. D. Fricker, submitted). Preliminary studies have found a similar cell surface expression and recycling of CPD in NIT-1 cells (data not shown).
Studies on the processing of proinsulin in NIT-2 and -3 cells show only a moderate accumulation of arginine-extended forms of insulin. This result is generally similar to that found for insulin processing in the pancreatic islets of the Cpefat/Cpefat mouse (7) and for the processing of other peptides in various tissues of these mice (8, 9, 32). In all cases, there is a decrease in the levels of correctly processed peptide and an accumulation of the substrate for the carboxypeptidase reaction. These results are consistent with a role for CPE in the processing of the various peptides. However, the presence of correctly processed peptide indicates that another enzyme can substitute for the defective CPE in the Cpefat/Cpefat mice. CPD is a potential candidate for this alternative enzyme. The levels of CPD protein do not appear elevated in the Cpefat/Cpefat mouse (8) or in NIT-2 and -3 cells compared with those in NIT-1 cells. Even though NIT-2 and -3 cells appear to express similar levels of immunoreactive CPD, these two cell lines differ in the relative amount of Arg-extended insulin. This finding suggests that factors other than the level of immunoreactive CPD protein can affect the C-terminal processing of Arg-extended insulin.
The relative increase in proinsulin-like immunoreactive material in
NIT-1 cells shown in Table 1 (
30% in contrast to <5% in normal
islets) probably reflects the increased proportion of ER-derived
material in relation to diminished granule stores in these transformed
ß-cells. If this amount is deducted from the proinsulin percentages
shown in Table 1, the values for stored proinsulin in NIT-2 and NIT-3
cells range from 10–30%, a somewhat lower value than that for
proinsulin retention in the
Cpefat/Cpefat mouse
pancreatic extracts (40%) (7). Although the accumulation of
arginine-extended forms of insulin were predicted for an inactivating
mutation of CPE, the increased levels of proinsulin were unexpected.
The present finding that this increase occurs in cultured NIT-2 and -3
cells indicates that the defect in CPE is responsible, and that the
effect is not merely due to increased secretory demand on the ß-cells
of the Cpefat/Cpefat
mice. The difference in accumulation of proinsulin in NIT-2 and NIT-3
cells may be due to levels of the endoproteases; NIT-2 cells have lower
levels of both PC2 and PC3 than NIT-3 cells (Lindberg, I., personal
communication). However, variability in the endopeptidases does not
account for the accumulation of proinsulin in NIT-3 cells, as these
cells have similar levels of PC2 and PC3 as NIT-1 cells (Lindberg, I.,
personal communication).
The mechanism by which the defect in CPE leads to abnormal proinsulin processing in Cpefat/Cpefat mice is unclear, and two models have been proposed (10). In one model, the absence of CPE activity leads to the accumulation of a peptide with C-terminal basic residues, and this peptide then inhibits the proinsulin-processing endopeptidases. Evidence in favor of this model is the finding that a fragment of 7B2 with C-terminal lysines is a potent inhibitor of PC2, and this inhibition is eliminated when CPE is added to the reaction (33). The other model is that CPE protein is required for the sorting of proinsulin into the regulated secretory pathway, and the absence of CPE from the TGN leads to the abnormal sorting of proinsulin. Our finding that NIT-2 and -3 cells are capable of secreting insulin/proinsulin via the regulated pathway indicates that CPE is not obligatory for the proper targeting of proinsulin. The magnitude of the glucose- and KCl-stimulated secretion of insulin/proinsulin from NIT-2 and -3 cells is lower than that from NIT-1 cells, but in the same range as that in other mouse pancreatic ß-cell lines produced by the same method (24). Thus, it is possible that CPE contributes to the regulated pathway sorting of proinsulin; due to inherent variability between cell lines, this could not be addressed in the present study. Evidence supporting this latter model is the recent finding that CPE binds to POMC and is required for the efficient sorting of this prohormone to the regulated secretory pathway (16). A key finding was that pituitary cells cultured from Cpefat/Cpefat mice did not secrete POMC-derived peptides via a regulated pathway (16). Cool et al. proposed that CPE may also be involved in the sorting of proinsulin based on the ability of these two proteins to interact (16). However, CPE is clearly not essential for the targeting of proinsulin in the TGN.
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Acknowledgments |
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Footnotes |
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Received April 10, 1997.
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