Biosynthesis of Islet Amyloid Polypeptide ELEVATED EXPRESSION IN MOUSE PTC3 CELLS*

Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse PTC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in BTC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in BTC3 cells. How- ever, the ratio of synthesis of insulin to IAPP was approximately 6: 1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of BTC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent M, 7400 and a mature form (IAPP) of apparent M, 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The tlh for conversion of pro-IAPP to IAPP was about 25 min, faster than the tlh for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from PTC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in BTC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone.

Islet amyloid polypeptide (IAPP) messenger RNA levels, biosynthesis, processing, and secretion were studied in cultured mouse PTC3 insulinoma cells. Northern blot analysis revealed that the size of IAPP mRNA (0.9 kb) in BTC3 cells was the same as that in normal mouse islets; IAPP mRNA was approximately 60% of the level of insulin mRNA in BTC3 cells. However, the ratio of synthesis of insulin to IAPP was approximately 6: 1, suggesting that IAPP mRNA is not translated efficiently in these cells. Metabolic labeling of BTC3 cells with [3H]leucine revealed the synthesis of both a precursor form of IAPP (pro-IAPP) of apparent M, 7400 and a mature form (IAPP) of apparent M, 3900. In pulse-chase experiments, pro-IAPP could be shown to be processed to IAPP in a manner similar to proinsulin. The tlh for conversion of pro-IAPP to IAPP was about 25 min, faster than the tlh for proinsulin to insulin of 70 min. A significant proportion of newly synthesized IAPP and insulin precursors were secreted via a constitutive pathway from PTC3 cells. Possible effects of dexamethasone and forskolin on IAPP mRNA levels and biosynthesis were examined but no effects were observed. In conclusion, the IAPP gene is strongly expressed in BTC3 cells leading to the biosynthesis, proteolytic processing, and secretion of IAPP, a putative islet hormone.
Amyloid deposits are found in the pancreas of >90% of NIDDM patients and are also present in >50% of insulinproducing tumors (1-3). Although the insolubility and low concentration of islet amyloid hampered its biochemical analysis, the recent isolation and sequence analysis of this material revealed that a major component of the deposits is a peptide, termed amyloid polypeptide (IAPP),' which consists of 37 amino acids and is structurally related to calcitonin gene-related polypeptide (4).
Recently, several laboratories have succeeded in isolating cDNAs encoding human and/or rat IAPP precursors (5-7). These studies have shown that prepro-IAPP has a typical signal peptide followed by a relatively short prohormone-like sequence which contains the IAPP sequence in its central region. IAPP has been shown to be present in normal pancreatic islets in measurable amounts (8) and has been localized * Work from our laboratory has been supported by Grants DK-13914 and DK-20595 and by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' The abbreviations used are: IAPP, islet amyloid polypeptide; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid kb, kilohase(s). by immunocytochemistry to the secretory granules of the B cells in normal adult islets (9-12). Thus, it is postulated that IAPP is synthesized as a prepropeptide in islet B cells and then processed proteolytically at diabasic residues in the early secretory granules, in a manner similar to that for proinsulin. However, the low content of IAPP in normal rat islets (13) requires that studies of its biosynthesis be carried out by immune precipitation with specific antisera.
In the present study, we found that mouse PTC3 cells, a clonal cell line derived from SV40 T antigen-induced insulinoma in transgenic mice (14), express high levels of IAPP mRNA and synthesize easily detectable levels of IAPP, which has enabled us to study its biosynthesis, processing and secretion in considerable detail.

EXPERIMENTAL PROCEDURES
Materials-L-[4,5-:'H]Leucine (145 Ci/mmol), Amplify", and a kit for nick translation were from Amersham Corp. [a-'"P]dCTP was purchased from Du Pont-New England Nuclear. Protein A-Sepharose was from Pierce Chemical Co. Nitrocellulose membranes were from Schleicher & Schuell. Dexamethasone and forskolin were purchased from Sigma.
Cell Culture-The BTC3 cell line was kindly provided by D. Hanahan (University of California at San Francisco, CA), and grown at 37 "C with Dulbecco's minimal essential medium supplemented to a final glucose concentration of 4 mg/ml containing 10% fetal bovine serum, 25 mM Hepes, 1 mM L-glutamine, minimal essential mediumnonessential amino acids, and 0.5 mg/ml gentamicin.
Labeling of Cells-After cells were incubated in RPMI 1640 medium (v/v 10% fetal bovine serum) under the conditions described in each figure legend, lo6 cells in 1.0 ml were pulsed with [''H]leucine (300 pCi/ml) in leucine-deficient RPMI 1640 medium, as described in each figure legend. For chase analyses, the pulse-labeled cells were washed twice in complete medium and incubated in complete medium appropriate time intervals.
IAPP Biosynthesis-Cells were lysed by the freeze and thaw method, lyophilized, and dissolved in 0.5 ml of TAS buffer (0.1 M Tris-HC1, 0.05 M NaC1, 0.25% bovine serum albumin, pH 7.6) containing proteinase inhibitors (5 X M phenylmethylsulfonyl fluoride, 50 pg/ml pepstatin, 50 pg/ml trasylol, 1 pg/ml leupeptin, and 5 X M EP-459). The cell lysate and supernatant after the chase incubation were immunoprecipitated in a volume of 0.4 ml with 1 p1 of rabbit anti-rat IAPP antiserum (Peninsula Laboratories) and pelleted with Protein A-Sepharose after prior absorption with normal rabbit serum. The immunoprecipitates were washed once in 0.6 M NaC1, 10 mM Tris (pH 8.6), 0.05% Nonidet P-40, and 0.1% SDS before electrophoresis on 17.5% polyacrylamide slab gels according to Laemmli (15) with minor modifications. The gels were then treated with Amplify'" and dried, and the radioactivity was visualized by autoradiography after 7 days in -80 "C.
Insulin Biosynthesis-Immunoprecipitation of insulin and proinsulin was performed with guinea pig anti-insulin antiserum as described elsewhere (16). Proinsulin and insulin were separated on 1-X 50-cm Bio-Gel P-30 column in 3 M acetic acid as described elsewhere (17).
RNA Analysis-Total cellular RNA was prepared from BTC3 cells by the guanidine isothiocyanate procedure (18). RNA was analyzed by electrophoresis through 1.5% agarose, 0.66 M formaldehyde gel, hlotted onto nitrocellulose lilters, and hybridized with nick-translated mouse insulin or mouse IAPI' cDNAs, which were prepared by polymerase chain reaction using specific oligonucleotide primers hased on the puhlished sequences (19,20). Filters were washed under high stringency conditions (0.1 X SSC. O.lri sodium dodecyl sulfate, 65 "C) and autoradiographed with intensifying screens. RESULTS AND DISCUSSION IAPP Gene Expression-To examine the level of IAPP expression in PTC3 cells tot,al RNA was extracted from both freshly isolated mouse islets and PTC3 cells maintained in DMEM containing 22 mM glucose for 24 h, and subjected to Northern analysis. As shown in Fig. 1, the 0.9-kb IAPP transcript in PTC3 cells was the same size as that in normal mouse islets. However, the ratio of IAPP to insulin mRNA in /jTC3 cells appeared to be relatively higher than in normal mouse islets. To more accurately estimate this ratio in PTC3 cells or mouse islets equal amounts of mouse IAPP and insulin cDNAs were nick-translated together to obtain probes having similar specific activities. When 50-pg-1-ng amounts of both unlabeled mouse IAPP and insulin cDNAs in the cloned plasmid PGEM 42 were dot-blotted as standards (Fig. 2), the hybridization signal with insulin cDNA was approximately 2fold stronger than that with IAPP cDNA based on densitometric analysis. Northern analysis (also shown in Fig. 2) revealed that IAPP mRNA (0.9 kb) was adequately separated from insulin mRNA (0.6 kb) in a 1.5% agarose/formaldehyde gel. The estimated concentration of IAPP mRNA In PTC3 cells was about 60% that of insulin mRNA based on the standards, while in mouse islets, the content of IAPP mRNA was approximately 20% that of insulin mRNA. Thus, the BTC3 cell line produced a much higher level of IAPP mRNA compared with that, in normal mouse islets.
Biosynthesis of ZAPP-Mouse PTC3 cells were labeled with ["Hlleucine for 3 h in either 0 or 11 mM glucose medium after 48-h preincubation of cells in RPMI 1640 medium containing either 0 or 11 mM glucose, respectively. Radioautography of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of proteins immunoprecipitated from labeled cell extracts with a rabbit anti-rat IAPP antiserum revealed two major bands (Fig. 3). These proteins were identified as pro-IAPP and mature IAPP on the basis of the following criteria: 1) they migrated as 7.4-kDa and 3.9-kDa proteins, respectively, cells or mouse islets by a blot hybridization assay using cloned complementary DNAs as standards. A, as the standards, mouse IAPP and insulin cDNAs were denatured at. 70 "C for 15 min and applied to nitrocellulose under a weak vacuum. The blot was hyhridized with a nick-translated probe, which was prepared using equal amounts of both mouse IAPP and insulin cDNAs in the same tube.
Lanes: I , 50 pg; 2, 100 pg; 3, 250 pg; 4 , 500 pg; 5 , 1 ng. R, Northern blot analysis of total cellular RNA from 8TC3 cells (lone I , 10 pg; lone 2, 15 pa) or normal mouse islets (lane 3, 10 p g ) . The blot was hybridized with the probes described above. The film was exposed for 2 h at -80 "C. RPMI 1640 medium supplemented with 10% fetal bovine serum in the presence of either 0 or 11 mM glucose for 48 h, and then pulse laheled for 3 h with ["HI leucine under the same conditions. Cells were lysed and immunoprecipitated with rabbit anti-rat IAPP serum as described under "Experimental Procedures." The immunoprecipitates were analyzed on 17.5% SDS-PAGE after fluorography with Amplify'" and autoradiography for 7 days at -80 "C.
as expected on the basis of their predicted M,; 2) they were not immunoprecipitated with a nonimmune control serum; 3) the addition of 10 pg of unlabeled rat IAPP displaced both bands (see Fig. 7); 4) the 3.9-kDa band comigrated with iodinated rat IAPP (data not shown); and 5) the 7.4-kDa band became labeled earlier in pulse chase experiments (see next section).
The lack of any effect of glucose on IAPP biosynthesis may be due to the high passage level of this cell line. In earlier experiments we found that the synthesis of insulin and trichloroacetic acid-extractable total protein in this cell line was regulated by glucose,2 but this responsiveness was lost during progressive passages of the cell line. In a parallel experiment, the synthesis of both IAPP and insulin was measured as shown in Table I. Interestingly, the rate of insulin synthesis was about 6-fold higher than that of IAPP, despite the finding (Fig. 2) that the mRNA level for insulin is only about 1.7-fold higher than that for IAPP.

TABLE I
Biosynthesis of pro-IAPP and proinsulin pTC3 cells (lo6 cells per plate) were pulse labeled in triplicate for 30 min with ['Hlleucine and then immunoprecipitated with anti-insulin or anti-IAPP serum as described under "Experimental Procedures." Biosynthesis of pro-IAPP and proinsulin were determined by subtracting the radioactivity in the control immunoprecipitates from antiinsulin or IAPP immunoprecipitates of the labeled cell extracts (control immunoprecipitates were less than 10% of experimental values.) " Based on the number of leucine residues.

Pro-IAPP biosynthesis
r Cells I Medium I In normal isolated rat islets, it was reported that the content of IAPP mRNA is approximately 10% that of insulin mRNA (6), and that the amount of IAPP is about 1-2% that of insulin (13). Thus, it seems possible that the translational efficiency of IAPP mRNA is relatively low compared with that of insulin in both normal mouse islets and PTC3 cells. The reasons for this difference are not known.
Processing and Secretion of ZAPP-To follow the proteolytic processing of IAPP, cells were pulse-labeled with ["HI leucine for 30 min, and then chased for 30 and 60 min. After the 30-min pulse, a protein with an M , of 7400 (pro-IAPP) was the main labeled component (Fig. 4), while at the end of the 60-min chase, most of the labeled protein migrated with an M, of 3900 (IAPP). Based on densitometric analysis of the intensities of the 7.4-and 3.9-kDa protein bands shown in Fig. 4, the conversion of pro-IAPP to mature IAPP appeared to follow pseudo first-order kinetics (Fig. 5), as previously described for proinsulin conversion (17). The tM of about 25 min for IAPP conversion was faster than the tnh of about 70 min for proinsulin cleavage measured in the same experiments. As shown in Fig. 5, the processing of these proteins began at almost the same time (30 min). Since IAPP and insulin are known to coexist in the insulin secretory granule (11, 12), it seems likely that pro-IAPP is processed within maturing secretory granules by the same converting enzymes as is proinsulin. Since the biosynthesis of IAPP was only 17% relative to that of insulin in this cell line, the molar ratio of pro-IAPP to converting enzyme in the secretory granules is lower than that of proinsulin, which may allow for more rapid conversion of pro-IAPP. On the other hand, it is probable l zzzzBa  Fig. 4 were analyzed by densitometric scanning and then the intensities of the mature IAPP (3.9 kDa) bands were multiplied by 6/4 to correct for the difference in leucine content between mature and pro-IAPP. For proinsulin conversion, the immunoprecipitates of insulin in the same experiments of Fig. 4 were analyzed by Bio-Gel P-30 column. The radioactivity eluting from the Bio-Gel P-30 column in the insulin peak was multiplied by 11/6 to correct for the loss of C-peptide. Slopes of lines (0, IAPP; 0, insulin) were determined by linear regression. The t H for conversion of pro-IAPP or proinsulin were about 25 min or 70 min, respectively. that pro-IAPP is processed preferentially by one of the putative prohormone processing enzymes in the early secretory granules (presumably the relatively Lys-Arg-specific Type I1 enzyme (21)) while the other (Type I) may be rate limiting for proinsulin processing.
As shown in Fig. 4, only pro-IAPP was found in the medium after either 30 or 60 min of chase incubation. Since the conversion rate of IAPP is rapid and IAPP is cosecreted along with insulin (22,23), mature IAPP must be retained in secretory granules for a t least 60 min during the chase, prior to their regulated release. However, Fig. 6 shows that up to 60% of the total labeled pool of either proinsulin or pro-IAPP was secreted from those cells during the 90-min pulse-chase period, while only about 2% of the total proinsulin was secreted from normal mouse islets during a 2-h chase (24). These data suggest that a significant fraction of both newly synthesized proinsulin and pro-IAPP is secreted via an unregulated, or constitutive, pathway, as is also the case in the pituitary corticotroph AtT2O cell line transfected with a human preproinsulin cDNA (25), as well as isolated islets from transgenic mice expressing an Asp B10 mutant human proinsulin gene (24).
Effects of Dexamethasone and Forskolin on Gene Expression and Biosynthesis of ZAPP-Glucocorticoids alter transcriptional rates of specific genes and have been reported to bind tRNA species required for general protein synthesis (26). Glucocorticoids have been shown to increase pancreatic islet insulin mRNA content (27)(28)(29). However, in the HIT cell line, dexamethasone inhibits proinsulin biosynthesis (30). Recently it has been reported that dexamethasone-treated rats showed an increase in islet IAPP mRNA levels (31). On the other hand, insulin gene transcription is known to be partly regulated via cyclic AMP (32). Indeed, in both HIT and RIN cells, cyclic AMP enhanced the levels of preproinsulin mRNA (32,33). In this study, we tested the effects of both dexamethasone and forskolin, which directly stimulate adenylate cyclase, on IAPP gene expression and the biosynthesis of IAPP in PTC3 cells. After cells were exposed to these compounds for 48 h in RPMI 1640 containing 11 mM glucose, levels of IAPP mRNA and rates of biosynthesis of I A P P were measured, as described earlier. However, 1 PM dexamethasone and 20 PM forskolin failed to affect these parameters (Fig. 7). Proinsulin biosynthesis also was not affected by either dexamethasone or forskolin in BTC3 cells (data not shown).
In summary, we have studied IAPP mRNA levels and the biosynthesis, processing, and secretion of IAPP in mouse PTC3 cells. These transformed p cells exhibit a number of significant, differences from normal islet [ ? cells. T h e level of expression of IAPP mRNA is considerably higher than in normal @ cells which is consistent with observations on various clonal derivatives of a rat insulinoma which are heterogeneous with respect to insulin and IAPP expression ( 3 5 ) . Taken all together these findings indicate that the expression of IAPP and insulin are not tightly linked and thus some insulinomas may express unusually high or low levels of IAPP. Although BTC3 cells expressed abundant IAPP mRNA, the biosynthesis of IAPP was lower than for insulin, probably due to less efficient translation of the IAPP mRNA. The t L i of conversion of pro-IAPP to mature IAPP was about 25 min. Newly synthesized pro-IAPP was partly secreted via a constitutive pathway in PTC3 cells, unlike normal @ cells which release almost no prohormone via unregulated pathways. Finally, no effects of dexamethasone or forskolin were observed on either IAPP gene expression or biosynthesis. Thus, @TC3, like other transformed neuroendocrine cells differ in several differentiated properties including levels of hormone expression, efficiency of prohormone sorting and regulation of both translation and secretion.