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Am J Physiol Endocrinol Metab 292: E845-E852, 2007. First published November 22, 2006; doi:10.1152/ajpendo.00103.2006
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Inhibitory role of Src family tyrosine kinases on Ca2+-dependent insulin release

Haiying Cheng, Susanne G. Straub, and Geoffrey W. G. Sharp

Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, New York

Submitted 6 March 2006 ; accepted in final form 3 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Both neurotransmitter release and insulin secretion occur via regulated exocytosis and share a variety of similar regulatory mechanisms. It has been suggested that Src family tyrosine kinases inhibit neurotransmitter release from neuronal cells (H. Ohnishi, S. Yamamori, K. Ono, K. Aoyagi, S. Kondo, and M. Takahashi. Proc Natl Acad Sci USA 98: 10930–10935, 2001). Thus the potential role of Src family kinases in the regulation of insulin secretion was investigated in this study. Two structurally different inhibitors of Src family kinases, SU-6656 and PP2, but not the inactive compound, PP3, enhanced Ca2+-induced insulin secretion in both rat pancreatic islets and INS-1 cells in a concentration-dependent and time-dependent manner. Furthermore, Src family kinase-mediated insulin secretion appears to be dependent on elevated intracellular Ca2+ and independent of glucose metabolism, the ATP-dependent K+ channel, adenylyl cyclase, classical PKC isoforms, extracellular signal-regulated kinase 1/2, and insulin synthesis. The sites of action for Src family kinases seem to be distal to the elevation of intracellular Ca2+ level. These results indicate that one or more Src family tyrosine kinases exert a tonic inhibitory role on Ca2+-dependent insulin secretion.

rat pancreatic islets; beta-cell; signaling; Src family kinase

TYPE 2 DIABETES mainly results from the defects in insulin secretion from pancreatic beta-cells and insulin resistance in target tissues (37, 44, 49). Under normal conditions, insulin secretion is tightly controlled by a variety of stimulatory, modulatory, and inhibitory factors. Glucose is the primary physiological secretagogue, whereas norepinephrine and somatostatin are major physiological inhibitors of insulin secretion (38). Glucose generates both triggering and amplifying signals. The triggering pathway, also referred to as the ATP-dependent K+ (KATP) channel-dependent pathway, is well documented, whereas the mechanisms underlying the KATP channel-independent amplification pathway(s) have yet to be established (20, 41, 42).

There is increasing evidence to suggest that protein tyrosine kinases (PTKs) are involved in the regulation of insulin secretion (35, 40, 48). However, the precise function of tyrosine kinases in secretion is not known. Some studies have demonstrated that tyrosine kinase inhibitors induce large increases in insulin secretion (40, 48), although decreased release also has been reported (35). The controversial findings may result from the possibility that different PTKs have different or even opposite effects on insulin secretion.

Tyrosine kinases are divided into the following two major groups: receptor and nonreceptor. The Src family belongs to the nonreceptor tyrosine kinase group. Several lines of evidence suggest that the members of this family play a role in the highly regulated exocytotic process: 1) Src is expressed at high levels and concentrated on secretory vesicles and the plasma membrane in cells that are specialized for exocytosis, such as neuronal and endocrine cells (13, 15); 2) It has been shown that Src family tyrosine kinases exert an inhibitory effect on neurotransmitter release from neuronal cells (33). In particular, they may be specifically involved in the regulation of kiss-and-run mode of exocytosis in synaptosomes, likely by tonic inhibition of kiss-and-run mode of secretion (4); 3) Src kinases can be activated by protein tyrosine phosphatase-{alpha} (PTP{alpha}; see Ref. 54). In INS-1E cells, overexpression of the wild-type PTP{alpha} reduced insulin secretion, whereas overexpression of a phosphatase-inactive mutant of PTP{alpha} improved insulin secretion (21); 4) Src family kinases may be regulated by myristoylation and dynamic palmitoylation, and protein acylation has been implicated in the mechanism of inhibition of insulin secretion (9); and 5) Src kinase pathways are important for the extracellular Ca2+-dependent pancreatic enzyme secretion from pancreatic acini (47).

Neurotransmitter release and insulin secretion share many similar regulatory mechanisms (17). We hypothesized that Src family tyrosine kinases, which play important roles in neurotransmitter release (33), may also be involved in insulin secretion. Moreover, PTP{alpha}, an activator of Src kinase, has been shown to have an inhibitory role in insulin secretion (21). It is reasonable to propose that Src kinases may negatively regulate insulin secretion. Therefore, in the present study, we investigated the potential role of Src family tyrosine kinases in insulin secretion. We found that two structurally unrelated inhibitors of Src family tyrosine kinases, PP2 and SU-6656, potentiated Ca2+-dependent insulin secretion from both rat pancreatic islets and INS-1 cells. The results suggest that Src family tyrosine kinases exert an inhibitory effect on Ca2+-dependent insulin secretion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Glucose, norepinephrine, forskolin, 12-O-tetradecanoylphorbol 13-acetate (TPA), Gö-6976, protein G-agarose beads, and nitrendipine (NTD) were obtained from Sigma (St. Louis, MO). 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolol[3,4,d]pyrimidine (PP2), 4-amino-7-phenylpyrazol[3,4,d]pyrimidine (PP3), and PD-98059 were purchased from Calbiochem.

Antibodies. Anti-Src monoclonal antibody (mAb) 327 was kindly provided by Dr David I. Shalloway (Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY). Anti-Src[pY418]phosphospecific antibody was bought from Biosource.

Isolation of rat pancreatic islets. Male Sprague-Dawley rats (250–400 g) were used. Immediately after CO2 asphyxiation, the pancreases were removed, and the islets were isolated by a collagenase digestion technique (24). Krebs-Ringer bicarbonate HEPES buffer (KRBH) containing 129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 2.8 mM glucose, 10 mM HEPES, and 0.1% BSA at pH 7.4 was used for isolation and picking of the islets.

Measurements of insulin release from islets. Insulin release was measured under static incubation conditions at 37°C using batches of five size-matched islets per tube. Islets were first cultured in RPMI 1640 media that contains 5.6 mM glucose, supplemented with 10 mM HEPES, 10% FBS, 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µM beta-mercaptoethanol at 37°C in a 95% air plus 5% CO2 atmosphere for 3 h (or for the time indicated in the text) and preincubated in regular KRBH buffer for the following 60 min. The islets were then incubated for 15 or 30 min. PP2, PP3, and SU-6656 were included in both culture and preincubation periods. At the end of the incubations, samples were taken and kept at –20°C until radioimmunoassayed. Insulin secretion was usually expressed as fractional release, i.e., the percentage of the insulin content of the islets that was released.

Cell culture and measurements of insulin release for clonal cells. INS-1 cells were grown and maintained in monolayer culture in RPMI 1640 media that contains 11.1 mM glucose, supplemented with 10 mM HEPES, 10% FBS, 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, and 50 µM beta-mercaptoethanol at 37°C in a 95% air plus 5% CO2 atmosphere. Under static incubation conditions, cells were grown in 16-mm-diameter wells until confluency was reached. The experimental conditions used were the same as those used for rat islets with the following three exceptions: 1) the total treatment time with PP2, PP3, SU-6656 and PD-98059 was 2 h, 2) for PP2 and PP3, the RPMI 1640 medium contained 11.1 mM glucose, and 3) for SU-6656 and PD-98059, cells were not cultured in RPMI medium, instead they were treated in KRBH buffer for 2 h in the presence or absence of SU-6656 and PD-98059 before incubation.

Treatment of INS-1 cells before kinase assay or immunoblotting. Tissue culture plates (60 or 35 mm in diameter) of INS-1 cells were washed two times in KRBH buffer. Preincubation was carried out at 37°C in KRBH buffer for 60 min (PP2 or PP3 were present in RPMI medium for 1 h and then in regular KRBH buffer for preincubation for another hour). After the medium was aspirated out, fresh KRBH buffer with or without test agents was used for the incubation conditions (KRBH alone with basal glucose conditions was used as the control). The timing of the incubations was 15 min. The reactions were stopped by removing the medium and washing the cells two times with cold PBS. After any leftover solution was completely aspirated out, the plates were incubated in lysis buffer (10 mM Tris, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 60 µg/ml aprotinin) and rocked at 4°C for 60 min. The samples were collected in 1.5-ml Eppendorf tubes and centrifuged at 15,000 revolutions/min for 15 min at 4°C, and the resulting supernatants were used in the experiments as the cell lysate.

SDS-PAGE and immunoblot analysis. Western blotting was done according to Biosource instructions with minor modifications as follows. Lysate was resuspended with an equal volume of Laemmli sample buffer (125 mM Tris, pH 6.8, 10% SDS, 10% glycerol, 0.006% bromphenol blue, 130 mM dithiothreitol, and 5% 2-mercaptoethanol) and boiled for 90 s. The samples (20–40 µg/lane with equal amounts of protein used for all samples in the same experiment) were then separated by SDS-PAGE and transferred electrophoretically to a polyvinylidene fluoride (PVDF) membrane. The transfer solution contained 20 mM Tris, 230 mM glycine, 0.01% SDS, and 20% methanol. The membrane was then blocked overnight at 4°C in Tris-buffered saline containing 20 mM Tris-Cl, pH 7.4, 0.9% NaCl, and 0.1% Tween 20, plus 5% BSA (TBST). Primary antibody incubations were carried out for 2 h at room temperature. After being washed with TBST buffer four times for 10 min each, the membrane was incubated with horseradish peroxidase-conjugated anti-rabbit IgG from donkey or horseradish peroxidase-conjugated anti-mouse IgG from goat for 2 h at room temperature as appropriate. Washing of the membrane was carried out as before, and the bands were visualized by chemiluminescence. For experiments that required quantification, the density of bands was analyzed using {alpha} Image software.

Immunoprecipitation. The reactions were performed by first incubating anti-Src mAb327 with cell lysates of INS-1 cells, rat pancreatic islets, or NIH 3T3 cells (300 µg/lane) for 2 h at 4°C with shaking. The materials were centrifuged, and the supernatant was incubated with protein G agarose beads overnight at 4°C with shaking. After several washes with lysis buffer, the samples were boiled, and the beads were removed by centrifugation. The samples were then loaded on gels for analysis. The PVDF membranes were probed with anti-Src mAb327.

Src kinase assay. Src kinase activity was determined as described with modification (1, 39, 54). INS-1 cell lysates containing 1–2 mg of protein were first incubated with anti-Src mAb327 for 2 h at 4°C and then mixed with GammaBind Sepharose beads for another hour. Kinase activity in the immunoprecipitate was subsequently measured by using an Src assay kit (Upstate Biotechnology), following the manufacturer's instructions except that the reactions were terminated by adding 5x SDS sample buffer.

Glucose oxidation. Glucose oxidation was measured as previously reported with minor modification (51). In brief, groups of 25 islets (in duplicate) were cultured at 37°C for 3 h in RPMI medium containing 5.6 mM glucose in the presence or absence of PP2 as indicated. The islets were then preincubated in KRBH buffer in the presence or absence of PP2 for 1 h. After preincubation, islets were incubated in 150 µl of KRBH buffer, to which 0.17 µCi D-[U-14C]glucose had been added as indicated for 30 min. At the end of the incubation, islet metabolism was terminated by adding 200 µl of 0.1 M HCl to the incubation mixture. 14CO2 was collected by 300 µl of 1 M NaOH for 2–4 h at 37°C and measured by liquid scintillation spectrometry.

Statistical methods. All data are shown as means ± SE. Statistical significance was evaluated by two-way ANOVA or t-tests as appropriate. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of Src kinase in pancreatic beta-cells. There are limited reports on the existence and function of Src family tyrosine kinases in pancreatic beta-cells. Ohnishi et al. (34) showed that the expression of Src gradually increased with postnatal development in rat pancreatic islets by immunohistochemical analysis. In insulin-producing RINm5F cells, c-Src was shown to be involved in the anti-apoptotic action of nitric oxide (45). Activation of cellular (c)-Src was also implicated in glucagon-like peptide 1-induced pancreatic beta-cell proliferation in INS (832/13) cells (8). However, Src was reported as "not expressed" in rGIP-15, betaTC-3, and INS (832/13) cells by the Kinetworks KPKS 1.0 Western blotting analysis (12). Therefore, to clarify the confusion, it was necessary to examine the expression of Src kinase in our system before testing its function.

As demonstrated in Fig. 1, the expression of Src kinase in INS-1 cells and rat pancreatic islets was confirmed by immunoprecipitation and immunoblotting analysis. In Fig. 1A, lysates of INS-1 cells, rat islets, and NIH 3T3 cells (which served as a positive control for the expression of Src kinase) were subjected to immunoprecipitation with anti-Src mAb327. Next, the immunoprecipitated protein was subjected to immunoblotting with anti-Src mAb327. In Fig. 1B, cell lysates were directly subjected to immunoblotting with anti-Src mAb327. A band corresponding to Src was present in the lysates of rat pancreatic islets, INS-1 cells, and NIH 3T3 cells but not in the absence of lysate or anti-Src mAb327. Taken together, both immunoprecipitation and the Src kinase activity assay confirmed the expression of Src kinase in insulin-secreting beta-cells.


Figure 1
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  		Fig. 1. Expression of Src kinase in INS-1 cells and rat pancreatic islets. A: 300 µg of lysates of INS-1 cells, rat islets, or NIH 3T3 cells were subjected to immunoprecipitation with anti-Src monoclonal antibody (mAb) 327. Next, the immunoprecipitated protein was subjected to immunoblotting with anti-Src mAb327. The anti-Src mAb327 bound protein G-agarose beads or mixture of beads, and cell lysates were used as controls (lanes 1 and 2, respectively). The bands of Src are indicated. The two bands below Src in lanes 1, 3, 4, and 5 are the heavy chain (seen immediately below the Src band) and the light chain of IgG. B: 25 µg lysates of cells in A were directly subjected to immunoblotting with anti-Src mAb327. The detailed procedures are described in MATERIALS AND METHODS.

 
Effects of Src family tyrosine kinase inhibitors on insulin secretion in INS-1 cells and Src kinase activity. The effects of PP2, a selective Src family tyrosine kinase inhibitor (19), were examined in INS-1 cells. PP3, structurally related to PP2, does not inhibit Src family kinases and was used to control for potential nonspecific effects of PP2 (26). Autophosphorylation of Tyr418 is critical for the full activation of Src family tyrosine kinases (50). The presence of activated c-Src in unstimulated INS-1 cells was detected by immunoblotting with a specific antibody for phospho-Tyr418-containing-Src (pY418). KCl (40 mM) was used to depolarize the beta-cell, stimulate Ca2+ influx, and consequently induce Ca2+-dependent insulin secretion. As shown in Fig. 2, in the presence of KCl, 20 µM PP2 significantly decreased the phosphorylation of Tyr418 (control, 1.4 ± 0.2 vs. PP2, 0.6 ± 0.1 of fold stimulation; {Delta} = –0.8 ± 0.3; P < 0.05), whereas its negative control, PP3, did not change the phosphorylation level [control, 1.4 ± 0.2 vs. PP3, 1.5 ± 0.2 of fold stimulation; {Delta} = 0.1 ± 0.2; not significant (NS)]. Accordingly, PP2 increased KCl-induced insulin secretion in a concentration-dependent manner (data not shown), and the maximal effects were obtained at 20 µM (control, 179 ± 28 vs. 20 µM PP2, 326 ± 60 pg·mg protein–1·min–1; {Delta} = 146 ± 44; P < 0.05), whereas 20 µM PP3 had no effects (control, 179 ± 28 vs. 20 µM PP3, 201 ± 13.35 pg·mg protein–1·min–1; {Delta} = 21 ± 27; NS). The effects of 20 µM PP2 on secretion seem to be dependent on an increase in intracellular Ca2+, because it only enhanced Ca2+-dependent insulin secretion evoked by KCl and not basal insulin release (control, 36 ± 5 vs. PP2, 43 ± 7 pg·mg protein–1·min–1; {Delta} = 7 ± 3; P = 0.07). The results indicate that decreased activation of Src family tyrosine kinases resulted in an enhancement of stimulated insulin secretion from INS-1 cells.


Figure 2
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  		Fig. 2. Effects of PP2 and PP3 on Src activity and insulin secretion in INS-1 cells. The activated c-Src and Src activity in INS-1 cells were detected in cell lysate by immunoblotting with a specific antibody for phospho-Tyr418-containing Src (pY418). Immunoblots with polyclonal anti-c-Src (N-16) are also shown. Insulin secretion was evoked by 40 mM KCl. The detailed procedures are described in MATERIALS AND METHODS. Values are shown as means ± SE (n = 4). *P < 0.05.

 
The effects of PP2 were also investigated with an Src kinase activity assay as mentioned above. PP2 (20 µM) decreased Src activities in the presence of 2.8 mM glucose, 40 mM KCl, and also 20 mM glucose (2.8 mM glucose, 2.3 ± 0.32 vs. 2.8 mM glucose + PP2, 1.0 ± 0.20; P < 0.005; KCl, 2.1 ± 0.29 vs. KCl + PP2, 0.71 ± 0.20; P < 0.005; 20 mM glucose, 1.9 ± 0.25 vs. 20 mM glucose + PP2, 0.9 ± 0.15 pmol 32PO4·min–1·mg lysate–1; P < 0.05.), whereas the inactive analog, 20 µM PP3, had no significant effects on Src kinase activity under the same conditions (data not shown). Moreover, Src kinase activity was reduced by high glucose, a stimulator of insulin release (2.8 mM glucose, 2.3 ± 0.32 vs. 20 mM glucose, 1.9 ± 0.25 pmol 32PO4·min–1·mg lysate–1; {Delta} = –0.4 ± 0.11; P < 0.05).

SU-6656, structurally different from PP2, is another selective Src family kinase inhibitor (5). Figure 3A shows that SU-6656 reduced the level of active c-Src in INS-1 cells as detected with a specific antibody. As shown in Fig. 3B, SU-6656 did not change basal secretion (control, 54 ± 5 vs. SU-6656, 58 ± 4 pg·mg protein–1·min–1; {Delta} = 4 ± 5; NS) but increased KCl-induced insulin secretion. The maximal effect was obtained at 10 µM (control, 133 ± 12 vs. SU-6656, 317 ± 60 pg·mg protein–1·min–1; {Delta} = 185 ± 57; P < 0.05). The data presented so far with PP2 and SU-6656, two structurally different inhibitors of Src family kinases, suggest that inhibition of Src family kinases potentiates KCl-induced Ca2+-dependent insulin secretion.


Figure 3
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  		Fig. 3. Effects of SU-6656 on Src activity and insulin secretion in INS-1 cells. The activated c-Src and Src activity in INS-1 cells were detected in cell lysates by immunoblotting with a specific antibody for phospho-Tyr418-containing Src (pY418). Insulin secretion was evoked by 40 mM KCl. The detailed procedures are described in MATERIALS AND METHODS. Values are shown as means ± SE (n = 4). *P < 0.05.

 
Effects of inhibition of extracellular signal-regulated kinase 1/2 on insulin secretion in INS-1 cells. The extracellular signal-regulated kinases (ERK1/2, also called mitogen-activated protein kinase) can be stimulated by glucose, KCl, and other secretagogues in pancreatic beta-cells (16). ERK1/2 plays an important role in cell proliferation and differentiation (30). Their effects on insulin secretion are less well defined. Because Src has been reported to be an upstream activator of ERK1/2 (28), the next series of experiments was performed to determine whether the effects of Src family kinases on insulin secretion were through the Src-ERK pathway. We tested the effects of PD-98059, a specific inhibitor of ERK on insulin secretion (11). PD-98059 binds to the inactive form of ERK and thus prevents its activation by upstream activators, such as Src. We found that 100 µM PD-98059 markedly decreased the level of KCl-activated ERK as expected. However, PD-98059 (3–100 µM) had no effect on KCl-induced insulin secretion [KCl control, 91 ± 13 vs. 3 µM PD-98059, 106 ± 14 (NS), vs. 10 µM PD-98059, 112 ± 15 (NS), vs. 30 µM PD-98059, 113 ± 10 (NS), vs. 100 µM PD-98059, 113 ± 16 pg·mg protein–1·min–1 (NS)] and did not significantly change the insulin content of INS-1 cells (data not shown).

Effects of Src family tyrosine kinase inhibitors on insulin secretion in rat pancreatic islets. The INS-1 cell is a rat-derived insulin-secreting cell line. Although it somewhat reflects the situation in normal rat pancreatic islets, some aspects of protein expression and exocytotic characteristics differ between this cell line and islets. Thus the effects of PP2 were further tested in islets. As indicated in Fig. 4, PP2 potentiated KCl-induced insulin secretion in a time-dependent and concentration-dependent manner. The maximal effect was obtained at 20 µM with a 4-h pretreatment with PP2. Under these conditions, PP2 caused a 2.1-fold increase in KCl-induced secretion (control, 0.7 ± 0.1 vs. PP2, 2.2 ± 0.5% of content 15 min–1; {Delta} = 1.5 ± 0.6; P < 0.05) and did not significantly change the insulin content of the islets (control, 178 ± 19 vs. PP2, 175 ± 21 ng/islet; {Delta} = –3 ± 1; NS).


Figure 4
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  		Fig. 4. Time course and concentration dependence of the effects of PP2 on KCl-induced insulin secretion in islets. Insulin secretion was evoked by 40 mM KCl. The detailed procedures are described in MATERIALS AND METHODS. A: 20 µM PP2. B: 4 h pretreatment with PP2. IR, insulin release; FR, fractional insulin release. Values are shown as means ± SE (n = 4–6). *P < 0.05.

 
The next experiments were carried out to test the effects of PP2 on glucose-stimulated insulin secretion from islets. As presented in Fig. 5, PP2 augmented insulin secretion evoked by either 8.3 mM glucose (control, 2.1 ± 0.25 vs. PP2, 7.0 ± 0.99% of content 30 min–1; {Delta} = 4.8 ± 1.01; P < 0.01) or 20 mM glucose (control, 5.3 ± 0.54 vs. PP2, 7.7 ± 0.98% of content 30 min–1; {Delta} = 2.4 ± 0.59; P < 0.05). As with secretion in INS-1 cells, PP2 did not affect basal secretion in islets. The effects of PP2 were further examined on the KATP channel-independent pathway of beta-cell signaling by incubating islets with KCl and diazoxide (27). In the presence of KCl and diazoxide, PP2 increased 2.8 and 8.3 mM but not 20 mM glucose-stimulated insulin secretion. The result indicates that the augmenting effects of PP2 on insulin release are distal to the KATP channel since PP2 could still increase insulin secretion when the KATP channels were "clamped" open by diazoxide.


Figure 5
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  		Fig. 5. Effects of 20 µM PP2 on glucose-induced insulin secretion in islets. Insulin secretion was stimulated in the presence and absence of 40 mM KCl and 250 µM diazoxide. The detailed procedures are described in MATERIALS AND METHODS. Values are shown as means ± SE (n = 5). *P < 0.05 and **P < 0.01.

 
Another selective Src family tyrosine kinase inhibitor, SU-6656, also augmented KCl-induced insulin secretion from islets in a concentration-dependent manner (Fig. 6). The EC50 for SU-6656 was 2 µM. The maximal effect was seen with 10 µM at which concentration it potentiated KCl-induced secretion 1.4-fold (control, 0.5 ± 0.03 vs. SU-6656, 1.2 ± 0.10% of content 15 min–1; {Delta} = 0.7 ± 0.10; P < 0.005) and did not change the insulin contents of islets (control, 155 ± 9 vs. SU-6656, 175 ± 19 ng/islet; {Delta} = 20 ± 16; NS). In separate experiments, basal secretion in the presence of 2.8 mM glucose was not significantly affected by 10 µM SU-6656 (control, 0.6 ± 0.10 vs. SU-6656, 0.7 ± 0.31% of content 15 min–1; {Delta} = 0.1 ± 0.26; NS).


Figure 6
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  		Fig. 6. Concentration dependence of the effect of SU-6656 on KCl-induced insulin secretion in islets. Insulin secretion was evoked by 40 mM KCl. The detailed procedures are described in MATERIALS AND METHODS. Values are shown as means ± SE (n = 4–6). *P < 0.05 and **P < 0.01.

 
Additional experiments were performed to test the effects of PP3 on islets under the same conditions as PP2. PP3 is structurally similar to PP2 but does not inhibit Src family tyrosine kinase activity (see Fig. 2). As expected, PP3 did not affect basal insulin secretion (control, 1.1 ± 0.12 vs. PP3, 1.1 ± 0.08% of content 30 min–1; {Delta} = 0.0 ± 0.16; NS), 40 mM KCl-induced insulin secretion (control, 2.2 ± 0.71 vs. PP3, 1.9 ± 0.38% of content 30 min–1; {Delta} = –0.3 ± 0.38; NS), or 8.3 mM glucose-induced insulin secretion (control, 3.9 ± 1.27 vs. PP3, 4.3 ± 0.99% of content 30 min–1; {Delta} = 0.4 ± 0.30; NS) from islets.

Ca2+ dependency of PP2 effects on insulin secretion. Our next experiments addressed the role of Ca2+ influx on the effects of PP2. As shown in Fig. 7, NTD, a Ca2+ channel blocker, completely blocked the amplifying effects of PP2 on 8.3 mM glucose-stimulated insulin secretion (8.3 mM glucose + PP2, 5.9 ± 0.45 vs. NTD + 8.3 mM glucose + PP2, 0.8 ± 0.26% of content 30 min–1; {Delta} = –5.0 ± 0.54; P < 0.01).


Figure 7
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  		Fig. 7. Effects of PP2 on insulin secretion in the presence of nitrendipine (NTD), TPA, Gö-6976 (Gö), and forskolin (Fsk) in islets. NTD, TPA, and Fsk were only present in 30-min incubation period. Gö-6976 was present in both 60-min preincubation and 30-min incubation period. The detailed procedures are described in MATERIALS AND METHODS. Values are shown as means ± SE (n = 4).

 
Protein kinase C or protein kinase A activation and subsequent phosphorylation of targets in beta-cells can potentiate insulin release. PP2 was reported to mildly inhibit protein kinase A (PKA) and protein kinase C (PKC) activity in an in vitro assay (3). Thus further experiments were performed to test whether PP2 also influenced insulin secretion in the presence of activated PKC or PKA. As shown in Fig. 7, PP2 changed neither TPA- nor forskolin-induced insulin secretion in the presence of 2.8 mM glucose. Moreover, the classical PKC (cPKC) inhibitor Gö-6976 (1 µM) did not significantly affect the augmenting effects of PP2 on glucose-stimulated insulin release (control, 5.9 ± 0.45 vs. Gö-6976, 5.1 ± 0.59% of content 30 min–1; {Delta} = –0.8 ± 0.84; P = 0.41). This further suggests that cPKC is not involved in PP2 effects. Taken together, the data indicate that the amplifying effect of PP2 on insulin secretion is exerted solely on Ca2+-stimulated secretion.

Effects of PP2 on glucose metabolism. In further studies on the mechanism underlying the effects of PP2 on insulin secretion, glucose oxidation was measured in rat pancreatic islets to investigate whether PP2 can drive glucose metabolism. As expected, 8.3 mM glucose significantly increased glucose oxidation compared with basal 2.8 mM glucose (2.8 mM glucose, 3.7 ± 0.19 vs. 8.3 mM glucose, 5.5 ± 0.17 pmol·islet–1·30 min–1; {Delta} = 1.2 ± 0.04; P < 0.05). However, 20 µM PP2 did not augment glucose oxidation. Rather, it slightly reduced it at both 2.8 mM (2.8 mM glucose, 3.7 ± 0.19 vs. PP2, 3.1 ± 0.04 pmol·islet–1·30 min–1; {Delta} = –0.7 ± 0.20; P < 0.05) and 8.3 mM glucose (8.3 mM glucose, 5.5 ± 0.17 vs. PP2, 4.4 ± 0.22 pmol·islet–1·30 min–1; {Delta} = –1.1 ± 0.08; P < 0.05) conditions.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Having established the expression of Src kinase in pancreatic islets and INS-1 cells, we sought to examine the potential roles of Src family kinases on insulin secretion. The presence of activated Src kinase in unstimulated beta-cells was first detected using an antibody recognizing the positive regulatory autophophorylated site (pY418). It was then found that, in INS-1 cells, the Src family kinase inhibitors PP2 and SU-6656, but not the inactive control compound PP3, reduced Src kinase activity and enhanced Ca2+-dependent but not basal insulin secretion. Likewise, and more importantly, in rat pancreatic islets, both PP2 and SU-6656, but not PP3, potentiated insulin secretion stimulated by glucose or KCl in a concentration- and time-dependent manner. This augmenting effect of the Src family kinase inhibitors was Ca2+ dependent and abolished by L-type Ca2+ channel blockade. Moreover, PP2 had no effects on insulin release evoked by PKA or PKC activation. The results indicate that Src family tyrosine kinases play an inhibitory role on Ca2+-dependent insulin secretion.

The major drawback of using inhibitors is their potential nonspecificity. To circumvent this problem, two structurally different inhibitors of Src family kinases, PP2 and SU-6656 (3), were tested, and similar effects on insulin secretion were demonstrated. Also, PP3, which is inactive but structurally similar to PP2, was used as a control for potential nonspecific effects throughout the present study.

We then tried to pinpoint the underlying mechanisms for Src family kinase inhibitors to potentiate insulin secretion. 1) The first was glucose metabolism. The effect of PP2 on glucose metabolism is unlikely to be the mechanism for PP2-potentiated insulin secretion since PP2 did not increase glucose oxidation to boost the production of metabolic coupling factors in stimulus-secretion pathways. 2) Second was the KATP channel. PP2 slightly reduced glucose oxidation, which could lead to a decrease in the ATP-to-ADP ratio and possible activation of the KATP channel. If anything, this would tend to inhibit the action of glucose. Furthermore, PP2 was still able to potentiate 2.8 and 8.3 mM glucose-induced insulin secretion under KATP channel-independent conditions by treating islets with KCl and diazoxide. It appears therefore that the augmenting effects of PP2 on insulin secretion are unlikely to be due to any interaction with the KATP channel. 3) Third was adenylyl cyclase. Neither the literature nor our data with forskolin indicate that Src kinases can regulate adenylyl cyclase activity. 4) Fourth was PKC. Neither the activation of cPKC and novel PKCs with TPA nor the inhibition of cPKCs with Gö-6976 changed the PP2 effects on insulin secretion, indicating that these PKC activities are not necessary for the PP2 action under these conditions. 5) Last was insulin synthesis. Src family kinases are thought to be involved in gene transcription (43). Nevertheless, their effects on insulin secretion seem not to be the result of changes in insulin synthesis because the insulin content was unchanged after either PP2 or SU-6656 treatment under our experimental conditions.

It appears that the main action of Src family kinase inhibitors on insulin secretion is distal to the elevation of intracellular Ca2+ concentration ([Ca2+]i). 1) The observation that 20 µM PP2 did not induce insulin secretion under basal conditions (2.8 mM glucose) from either rat pancreatic islets or INS-1 cells indicates that 20 µM PP2 could not evoke any significant Ca2+ entry to stimulate insulin exocytosis. 2) In a similar exocytotic system, rat brain synaptosomes, PP1 (another selective Src family kinase inhibitor, structurally and functionally similar to PP2) had no effect on the intracellular Ca2+ levels under either basal or KCl conditions but increased KCl-evoked Ca2+-dependent release of glutamate, again suggesting that the effects of PP1 on exocytosis are downstream of Ca2+ influx (4). Taken together, the potentiating effects of Src family kinase inhibitors on insulin secretion are likely because of their action at a site distal to the elevation of [Ca2+]i and are independent of glucose metabolism, KATP channel, adenylyl cyclase, cPKC and novel PKC isoforms, and insulin synthesis. Moreover, the augmenting effects are Ca2+ dependent.

We next attempted to decode the signaling pathway for Src family kinase-mediated inhibition of insulin secretion. Src family kinases interact with a variety of protein targets such as ERK1/2, phosphatidylinositol 3-kinase (PI 3-kinase), dynamin, synapsin, Shc, focal adhesion kinase, and paxillin. Among them, ERK1/2 can be activated by glucose, glucagon-like peptide-1, KCl, insulin, and phorbol esters and inhibited by epinephrine (2, 6, 18, 23). It was shown that high glucose acutely increased the rate of insulin gene transcription, but chronically (from 24 h) inhibited it; both effects were found to be ERK1/2 dependent (25). In our experiments, consistent with the study by Khoo and Cobb (22), we did not find any effects of ERK1/2 inhibition on insulin secretion in INS-1 cells. In contrast, under exactly the same condition, the Src family kinase inhibitor (SU-6656) significantly increased insulin exocytosis. These data exclude the Src-ERK pathway in the mechanisms responsible for the augmenting effects of Src family kinase inhibitors on insulin release.

PI 3-kinase, another downstream effector of c-Src, is a possible candidate since several studies have shown that inhibitors of PI 3-kinase amplify insulin secretion from INS 832/13 cells and mouse and rat pancreatic islets (10, 14, 29, 32, 53). Our own preliminary study also demonstrated that LY-294002, an inhibitor of PI 3-kinase, augmented glucose-stimulated insulin secretion in a concentration-dependent manner (data not shown). Other potential players include dynamin and synapsin, which interact with Src through SH3 domains (13, 15). Dynamin, a GTPase (36), has well-established roles in endocytosis and actin cytoskeleton remodeling (31). Even more relevant to the present study, Tsuboi et al. (46) reported that dynamin is critical for Ca2+-dependent "kiss-and-run" (cavicapture) exocytosis in insulin-secreting cells (46), whereas Baldwin et al. (4) suggested that Src family tyrosine kinase negatively regulates the "kiss-and-run" mode of exocytosis in rat brain synaptosomes. Therefore, it is quite likely that Src family tyrosine kinase negatively regulates insulin exocytosis in pancreatic beta-cells by interacting with dynamin, destabilizing its associations with adapter proteins and affecting cytoskeleton dynamics (7). Such a Src-dynamin interaction would explain the independence from upstream signaling pathways of insulin stimulus-secretion coupling, which we observed for Src family kinase-mediated inhibition of insulin secretion. Despite these data with dynamin, there is also evidence suggesting a potential role of Ca2+/calmodulin-dependent phosphorylation of synapsin I in insulin secretion (52). Thus the interaction that occurs between Src and synapsin provides another potential mechanism for how Src could influence Ca2+-dependent insulin secretion.

In conclusion, using selective inhibitors of the Src family kinase, we demonstrated that reduced Src family kinase activity led to increased Ca2+-dependent insulin secretion. It suggests that, under normal conditions, the Src family kinases are involved in a tonic inhibition of insulin secretion. This pathway is likely acting at a point distal to the elevation of [Ca2+]i and independent of ERK1/2, PKC, and PKA. Future work is needed to determine which member or members of the Src family kinases are responsible for the inhibitory action on insulin exocytosis and their mode of action. The augmenting effects of Src family kinase inhibitors on insulin secretion may shed some light on the design of new therapeutic agents for type 2 diabetes mellitus.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-42063 (to G. W. G. Sharp); by a Career Development Award from the Juvenile Diabetes Association (to S. G. Straub), and by a predoctoral Fellowship from the Pharmaceutical and Research Manufacturers Association (to H. Cheng).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Xiaoyang Wu and Dr. Jun-Lin Guan for providing reagents and helpful suggestions, to Dr. Mossaad Abdel-Ghany for excellent advice during the course of this study, to Dr. David I. Shalloway for Anti-Src mAb327, and to Dr. Troitza Bratanova-Tochkova for expert technical assistance.

Current address for H. Cheng: Dept. of Medicine, Univ. of Massachusetts Medical School, Worcester, MA 01655.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. W. G. Sharp, Dept. of Molecular Medicine, College of Veterinary Medicine, Cornell Univ., Ithaca, NY 14853-6401 (e-mail: gws2{at}cornell.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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