Proprotein-processing Endoprotease Furin Decreases Regulated Secretory Pathway-specific Proteins in the Pancreatic (cid:98) Cell Line MIN6*

Prohormone convertases PC2 and PC3, yeast Kex2- family endoproteases specific to the regulated secretory pathway, cleave proinsulin to insulin in the secretory granules of pancreatic (cid:98) cells. The well-differentiated (cid:98) cell line MIN6 expresses PC2 and PC3 and another reg- ulated secretory pathway-specific protein chromogranin A. Furin, another yeast Kex2 endoprotease, exists in the trans -Golgi networks of many cell types. The (cid:98) cell line RINm5F (a cell line that is less differentiated than the MIN6 cell line) does not express the regulated path-way-specific proteins, but strongly expresses furin. We suspected that furin expression may cause the decrement of regulated secretory pathway-specific proteins. To test this hypothesis, we expressed a furin cDNA with a metallothionein promoter in MIN6 cells. With Zn 2 (cid:49) stimulation of furin expression, the messages of PC2, PC3, and chromogranin A decreased, and the processing of proinsulin to mature insulin became less efficient. The furin-expressing MIN6 cells exhibited less insulin content and weakened insulin secretion in response to a high glucose concentration. The conditioned medium from furin-expressing MIN6 cells also exerted a de- crease of PC2 and PC3 expression in unaltered MIN6 cells. Thus, proteins

Pancreatic ␤ cells possess well-differentiated characteristics, such as the formation of insulin secretory granules, the processing of proinsulin to mature insulin, and the glucose-stimulated secretion of insulin. ␤ cells synthesize insulin as proinsulin, a precursor peptide, at the rough endoplasmic reticulum, from which transport vesicles carry the proinsulin to the trans-Golgi networks. At the trans-Golgi networks, proinsulin is sorted to a secretory pathway that is well regulated by an extracellular stimulus, such as glucose (1). Through this pathway, proinsulin (B chain-Arg-Arg-C peptide-Lys-Arg-A chain) is packaged into immature granules and processed to mature insulin (B chain-S-S-A chain), which is then stored in densecore granules (2). When this secretory system is stimulated, insulin molecules stored in the dense-core granules are secreted into the extracellular space. These granules contain at least three distinct groups of processing enzymes: the prohormone convertases PC2 and PC3 (also called PC1), carboxypeptidase H, and amidation enzymes (3)(4)(5). Processing of proinsulin into mature insulin requires cleavage by PC2 and PC3 at the paired basic residues and their subsequent removal by carboxypeptidase H (3,4,6).
In addition to the regulated secretory pathway, ␤ cells are equipped with a secretory pathway that is not regulated by an extracellular stimulus. Along this constitutive secretory pathway, proteins are transported from the trans-Golgi networks to plasma membranes by small transport vesicles (1,7). Furin, from the family of yeast Kex2 endoproteases to which PC2 and PC3 also belong (8,9), is located at the trans-Golgi network (10). While PC2 and PC3 are uniquely expressed in the regulatory pathway of neuroendocrine cells, furin is thought to be present in the constitutive pathway of virtually all cell types (11). Furin is specific for the unique cleavage sequence -Arg Ϫ4 -X Ϫ3 -(Lys/Arg Ϫ2 )-Arg Ϫ1 (RX(K/R)R) (12,13). This furin-specific RX(K/R)R motif is found in a number of growth factor precursors (12), including nerve growth factor, platelet-derived growth factor, transforming growth factor ␤, activin A, the adhesion molecule cadherin, metalloproteinases such as stromelysin 3 (14), and matrix metalloproteinase X1 (15), and, in some growth factor proreceptors (12) including those for insulin receptor, insulin-like growth factor 1 receptor, and hepatocyte growth factor receptor (oncoprotein MET). Several reports indicate that furin is responsible for the formation of bioactive proteins such as transforming growth factor ␤ (16), stromelysin 3 (14), and insulin receptor (17). We previously found that when mutant proinsulin with furin cleavage sites was expressed in the mouse fibroblast-like cell line NIH3T3, containing high levels of furin, and the Chinese hamster ovaryderived cell line CHO which expressed low levels of furin, NIH3T3 cells produced more bioactive insulin than did CHO cells (18). Thus, while furin is localized in virtually all cell types (11), increased expression of furin may generate more bioactive proteins from their precursors, and the increased protein levels may induce a change in cell growth as well as differentiation of cell functions. Furin expression in mature ␤ cells may induce notable changes in their highly differentiated characteristics, such as the formation of insulin secretory granules, the processing of proinsulin to mature insulin, and glucose-stimulated secretion of insulin.
To investigate this possibility, we chose one well-differentiated ␤ cell line, MIN6 (19,20), and two less-differentiated cell lines, HIT-T15 (21) and RINm5F (22). We compared the ex-pression of the prohormone convertases PC2 and PC3, the regulatory pathway marker protein chromogranin A (23), and the constitutive pathway enzyme furin among the three ␤ cell lines. Since furin expression appears to be correlated with an undifferentiated state of ␤ cell function, we prepared a furinexpressing MIN6 cell line, MIN6-mf, under the control of a metallothionein promoter. The present study investigates the decrement of the differentiated characteristics of ␤ cells using the MIN6-mf cell line.
Radioimmunoassay-Immunoreactive insulin (IRI) was measured using an insulin immunoassay kit (Amersham Japan, Tokyo), according to the manufacturer's instruction. The antibody in this kit recognized both proinsulin and mature insulin equally on a molar basis. Assay samples were prepared by collecting the cell culture medium, obtaining acid-ethanol extracts from the islets or cells, and fractionating the solutions by gel filtration chromatography.
Gel Filtration-Since the IRI concentration in the culture medium secreted from the RINm5F cells was too low for the gel filtration analysis, the RINm5F cells were permanently transformed with the rat insulin I gene inserted into the pCXN2 vector (24). Expression with this vector is based on the chicken ␤-actin promoter along with the cytomegalovirus immediate early region enhancer.
The media from the rat pancreatic islets, and the three cell lines MIN6, HIT-T15, and rat insulin I gene-expressing RINm5F were applied to a 1.0 ϫ 120-cm column of Sephadex G-50 superfine gel (Pharmacia Biotech Inc.) equilibrated with 100 mM acetic acid, as described previously (18). The proportion of proinsulin that was converted to insulin was quantified by measuring the areas of proinsulin and insulin fractions on the gel chromatogram, and the area proinsulin /area proinsulin ϩ area insulin was calculated.
Northern Blot Analysis-The cells were lysed using the guanidinium thiocyanate hot phenol method, as described previously (18). For Northern blot analysis, 10 g of total cellular RNA was electrophoresed on a 1.0% agarose gel and transferred to a nylon membrane (Hybond-N, Amersham Japan). Hybridization was carried out as described previously (18), using the following cDNA probes: mouse PC2, a 720-base pair (bp) fragment cut with BamHI (25); mouse PC3, a 231-bp fragment cut with KpnI and HindIII (26); mouse furin, a 924-bp fragment cut with BamHI (11); rat chromogranin A, a 1.8-kilobase pair fragment cut with EcoRI (27); and mouse ␤-actin.
Immunocytochemical Studies-Antibody against furin was raised by inoculating rabbits with the synthetic oligopeptide QDPDPQPRY-TQMNDNRHGTRC which spans the mouse furin sequence of 21 amino acids from position 178 to 198 (11). Histidine, the fifth amino acid from the end, consists of an essential active site of Kex2 endoproteases with two other amino acids, aspartic acid and serine. Since we needed the last amino acid to be cystine for the covalent conjugation with the carrier protein keyhole limpet hemocyanin, we added the last sequence of five amino acids, which shares a strong homology with other Kex2 endoproteases. The first sequence of 16 amino acids shared 50% homology with PC2 (25), PC3 (26), and PACE4C, another furin-like endoprotease (28). The oligopeptide-protein conjugate was emulsified with Freund's complete adjuvant and injected into three New Zealand White rabbits. After several booster injections, all three rabbits developed antibodies against the oligopeptide.
Immunofluorescence was studied using MIN6 and RINm5F cells cultured on poly-L-lysine-coated glass coverslips for 2 to 3 days and fixed with 4% paraformaldehyde in a 0.1 M phosphate buffer at a pH of 7.4 for 1 h at room temperature. These cells were then treated with 50 mM NH 4 Cl in phosphate-buffered saline (PBS) for 20 min to quench any free aldehyde and made permeable by treatment with PBS containing 0.1% saponin and 0.4% bovine serum albumin (BSA) for 30 min. The specimens were incubated with 1% BSA-PBS for 30 min, followed by another 12-h incubation at 4°C with anti-furin serum diluted at 1:4000. After the cells were washed with PBS, they were incubated for 1 h with indocarbocyanine-conjugated affinity-purified donkey anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA), diluted with 1% BSA-PBS, and mounted with PermaFluor (Immuron, Pittsburgh, PA). The specimens were examined through a Olympus BX50 microscope equipped with a BX-FLA incident illuminator.
Establishment of Furin-expressing MIN6 Cells-Mouse furin cDNA (11) was inserted between the HindIII and NotI sites of the expression vector pMEP4 (Invitrogen), to be expressed under the control of a metallothionein promoter. This vector carries a hygromycin-resistant gene as a selectable marker. MIN6 cells were transformed with this furin expression vector or empty vector pMEP4 alone using liposome N- [1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Boehringer Mannheim). The furin-expressing MIN6-mf cells and the empty vector-introduced MIN6-0 cells were selected by maintaining the culture in a medium containing hygromycin B (0.1 mg/ml) (Sigma).
Glucose-stimulated Insulin Secretion-MIN6 and MIN6-mf cells were cultured separately in 1-ml aliquots of Dulbecco's modified Eagle's medium without FBS and glucose in 24-well plates. After the cells were washed twice with PBS, they were placed in Dulbecco's modified Eagle's medium containing 5% FBS with 1.0, 5.0, or 25.0 mM glucose for 12 h, as described previously (19). Insulin secreted into the buffer was measured using radioimmunoassay.

RESULTS
Insulin Content in Each ␤ Cell Line-The IRI content was highest in the MIN6 cells, followed by the HIT-T15 and RINm5F cells (Table I). The insulin content of ␤ cells in rat islets has been reported by other researchers to be approximately 8 nmol/10 6 cells (29, 30), and we have previously found it to be 5.2 Ϯ 0.3 nmol/10 6 cells. In the present study, the insulin content of the MIN6 cells (320 pmol/10 6 cells) was one order of magnitude lower than that found in ␤ cells of the rat islet. This difference is consistent with studies using the electron microscope that indicate that although MIN6 cells contained a fair number of dense-core secretory granules, mature islet ␤ cells contain far more dense-core granules than do MIN6 cells (19). No secretory granules were observed in HIT-T15 and RINm5F cells (data not shown). Their insulin content was three orders of magnitude lower than that of MIN6 cells (Table I).
Conversion Ratio of Proinsulin to Insulin-We examined the extent of conversion of proinsulin to mature insulin in each ␤ cell line. The culture medium from each cell line was subjected to gel filtration on a Sephadex G-50 column. IRI was eluted first at the proinsulin position and then at a peak corresponding to the mature insulin position (18). The ratio of proinsulin to total IRI was approximately 12.7% in MIN6 cells, 44.8% in HIT-T15 cells, and 64.6% in RINm5F cells (Table II). The percentage of proinsulin found in MIN6 cells was similar to the percentage of proinsulin found in the culture medium of ma- Northern Blot Analysis of Secretory Pathway Protein mRNAs-We evaluated the predominance of the proteins involved in the two secretory pathways in each ␤ cell line and we studied the prohormone convertases PC2 and PC3 along with the secretory granule-specific protein chromogranin A as representative proteins of the regulated secretory pathway. We also studied the endoprotease furin as a representative protein of the constitutive secretory pathway. As shown in Fig. 1, the messages for the three regulated pathway proteins were strongly expressed, in MIN6 cells, while the furin message was not. On the other hand, the furin message was found at high levels in RINm5F cells and at lower levels in HIT-T15 cells.
HIT-T15 cells also contained message for PC2, but RINm5F cells did not contain any of the three regulated pathway-specific protein messages. Although RINm5F cells were previously reported to contain a PC2 message (31, 32), we could not detect it on Northern blot. Since our RIN cells contained a trace level of insulin, the cell line may have been dedifferentiated and lost PC2 message.
Immunocytochemical Staining of Furin-Since the furin message was abundant in the RINm5F cells and absent in the MIN6 cells, we studied these two cell lines for furin localization. Furin was found to be present in RINm5F cells along the perinuclear region, shown by a number of stained granular dots ( Fig. 2A). The amino acid structure of furin suggests that there is a transmembrane-spanning region at its carboxyl terminus and furin is thought to be located in the trans-Golgi network (10,33). However, these stained dots appear to be distributed from the trans-Golgi network to whole Golgi complex. On the other hand, MIN6 cells showed only slight immunostaining scattered in the cytoplasm (Fig. 2B), indicating that MIN6 cells contain a very low level of furin. Both RINm5F and MIN6 cells were completely negative when stained with nonimmunized serum.
Characteristics of MIN6 Cells with a Different Passage Number-We noticed that with increasing passages, MIN6 cells began to exhibit visible furin messages in Northern blots of their RNAs. To confirm this phenomenon, we compared the Northern blot pattern using the cells with passage numbers 16, 25, and 35. Fig. 3 demonstrates that the furin message became stronger with increasing cell line passages. In contrast, PC2 message extensively and PC3 message slightly decreased with increasing cell line passages.
We next compared insulin content and glucose-responsive insulin secretion between the MIN6 cells with the passage numbers 16 and 35. Insulin content was almost 2-fold higher in the cells at passage 16 than in the cells at passage 35 (Table  III). Insulin secretion in response to 1 mM and 5 mM glucose was similar in MIN6 cells at 16 and 35 passages. However, when the glucose concentration was raised form 5 mM to 25 mM, the cells at passage 16 showed nearly a 2-fold increase in insulin secretion, while the cells at passage 35 showed an increase of only 1.2-fold (Fig. 4). Thus, PC2 and PC3 expression, insulin content, and glucose-responsive insulin secretion decreased with increasing passage number, whereas furin expression increased.
Characteristics of MIN6-mf Cells-Since MIN6 cells exhibit  1. Northern blot analysis of regulatory pathway prohormone convertases PC2, PC3, and the regulatory pathway protein chromogranin A (CgA), and the constitutive pathway enzyme furin. 18 S (Ϫ) and 28 S (Ϫ*) represent the corresponding sizes of ribosomal RNAs. Note that the bands of the three regulatory pathway protein mRNAs were clearly visible, but a furin mRNA band was not found in the MIN6 cells. The furin was observed strongly in RINm5F cells, which did not contain any of the three regulatory pathway protein messages. increasing furin levels as the passage number increases (Fig.  3), we engineered a new MIN6 cell line with a furin-expression system that could be regulated. By regulating furin expression, we were able to compare the induced and noninduced furin expression states in MIN6 cells of the same passage. We introduced furin cDNA into MIN6 cells under the control of a metallothionein promoter that can be induced with Zn 2ϩ . The furin-expressing MIN6-mf cells and the empty vector-introduced MIN6 -0 cells were selected by maintaining the culture in a medium containing hygromycin B. At selection, the passage number had already reached 26, and on a Northern blot, the cells exhibited a faint band of furin expressed without Zn 2ϩ stimulation. The MIN6 cells either with or without the empty vector were not affected in cell viability by both 50 and 100 M ZnCl 2 . The MIN6-mf cells exhibited an increase in furin message linearly with an increasing concentration of Zn 2ϩ (0, 25, 50, and 100 M) on Northern blots (data not shown). Since none of the Zn 2ϩ concentrations showed any toxic effect on the cells, we used 100 M Zn 2ϩ for the time course experiment of furin expression. Fig. 5A illustrates the higher furin expression in the MIN6-mf at 48 and 72 h after the addition of Zn 2ϩ into the culture medium. On the other hand, the MIN6-0 cells with the empty vector pMEP4 alone exhibited a faintly low level of furin expression in the presence of Zn 2ϩ in the culture medium (Fig.  5B). Since Zn 2ϩ is known to inhibit yeast Kex2 endoprotease and furin (13,34), we examined the effect of Zn 2ϩ on the expression of PC2 and PC3 transcripts. As shown in Fig. 5B, both PC2 and PC3 were not affected in their expression by the presence of Zn 2ϩ .
Upon immunocytochemical staining of furin, stained granular dots were amassed in the perinuclear region in the Zn 2ϩstimulated cells, unlike in the unstimulated cells (Fig. 6, A and  B). As mentioned above, furin staining was visible even in unstimulated cells because these cells were at passage 26 and had began to express furin as shown in Fig. 5, A and B.
In contrast with the increase in furin expression, the expres-sion of PC2 and PC3 decreased extensively in Zn 2ϩ -stimulated MIN6-mf cells cultured for 48 and 72 h (Fig. 7). This decrease of PC2 and PC3 messages by Zn 2ϩ was not observed in the empty vector-introduced MIN6 -0 cells (Fig. 5B). MIN6-mf cells cultured for 72 h also showed a decrease in chromogranin A message. Thus, it appears that increased furin induction decreased the expression of the regulated pathway proteins PC2, PC3, and chromogranin A. As expected from the decrement of PC2 and PC3, Zn 2ϩ -stimulated MIN6-mf cells produced a higher proportion of proinsulin to the total IRI than did unstimulated MIN6-mf cells (54% versus 13%) (Fig. 8). The Zn 2ϩ -stimulated MIN6-mf cells (high furin induction) exhibited less insulin content than unstimulated MIN6-mf (low furin induction) at 72 h after the Zn 2ϩ addition (Table IV). This is consistent with the decrease in insulin content as unaltered MIN6 cells progress from passage 16 (low furin induction) to passage 35 (moderate furin induction) as shown in Table III.  With the decrement of regulated pathway-specific proteins, glucose-responsive insulin secretion was diminished. The MIN6 cells at passage 16 secreted a 2.0-fold higher level of insulin in a 25 mM glucose medium than in a 1 mM glucose medium (Fig. 4). This glucose-dependent increase became only 1.6-fold in MIN6-mf cells without Zn 2ϩ stimulation, probably due to the increase in cell line passage (passage 26) (Fig. 9). The glucose-induced secretion was further reduced when the cells were stimulated with 100 M Zn 2ϩ for 48 h. Zn 2ϩ -stimulated MIN6-mf cells exhibited only a 1.2-fold increase in insulin secretion when the glucose concentration was raised from 5 mM to 25 mM (Fig. 9). However, the increase from 1 mM to 5 mM glucose resulted in similar increases in furin expression (2.1-to 2.3-fold) in all three cell conditions, MIN6 cells at passage 16 and MIN6-mf cells with and without Zn 2ϩ stimulation. Thus, the greatest change in glucose-responsive insulin secretion was a marked decrease observed in Zn 2ϩ -stimulated MIN6-mf cells when glucose concentrations were shifted from 5 mM to 25 mM.
Effect of Conditioned Medium on PC2 and PC3 Expression-Furin generates bioactive proteins from their precursor forms which then may be released into the extracellular space. We investigated the possibility that furin-expressing MIN6-mf cells secrete these proteins into the culture medium. To address this question, we used MIN6-mf cells to prepare a conditioned medium and tested for the presence of such extracellular proteins. As shown in Fig. 10, the conditioned medium from Zn 2ϩstimulated MIN6-mf cells induced a marked decrease of PC2 and PC3 expression in unaltered MIN6 cells compared with the medium from unstimulated cells. Since the conditioned medium of Zn 2ϩ -stimulated MIN6-mf cells contained Zn 2ϩ , we dialyzed the medium using Amicon filter to exclude the Zn 2ϩ effect and examined then the effect of Zn 2ϩ -free medium on PC2 and PC3 expression. The dialyzed medium from Zn 2ϩstimulated MIN6-mf cells also caused a decreased expression of PC2 and PC3 similar to the nondialyzed conditioned medium, whereas the dialyzed medium from nonstimulated cells did not induce any change in the expression. The result suggests that the conditioned medium may contain bioactive protein(s) cleaved by furin. The cleavage may take place intracellularly or extracellularly, because membrane-bound furin is localized in the trans-Golgi network while truncated furin is secreted into the culture medium (33). In either case, furin induced by Zn 2ϩ in MIN6-mf cells may convert some precursor proteins to their bioactive forms which consequently mediate the decrement of PC2 and PC3 expression. DISCUSSION The present results demonstrate that when furin is induced in the pancreatic ␤ cell line MIN6, expression of the regulatory pathway-specific prohormone convertases PC2 and PC3 and the secretory granule-specific protein chromogranin A, insulin content, and glucose-stimulated insulin secretion are decreased. MIN6 cells exhibited typical characteristics of mature ␤ cells, including the presence of the three regulatory pathway- Culture medium was collected every 24 h. IRI in the culture medium collected between 48 and 72 h was separated with a Sephadex G-50 superfine gel column. The proinsulin peak of MIN6-mf cells stimulated by Zn 2ϩ was larger than that of unstimulated MIN6-mf cells. Molecular size was calibrated with blue dextran (V o ), potassium ferricyanide (V t ), and synthetic human insulin. Similar elution profiles were obtained in three other chromatography tests.

TABLE IV
Comparison of IRI content between MIN6-mf cell lines cultured in the presence and absence of Zn 2ϩ Cells were sonicated in an acid-ethanol solution for homogenization. After the homogenate was centrifuged, its supernatant was evaporated and the remaining pellet was reconstituted with an assay buffer. IRI was measured using an immunoassay kit. The values represent the mean Ϯ S.D. specific proteins PC2, PC3, and chromogranin A, and a conversion ratio of proinsulin to insulin similar to that found in mature islet ␤ cells. The cells also retain an insulin secretion capacity that can be regulated by a physiological range of glucose concentrations (19,20). A previous study observed that pancreatic ␤ cells purified by a fluorescence-activated cell-sorting system exhibited only 1.5-to 2.0-fold insulin secretion when the glucose concentration was raised from 3 mM to 20 mM, although the addition of cAMP-generating secretagogues together with glucose strongly facilitates the insulin secretion (35). Thus, the glucose-responsive insulin-secreting capacity of MIN6 cells is well-maintained, although it is considerably lower than that of pancreatic ␤ cells exhibited in the islet (36). In contrast, RINm5F cells illustrated characteristics opposite to those of the well-differentiated ␤ cells. These characteristics included a low level of insulin, virtually no regulated secretory pathway protein messages, no glucose-responsive insulin secretion (36,37), and an especially high level of furin expression. The presence of furin in RINm5F suggests that this enzyme may be expressed in the less differentiated state of ␤ cells. Indeed, rat fetal and infant ␤ cells showed strong furin immunostaining (data not shown), but adult ␤ cells stained only faintly (38).
Since furin appeared to be expressed in less-differentiated ␤ cells, we examined the furin expression in MIN6 cells of different passage numbers. For studies such as these, MIN6 is the ␤ cell line of choice because the cells retain their glucose-responsive insulin-secreting capacity (19,20). In many well-differentiated ␤ cell lines, this glucose-responsive insulin secretion is known to be gradually diminished with the increase of cell line passages (39,40), as seen in MIN6. With increasing MIN6 cell line passages and the resulting decline in secretion capacity, PC2 and PC3 messages also decreased and insulin content was diminished. In contrast, furin expression increased with increasing cell passages.
Proinsulin is converted to mature insulin through the regulated secretory pathway (1). On the other hand, furin is thought to cleave precursor proteins that are secreted through a constitutive secretory pathway. Thus, furin probably has a minor role in processing proinsulin. Furin cleaves the consensus processing site -Arg Ϫ4 -X Ϫ3 -(Lys/Arg Ϫ2 )-Arg Ϫ1 2 (12,13). For cleavage to occur properly, more basic amino acids are required ahead of the general cleavage site of most propeptide hormones, -(Lys/Arg Ϫ2 )-Arg Ϫ1 2. If furin is highly expressed in ␤ cells, the bioactive proteins cleaved from their precursors would increase in concentration. To examine whether furin is a primary cause for the decline of PC2 andPC3 expression and for the diminished glucose-responsive insulin secretion, we developed a new cell line MIN6-mf with a furin expression system that was regulated by Zn 2ϩ . With an increase in furin expression, all three regulatory pathway-specific proteins (PC2, PC3, and chromogranin A) were diminished. The MIN6-mf cells stimulated with Zn 2ϩ generated a larger proportion of proinsulin to total IRI than did MIN6-mf cells without Zn 2ϩ stimulation. Two reasons may be considered for the increase in proinsulin proportion. First, the decrease of prohormone convertases PC2 and PC3 may have increased the proportion of unprocessed proinsulin. Second, the decrease of regulated pathway proteins may have eventually driven proinsulin to a constitutive pathway. Since furin is known to recycle between the trans-Golgi network and plasma membrane (33,41), the furin recycling may increase a protein flow of the constitutive pathway, which results in an augmented transport of proinsulin molecules outside the cells. In a mouse pituitary tumorderived cell line AtT20, which carries both regulatory and constitutive pathways, a part of human proinsulin expressed in this cell line was sorted to a constitutive pathway where proinsulin is processed to an intermediate form des-31,32-split proinsulin (42). In the MIN6-mf cell line, it remains to be investigated whether proinsulin is processed to this intermediate form.
Insulin content and glucose-stimulated insulin secretion were also diminished in Zn 2ϩ -stimulated MIN6-mf cells. Proinsulin may have escaped by way of a rapid secretion of newly formed immature granules or through the constitutive pathway to the culture medium, which may result in the decrease of insulin content in MIN6 cells. The resulting diminished insulin content may lead to the decrease of insulin secretion in response to glucose. In addition to this possibility, we need to assess the impairment of the glucose-sensing system, namely the decreased expression of type 2 glucose transporter and glucokinase in furin-expressing MIN6-mf cells.
Furin generates secretory peptides such as platelet-derived growth factor, transforming growth factor ␤, and activin A as well as membrane-bound proteins such as cadherin and insulin receptor (12, 14 -17). To examine if extracellular peptides cleaved by furin cause the loss of differentiation in MIN6 cells, we prepared the conditioned medium from the Zn 2ϩ -stimulated and unstimulated MIN6-mf cells. The conditioned medium from the Zn 2ϩ -stimulated cells induced the decrement of PC2 and PC3 expression in the genetically unaltered MIN6 cells. Thus, we can say at least that some extracellular peptides cleaved by furin must have exerted the loss of differentiation in ␤ cells, although we cannot say whether furin cleaved precursor peptides intracellularly or extracellularly.
Activation of the constitutive secretory pathway appears to be correlated with furin expression. We do not know what factors are involved in the activation of the constitutive pathway, but once furin is induced, furin-cleaved bioactive proteins including a number of growth factors may lead well-differentiated ␤ cells to an undifferentiated state. Since the decline of ␤ cell functions are often observed in insulinomas (43,44) and non-insulin-dependent diabetes mellitus (45), understanding the physiological switching from a regulated secretory pathway to a constitutive pathway may aid in understanding the onset of such disorders. FIG. 10. Effect of conditioned media from Zn 2؉ -stimulated and unstimulated MIN6-mf cells on the expression of PC2 and PC3 in unaltered MIN6 cells. Conditioned medium A was derived from unstimulated MIN6-mf cells cultured for 48 h; conditioned medium B was derived from Zn 2ϩ -stimulated MIN6-mf cells cultured for 48 h. MIN6 cells were cultured for 48 h with either conditioned medium A or B. RNA was isolated from MIN6 cells cultured in each medium. Northern blot analysis was performed to examine PC2 and PC3 expression. Both PC2 and PC3 expressions were lower in the MIN6 cells cultured with the conditioned medium B than in those cultured with the conditioned medium A. This result was reproduced even with the dialyzed Zn 2ϩ -free media A and B.