Involvement of protein kinase C- in inositol hexakisphosphate-induced exocytosis in mouse pancreatic β-cells

Inositolhexakisphosphate (InsP6) plays a pivotal role in the pancreatic β-cell stimulus-secretion coupling. We have used capacitance measurements to study the effects of InsP6 on Ca2+-dependent exocytosis in single mouse pancreatic β-cells. In the presence of inhibitors of the protein phosphatase calcineurin to block endocytosis, intracellular application of InsP6 produced a dose-dependent stimulation of exocytosis, and half-maximal effect was observed at 22 μm. The stimulatory effect of InsP6 was dependent on protein kinase C (PKC) activity. Antisense oligonucleotides directed against specific PKC isoforms (α, βII, δ, ϵ, ξ) revealed the involvement of PKC-ϵ in InsP6-induced exocytosis. Furthermore, expression of dominant negative PKC-ϵ abolished InsP6-evoked exocytosis, whereas expression of wild-type PKC-ϵ led to a significant stimulation of InsP6-induced exocytosis. These data demonstrate that PKC-ϵ is involved in InsP6-induced exocytosis in pancreatic β-cells.

Inositol hexakisphosphate (InsP 6 ) 1 levels transiently increase in pancreatic ␤-cells following an elevation of the ambient glucose concentration (1). This elevation in InsP 6 levels plays an important role in controlling exocytosis of the insulincontaining granules and subsequent retrieval of membrane by endocytosis (2,3). These processes of exocytosis and endocytosis are dependent on protein kinase C (PKC) activity (2,3). Several PKC isoforms are expressed in pancreatic ␤-cells and include PKC-␣, -␤, -␦, -⑀, and - (4,5). However, the importance of a particular PKC isoform in controlling InsP 6 -evoked exocytosis is unknown. The present study was designed to examine which PKC isoform mediates the stimulatory action of InsP 6 on Ca 2ϩ -induced exocytosis. Using whole-cell patch clamp techniques and capacitance measurements of exocytosis, we dem-onstrate that PKC-⑀ regulates InsP 6 -induced exocytosis in single mouse pancreatic ␤-cells.

EXPERIMENTAL PROCEDURES
Preparation of Islet Cells-Pancreatic islets were isolated from fed female NMRI mice (18 -23 g) by collagenase digestion as described previously (3). The local ethical committee in Copenhagen approved the methods of euthanasia. The islets were dispersed into single cells by shaking in a Ca 2ϩ -free solution, and the resulting cell suspension was plated on Nunc Petri dishes and maintained for up to 3 days in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 international units/ml penicillin, and 100 g/ml streptomycin.
Plasmid Construction-The cDNAs for PKC-⑀ and PKC-␤I were kindly provided by Y. Nishizuka (Kobe University, Kobe, Japan). A dominant negative mutant of PKC-⑀ was obtained from K. Ridge (Northwestern University, Chicago, IL). Transfection of mouse ␤-cells was performed using 3 g of PKC cDNA and 1 g of green fluorescent protein using LipofectAMINE according to the manufacturer's instructions. Following transfection, cells were cultured for 48 h in RPMI 1640 supplemented as described above.
Data Analysis-Results are presented as mean values Ϯ S.E. for the indicated number of experiments. The exocytotic rate (⌬C m /⌬t) is presented as the increase in cell capacitance measured 30 -90 s following establishment of the whole-cell configuration. Statistical significance * This study was supported in part by the National Institutes of Health (Grant DK 58508), the Swedish Research Council, the Swedish Diabetes Association, the Novo Nordisk Foundation, the Juvenile Diabetes Foundation International, and the Funds of Karolinska Institutet. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A recipient of a scholarship from the Academy of Technical Sciences and Novo Nordisk A/S.  . 1A shows the exocytotic responses in single mouse pancreatic ␤-cells obtained using the standard whole-cell configuration in which the pipette-filling electrode solution replaces the cytosol. Exocytosis was stimulated by intracellular dialysis of an electrode solution with a Ca 2ϩ -EGTA buffer with a free Ca 2ϩ concentration of 0.87 M. Under control conditions, the rate of capacitance increase averaged 21 Ϯ 4 femtofarads/s (n ϭ 5). This corresponds to the release of seven granules/s as every granule contributes Ϸ3 femtofarads (10). Inclusion of 100 M InsP 6 in the pipette-filling solution produced a delayed (23 Ϯ 4 s; n ϭ 5) decrease in cell capacitance, an effect that we attribute to stimulation of endocytosis (3). On average, the increase in cell capacitance was reduced by 62% (p Ͻ 0.05; n ϭ 5) when measured after 30 -90 s. We have previously reported that InsP 6 -induced endocytosis is mediated by the protein phosphatase calcineurin (3). Interestingly, in cells pretreated for 15 min with 1.5 M of the calcineurin inhibitor cyclosporin A, rather different results were obtained (Fig. 1A). Under these conditions, InsP 6 (100 M) increased the exocytotic response by 86% (p Ͻ 0.01; n ϭ 6). InsP 6 also stimulated exocytosis in the presence of the calcineurin inhibitor deltamethrin (20 nM for 1.5 h) (96% stimulation; p Ͻ 0.01; n ϭ 5), an effect that was not mimicked by permethrin, an inactive analogue of deltamethrin (Fig. 1B). In the presence of permethrin, InsP 6 -induced exocytosis was inhibited by 65% (p Ͻ 0.05; n ϭ 5), which is not different from that observed under control conditions (Fig. 1B). Finally, okadaic acid, an inhibitor of protein phosphatases 1, 2A, and 3, did not affect the InsP 6 -induced reduction of exocytosis (Fig. 1B). These data suggest that InsP 6 stimulates both exocytosis and endocytosis in pancreatic ␤-cells and that the endocytotic process dominates under the actual experimental conditions as suggested by the overall decrease in the rate of capacitance increase. In this respect, it is important to emphasize that capacitance measurements reflect net changes in plasma membrane area resulting from the summed activity of all endocytotic and exocytotic processes. The stimulatory action of InsP 6 on exocytosis was revealed following inhibition of endocytosis.
The stimulatory action of InsP 6 on exocytosis was dependent on dose. No significant stimulation of exocytosis was observed at Ͻ10 M InsP 6 , but higher concentrations elicited a dose-dependent acceleration of secretion (Fig. 1C). Approximating the average data points to the Hill equation yielded a half-maximal stimulatory effect at 22 M and a cooperativity factor of 1.4. Maximal stimulation of exocytosis was seen at concentrations of InsP 6 Ն100 M (Fig. 1C), at which exocytosis was nearly doubled over the control rate of capacitance increase.
The ability of InsP 6 to stimulate exocytosis depends on the [Ca 2ϩ ] f in the pipette-filling solution (Fig. 1D). No stimulation was observed at 30 nM [Ca 2ϩ ] f , but increasing concentrations of Ca 2ϩ elicited a progressive increase in the rate of exocytosis, which was further accelerated in the presence of InsP 6 . The lack of effect of InsP 6 at low [Ca 2ϩ ] f contrasts previous findings in HIT insulinoma cells (2). This difference in Ca 2ϩ sensitivity of the stimulatory action of InsP 6 on exocytosis most likely reflects the use of a clonal cell line versus primary ␤-cells but could also result from different experimental approaches (permeabilized cells and single cell capacitance measurements). Fig. 1E shows that the stimulatory action of InsP 6 on exocytosis was also evoked, although to a lesser extent, by inositol tetrakisphosphate compounds and the inositol pentakisphosphate, Ins(1,3,4,5,6)P 5 . These inositol polyphosphates stimulated exocytosis by only 48 -66%, as compared with 86% increase in the presence of InsP 6 (Fig. 1D). It is noteworthy that the required stimulatory concentrations of InsP 4 , but not InsP 6 , are 50 -100 times higher than those measured in insulin-secreting cells (11). No stimulation of exocytosis was observed in the presence of 100 M Ins(1,3,4)P 3 and Ins(1,4,5)P 3 (Fig. 1D). These data suggest that a minimum of four phosphate groups are required for stimulation of exocytosis and that these phosphate groups can be randomly placed on the inositol ring. Furthermore, the data show that all six phosphate groups are required for maximal stimulation of exocytosis. InsP 6 stimulates exocytosis by activation of PKC in permeabilized insulinoma cells (2). Here we extend this observation to mouse ␤-cells since the stimulatory action of InsP 6 on exocytosis was abolished by the PKC inhibitors calphostin C (1.5 M for Ͼ15 min), bisindolylmaleimide (4 M for 20 min), and staurosporine (100 nM for Ͼ10 min) (Fig. 2). On the contrary, no effect was observed on the stimulatory action of InsP 6 in the presence of the protein kinase A inhibitor (R p )-cAMP (100 M for Ͼ30 min). Under these conditions, InsP 6 stimulated exocytosis by 90% (p Ͻ 0.05; n ϭ 5; Fig. 2).
To investigate which isoform of PKC mediates the stimulatory action of InsP 6 on exocytosis, cells were treated for 48 h with antisense oligonucleotides against PKC-␣, -␤II, -␦, -⑀, and -. Fig. 3A shows that InsP 6 failed to increase exocytosis in cells exposed to antisense oligonucleotides against PKC-⑀. However, InsP 6 retained its stimulatory action when cells were treated with antisense oligonucleotides against PKC-␣, -␤II, -␦, -, and sense PKC-⑀ oligonucleotides. These data argue that InsP 6 stimulates exocytosis by a mechanism that involves activation of PKC-⑀.
The involvement of PKC-⑀ in InsP 6 -induced exocytosis was further supported by studies in mouse ␤-cells transiently transfected with either wild-type or a dominant negative mutant of PKC-⑀. Fig. 3B shows that InsP 6 (100 M) evoked a robust increase in cell capacitance in mock-transfected cells and in cells expressing wild-type PKC-⑀. On the contrary, the dominant negative PKC-⑀ isoform abolished the ability of InsP 6 to stimulate exocytosis. Interestingly, the wild-type PKC-⑀ isoform significantly increased Ca 2ϩ -induced exocytosis, whereas the dominant negative isoform reduced the exocytotic response in the absence of InsP 6 by 50% (Fig. 3B). Transfection of the wild-type PKC-␤I isoform did not influence Ca 2ϩ -and InsP 6induced exocytosis (Fig. 3B).
The results from this study show for the first time that PKC-⑀ is involved in the stimulatory action of InsP 6 on Ca 2ϩevoked exocytosis. The molecular mechanism for the activation of PKC-⑀ by InsP 6 remains to be explored. However, it is pertinent to emphasize that an InsP 6 -activated protein kinase has been identified in rat brain (12) and that InsP 6 enhances L-type Ca 2ϩ channel activity in vascular smooth muscle cells by a PKC-dependent mechanism (13). The substrate(s) for PKC-⑀ are poorly understood, but recent data from our laboratories demonstrate that PKC-⑀ associates with insulin-containing granules in response to glucose and clinically used sulfonylureas. 2 These data suggest that PKC-⑀ represents an important component of the secretory network in pancreatic ␤-cells.
A model for the effects of glucose and InsP 6 on exocytosis and endocytosis is presented in Fig. 4. Glucose acts to stimulate ATP synthesis, leading to closure of ATP-sensitive K ϩ chan-nels, cell depolarization, and Ca 2ϩ influx. The resulting increase in cytoplasmic Ca 2ϩ concentration stimulates exocytosis. Not only activation of the phospholipase C system but also glucose stimulation result in InsP 6 production, which leads to enhanced PKC-⑀ activity. PKC-⑀ potentiates Ca 2ϩ -induced exocytosis. An increase in cytoplasmic free Ca 2ϩ concentration is also required for stimulation of endocytosis. This process involves activation of calcineurin and PKC-⑀, most likely resulting in a sequential phosphorylation and dephosphorylation of proteins involved in endocytosis (for details, see Ref. 3). This suggests that InsP 6 has an important integral role in pancreatic ␤-cell membrane trafficking, being part of the molecular mechanisms linking exocytosis and endocytosis.