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Endocrinology Vol. 141, No. 7 2350-2360
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Insulin Secretion by Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II in Insulinoma MIN6 Cells

Hirotaka Tabuchi, Hideyuki Yamamoto, Kazuya Matsumoto, Kenji Ebihara, Yusuke Takeuchi, Kohji Fukunaga, Hideji Hiraoka, Yasuharu Sasaki, Motoaki Shichiri and Eishichi Miyamoto

Departments of Pharmacology (H.T., H.Y., Y.T., K.F., E.M.) and Metabolic Medicine (H.T., K.M., K.E., M.S.), Kumamoto University School of Medicine, Kumamoto 860-0811; and Frontier 21 Project, Institute for Life Science Research, Asahi Chemical Industry (H.H., Y.S.), Samejima 2–1, Fuji, Shizuoka 416-8501, Japan

Address all correspondence and requests for reprints to: Eishichi Miyamoto, M. D., Department of Pharmacology, Kumamoto University School of Medicine, 2–2-1 Honjo, Kumamoto 860-0811, Japan. E-mail:emiyamot{at}gpo.kumamoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in Ca2+-induced insulin secretion. We have previously reported that treatment of insulinoma MIN6 cells with secretagogues activated CaM kinase II and increased the phosphorylation of synapsin I, followed by insulin secretion. Here, we identified isoforms of CaM kinase II in MIN6 cells and rat islets. Immunoblot analysis suggested that the major isoforms of CaM kinase II were ß'e and {delta}2 at the protein level in MIN6 cells. Only the ß'e isoform was detected in rat islets by both RT-PCR and immunoblot analysis. We transiently overexpressed ß'e and {delta}2 isoforms in MIN6 cells and confirmed that treatment of cells with tolbutamide and glucose activated the isoforms. Immunoblot analysis with an antibody against synapsin I phosphorylated by CaM kinase II demonstrated that treatment with tolbutamide and glucose rapidly increased phosphorylation of synapsin I and that phosphorylation was potentiated by overexpression of the isoforms. The secretagogue-induced insulin secretion was potentiated by overexpression of the isoforms. Our results further support our conclusion that activation of CaM kinase II and the concomitant phosphorylation of synapsin I contribute to insulin secretion from pancreatic ß-cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HIGH INTRACELLULAR Ca2+ concentrations ([Ca2+]i) play a critical role in secretagogue-induced insulin secretion (1). Stimulation of pancreatic ß-cells with secretagogues such as glucose and tolbutamide results in a rise in [Ca2+]i due to either Ca2+ influx from the extracellular space (2) or Ca2+ release from intracellular storage sites (3). Many effects of Ca2+ are mediated through Ca2+-binding proteins such as calmodulin (CaM). The effects of Ca2+/CaM may be mediated by Ca2+/CaM-dependent protein kinase II (CaM kinase II) (4, 5, 6). We previously reported that stimulation of MIN6 cells, one of the insulinoma cell lines, with secretagogues such as glucose, tolbutamide, and high K+ enhanced activation of CaM kinase II and phosphorylation of synapsin I with a concomitant rise in insulin secretion (7). Similar findings were reported previously using isolated rat pancreatic islets (8). Furthermore, the stimulatory effects of secretagogues were enhanced by inhibition of protein phosphatase 2B by cyclosporin A, confirming that activation of CaM kinase II and phosphorylation of synapsin I are required for insulin secretion (9).

Certain isoforms of CaM kinase II have been identified in rat islets (10, 11, 12) and insulinoma cell lines (7, 13). Previous studies have shown that W-7, a CaM inhibitor, inhibits the movement of insulin secretory granules and insulin release from ß-cells (14). Furthermore, CaM kinase II inhibitors such as KN-62 and KN-93 inhibit insulin release from insulinoma cells and rat pancreatic islets (11, 15). CaM kinase II contains four subunits, {alpha}, ß, {gamma}, and {delta}, encoded by distinct genes in eukaryotes (16, 17). Various isoforms of these subunits exist as different splicing variants. The localization of the isoforms of CaM kinase II differs from one cell to another (6, 16). The {delta}2 isoform has been detected at both messenger RNA (mRNA) and protein levels in rat insulinoma cell lines, RINm5F cells, whereas mRNA of the {delta}6 isoform has been detected in RINm5F cells (13).

CaM kinase II phosphorylates proteins that are thought to be involved in the traffic, docking, and fusion of insulin secretory granules. These include synapsin I (7, 18), microtubule-associated protein-2 (MAP-2) (19), N-ethylmaleimide-sensitive fusion protein, soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP), vesicle-associated membrane protein (VAMP/synaptobrevin) (20), {alpha}-SNAP (21), SNAP-25, and synaptotagmin (22). Among the substrates for CaM kinase II, synapsin I is one of the most attractive candidates for the regulation of insulin secretion. In the brain, CaM kinase II plays a critical role in regulation of the interaction between synaptic vesicles and the cytoskeleton via phosphorylation of synapsin I (23). We have identified synapsin I in rat islets, insulinoma cells, and several hormone-secreting cells (24). Cloning of synapsin I in MIN6 cells revealed that the major isoform in the cells was synapsin Ib (24). Interestingly, some isoforms of the {delta}-subunit of CaM kinase II were associated with synapsin Ib in insulin secretory granules (13, 24).

The present study is an extension to the above studies. We identified here the isoforms of CaM kinase II in MIN6 cells and rat islets and investigated the effects of overexpression of these isoforms on phosphorylation of synapsin I and insulin secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and chemicals
The following reagents and chemicals were obtained from the indicated sources: [{gamma}-32P]ATP and [32P]orthophosphate, NEN Life Science Products (Boston, MA); phosphocellulose paper, Whatman (Clifton, NJ); syntide-2, Bachem (Torrance, CA); DMEM, Nissui Pharmaceutical Co. (Tokyo, Japan); FBS, Hazleton Biologics (Lenexa, KS); tolbutamide, Sigma (St. Louis, MO); activated murine glutathione-S-transferase-p42 mitogen-activated protein (MAP) kinase, Upstate Biotechnology, Inc. (Lake Placid, NY); Phadeseph insulin, Kabi Pharmacia Diagnostics (Uppsala, Sweden), monoclonal antibodies (mAbs) to the {alpha}-subunit (CB{alpha}-2) and the ß-subunit (CBß-1) of CaM kinase II, Life Technology (Tokyo, Japan); and anti-CaM kinase IIß{gamma} antibody [CaMKII{gamma} (C-18)], Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). CaM (25) and synapsin I (26) were purified from bovine brain and rat brain, respectively. The IgG fraction of antibody to CaM kinase II (anti-brain CaM kinase II antibody) was prepared, and the concentration was adjusted to 1 mg/ml, as described previously (27). The antibody to the {delta}1-{delta}4 isoforms (anti-CaM kinase II{delta}1-{delta}4 antibody) was prepared by immunizing rabbits with a synthesized peptide corresponding to a 15-amino acid segment from the unique carboxyl-terminal ends of {delta}1-{delta}4 isoforms (28). The IgG fraction was prepared from the antiserum by ammonium sulfate fractionation (0–50%). The polyclonal antibodies to the synapsin I phosphorylated at the sites of serine 566 (pS566-Syn I-Ab) and serine 603 (pS603-Syn I-Ab) were prepared by immunizing rabbits with P-Ser566 peptide [ATRQAS(P)ISGPAPC] and P-Ser603 peptide [GPIRQAS(P)QAGPGP], respectively, coupled to hemocyanin from keyhole limpet. Serum titers of the antibodies were determined by enzyme-linked immunosorbent assay using P-Ser566 peptide and P-Ser603 peptide (10 µg/ml). The antibodies showed high titers (up to 1:10,000) against each phosphopeptide. Other chemicals used were of analytical grade.

Assay for CaM kinase II
Frozen MIN6 cells were scraped from the dishes and solubilized at 0 C in 0.25 ml of a homogenization buffer containing 30 mM HEPES (pH 7.5), 0.1% (vol/vol) Triton X-100, 4 mM EGTA, 10 mM EDTA, 100 mM ß-glycerophosphate, 0.1 mM leupeptin, 75 µM pepstatin A, 0.1 mg aprotinin/ml, and 1 mM dithiothreitol in the presence or absence of 15 mM Na4P2O7 and 25 mM NaF (7). After sonication with a Branson Sonifier 250 (Danbury, CT), the insoluble material was removed by centrifugation at 15,000 x g for 5 min. The standard kinase assay contained 50 mM HEPES (pH 7.5), 10 mM magnesium acetate, 0.1 mM [{gamma}-32P]ATP (3,000–5,000 cpm/pmol), and 1 mg BSA/ml in a final volume of 25 µl. Syntide-2 (40 µM) was used as substrate. Total CaM kinase II activity was determined in the presence of 1 mM CaCl2 and 3 µM CaM, whereas 1 mM EGTA was added to determine Ca2+/CaM-independent activity. Reactions were initiated by adding 2.0 µl of the cell extract. After incubation at 30 C for 4 min, 15 µl of each sample were spotted on a phosphocellulose paper square and processed as described previously (29).

Northern blot analysis
Total RNA was prepared from MIN6 cells and rat brains using Trizol LS reagent as recommended by the supplier (Life Technologies, Inc., Gaithersburg, MD). Total RNA was denatured with formaldehyde, electrophoresed on a 1% agarose gel, and transferred to a nylon membrane. Specific complementary DNA (cDNA) probes for the {alpha}-, ß-, {gamma}-, and {delta}-subunits of CaM kinase II were isolated as previously described (30). These probes were labeled to a specific activity of 108 cpm/µg by the random primer method. After hybridization overnight, the membranes were washed once in each of the following solutions before autoradiography: 1) 2 x SSC (standard saline citrate)-0.1% SDS for 10 min; 2) 1 x SSC-0.1% SDS for 10 min; and 3) 1 x SSC-0.1% SDS for 1 h at 65 C.

RT-PCR and sequencing
The experimental protocol was approved by the ethics review committee for animal experimentation of Kumamoto University School of Medicine. Islets were isolated by collagenase digestion from the pancreas of Wistar rats, as described previously (24). Total RNA was reverse transcribed using an oligo(deoxythymidine) primer (Promega Corp., Madison, WI) and Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.). PCR primers were designed based on the published sequence of the {alpha}, ß, {gamma}A, and {delta}1 isoforms of rat brains (31, 32, 33, 34). Primers used to amplify the whole coding region of each subunit are shown in Table 1Go. The PCR product was purified by low melting agarose gel electrophoresis after primary RT-PCR and used as a template in the nested PCR to amplify a fragment containing variable domains using the primers shown in Table 1B. PCR amplification was carried out using Gene Amp PCR system 2400 (Perkin-Elmer Corp., Norwalk, CT). After amplification, the final 10-min extension step was carried out at 72 C. Each PCR fragment from the nested PCR was purified by low melting agarose gel electrophoresis and subcloned into the pCR 2.1 cloning vector following the procedures provided in the TA Cloning kit (Invitrogen, San Diego, CA). DNA sequencing of double stranded plasmid DNAs was performed using an ABI PRISM 377 DNA sequencer (PE Applied Biosystems Japan, Chiba, Japan), according to the protocol recommended by the manufacturer. Both strands were sequenced.


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  		Table 1. PCR protocols

 
Mutagenesis of ß'e and {delta}2 isoforms and sequencing
The inactive mutants of the ß'e isoform [ß'e (K43A) mutant] and the {delta}2 isoform [{delta}2(K43A) mutant] were generated by mutation of lysine 43 to alanine (35). Oligonucleotide-directed mutagenesis was performed using the GeneEditor In Vitro Site-Directed Mutagenesis System (Promega Corp., Madison, WI). Mutation was confirmed by sequencing the mutated coding strand.

Overexpression of CaM kinase II in MIN6 cells
The pCAGGSneo expression vector was provided by Prof. J. Miyazaki (Osaka University, Osaka, Japan). Each PCR product of the ß'e and {delta}2 isoforms was purified by low melting agarose gel electrophoresis and subcloned into the pCR 2.1 cloning vector. Each cDNA of the ß'e and {delta}2 isoforms, the {delta}2(K43A) mutant, and the ß'e (K43A) mutant was excised with EcoRI, and each EcoRI fragment was inserted into the EcoRI site of the pCAGGSneo expression vector under the control of the chicken ß-actin promoter (28).

Culture and transfection of MIN6 cells
MIN6 cells, a mouse insulinoma cell line (36) obtained from Dr. J. Miyazaki, were cultured in DMEM supplemented with 25 mM glucose, 50 IU penicillin/ml, 50 µg streptomycin/ml, 5 µM 2-mercaptoethanol, and 15% heat-inactivated FBS, as previously described (7). Cells (5 x 105/dish) were plated on a 35-mm petri dish (Nunc, Roskilde, Denmark) for 24 h, then transfected with each CaM kinase II isoform cDNA in the pCAGGSneo expression vector (2 µg plasmid DNA), using 10 µl Lipofectamine (Life Technologies, Inc.) in 2 ml serum-free medium for 6 h. Subsequently, the cells were cultured with a fresh standard medium (DMEM containing 5% FBS) for 24 h on 35-mm dishes, then washed once with Krebs-Ringer-HEPES (KRH) containing 128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.2 mM MgSO4, 1 mM Na2HPO4, 20 mM HEPES (pH 7.4), and 3 mM glucose. After incubation for 30 min in KRH, the cells were further incubated in KRH at 37 C for specified time intervals without (control) or with tolbutamide or glucose. They were then quickly frozen in liquid N2 and used for the measurement of CaM kinase II activity and immunoblot analysis of synapsin I.

HIT-T15 cells were cultured in RPMI 1640 medium with 10% heat-treated FBS at 5% CO2 at 37 C (37). Two days before the experiment, 1–2 x 105 cells were plated on a 35-mm petri dish (Nunc, Naperville, IL).

Insulin secretion
Cells were preincubated at 37 C for 30 min in KRH with 3 mM glucose. After preincubation, the medium was removed, and cells were incubated at 37 C for the indicated time without (control) or with tolbutamide or glucose in 1 ml KRH. After incubation, the medium was collected and centrifuged at 12,000 x g for 2 min, then the supernatants were used for insulin assay. The concentration of insulin in the supernatant was determined by a double antibody RIA (38) using an insulin assay kit (Phadeseph Insulin, Kabi Pharmacia Diagnostics). The unit of insulin was based on the instructions in the assay kit used for the RIA. The cells in the dish were solubilized in a homogenization buffer containing 50 mM Tris-HCl (pH 7.5), 2 mM EGTA, 2 mM EDTA, 75 mM NaCl, and 0.05% Triton X-100. After sonication in a Branson Sonifier 250 (Danbury, CT), the solutions were centrifuged at 15,000 x g for 5 min, and 10 µl of each sample were used for determination of the protein concentration (39). The amount of insulin was corrected for protein concentration. The amount of insulin secreted per cell gradually decreased with continuous passages in HIT cells (9, 40). Because this was the case for MIN6 cells, the representative results of the experiments were shown.

Immunoprecipitation of 32P-CaM kinase II in MIN6 cells
MIN6 cells on a 35-mm dish transfected with pCAGGSneo plasmid alone or the ß'e or {delta}2 isoform were washed once with phosphate- and serum-free DMEM containing 5.6 mM glucose and labeled in 1.0 ml of the medium containing carrier-free [32P]orthophosphate (0.25 mCi/ml) for 5 h, as described previously (7). Cells were washed once in KRH and preincubated at 37 C for 30 min with 3 mM glucose in KRH. After incubation, cells were incubated at 37 C for 3 min with 0.37 mM tolbutamide in KRH, the medium was aspirated, and the cells were quickly frozen on liquid N2. The cells were solubilized in 400 µl 50 mM HEPES (pH 7.5), 0.1% Triton X-100, 4 mM EGTA, 10 mM EDTA, 15 mM ß-glycerophosphate, 25 mM NaF, 0.1 mM leupeptin, 75 µM pepstatin A, 50 µg/ml soybean trypsin inhibitor, 0.1% SDS, and 1 mM dithiothreitol. The insoluble material was removed by centrifugation at 15,000 x g for 15 min. The solution was incubated at 4 C for 4 h with the anti-brain CaM kinase II antibody (6 µg IgG protein) and 50 µl protein A-Sepharose CL-4B suspension (50%, vol/vol). After incubation, the immunocomplex immobilized on protein A-Sepharose CL-4B was precipitated by centrifugation at 12,000 x g for 2 min and washed three times with RIPA solution containing 50 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 0.5% Triton X-100, 10 mM EDTA, 1 mM Na3VO4, 30 mM Na4P2O7, 50 mM NaF, 4 mM EGTA, and 0.1% SDS. Immunoprecipitates were eluted from protein A-Sepharose CL-4B by adding SDS-sample buffer (41), boiling for 5 min, and centrifuging at 12,000 x g for 2 min, and the eluate was subjected to SDS-PAGE in 10% polyacrylamide, followed by autoradiography.

Other procedures
SDS-PAGE was performed according to the method of Laemmli (41). Immunoblot after SDS-PAGE was performed by the method of Towbin et al. (42) using the enhanced chemiluminescence detection kit (Amersham Pharmacia Biotech, Arlington Heights, IL) as directed by the instructions provided by the manufacturer. For reprobing, the membrane was submerged in stripping buffer [62.5 mM Tris-HCl (pH 6.7), 100 mM 2-mercaptoethanol, and 2% SDS] and incubated at 50 C for 30 min. The membrane was washed twice for 10 min in TTBS containing 100 mM Tris-HCl (pH 7.5), 0.9% NaCl, and 0.1% Tween-20 at room temperature and was subjected to immunoblot analysis. Immunostaining of MIN6 cells after transfection with the {delta}3 isoform was carried out as previously reported (28).

Statistical analysis
Data were expressed as the mean ± SEM. Differences between groups were examined for statistical significance using the one-way ANOVA plus Duncan’s multiple range test. P < 0.05 denoted the presence of a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of mRNAs of CaM kinase II isoforms in MIN6 cells and rat islets
To identify the subunits of CaM kinase II in MIN6 cells, total RNA isolated from MIN6 cells was subjected to Northern blot analysis with a probe specific for each subunit (Fig. 1AGo). RNA bands from MIN6 cells and rat brains were detected with {alpha}, ß, {gamma}, and {delta} probes. The mRNAs of ß- and {delta}-subunits were readily detected by Northern blot analysis, whereas those of {alpha}- and {gamma}-subunits were less evident. We amplified the full-length of {alpha}-, ß-, and {delta}-subunits of CaM kinase II by RT-PCR (Fig. 1BGo). With regard to the {gamma}-subunit, we could not obtain the PCR product using the primer that consisted of nucleotides 1602–1620 of the coding region of the {gamma}A isoform as downstream primer. On the other hand, when we used the primer of nucleotides 1250–1270, a 1270-bp product was obtained (Fig. 1BGo). Then, mRNAs of the isoforms were examined with total RNA from rat islets by RT-PCR. The band of the ß-subunit was clear, whereas the band of the {delta}-subunit was weak at a position higher than any reported isoforms (Fig. 1BGo). We could not detect PCR products of {alpha}- and {gamma}-subunits (Fig. 1BGo). The fragment of the {gamma}-subunit of 1270 bp was not obtained by PCR (data not shown).



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  		Figure 1. A, Northern blot analysis of CaM kinase II subunits in MIN6 cells and rat brains. Total RNAs from MIN6 cells (5 µg) and rat brains (2 µg) were subjected to Northern blot analysis. The specific radioactivities of the 32P-labeled cDNA probes for each subunit were adjusted to the same value. Size markers used were rat ribosomal RNAs of 18S and 28S. B, Total RNA prepared from MIN6 cells (15 ng) and rat islets (18 ng) were subjected to RT-PCR as described in Materials and Methods. PCR products were separated on a 0.8% agarose gel and visualized by ethidium bromide staining. {gamma}-Subunit ({gamma}1270) fragment, but not the whole coding region of the {gamma}-subunit, was detected by RT-PCR.

 
Each PCR product was inserted into the pCR 2.1 vector. Sequencing of the variable domains showed that the obtained cDNAs from MIN6 cells were {alpha} and {alpha}B (Fig. 2AGo), ß'e (Fig. 2BGo), {gamma}B and {gamma}C (Fig. 2CGo), and {delta}2 and {delta}6 (Fig. 2DGo) isoforms, whereas only the ß'e isoform (Fig. 2BGo) was obtained from rat islets. The sequencing of the {delta}-subunit fragment revealed that it was not related to the {delta}-subunit (data not shown). The deduced amino acids of each isoform were confirmed to be similar to those of previous reports (31, 32, 33, 34) (Fig. 2EGo).



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  		Figure 2. PCR products of each subunit and comparison of amino acid sequences of variable domains. Fragments containing variable and association domains of each subunit were amplified, as described in Materials and Methods. A–D, PCR products were separated on 1.5% agarose gel. E, Amino acid sequences of variable domains and neighboring residues of each isoform are indicated. Amino acid positions are numbered based on {alpha}B, ß'e, {gamma}B, and {delta}2 isoforms. Amino acids indicated by dots were sequenced and confirmed to be the same as in previous reports.

 
Immunoblot analysis of CaM kinase II isoforms in MIN6 cells and rat islets
In previous work we reported that the apparent molecular mass of the major subunit in MIN6 cells was 55 kDa (7). To compare the molecular masses of the isoforms in MIN6 cells by SDS-PAGE, we transfected MIN6 cells with the cDNAs of ß'e, {delta}2, and {delta}6 isoforms. Anti-brain CaM kinase II antibody detected a protein with an apparent molecular mass of 55 kDa in MIN6 cells transfected with pCAGGSneo plasmid alone (mock-transfected cells; Fig. 3AGo). It was interesting that the 55-kDa protein and overexpressed ß'e and {delta}2 isoforms comigrated at the same position. Overexpressed {delta}6 isoform migrated to a lower position than the 55-kDa protein (Fig. 3AGo). Anti-CaM kinase II{delta}1-{delta}4 antibody detected a 55-kDa protein and overexpressed the {delta}2 isoform, but not the overexpressed ß'e isoform (Fig. 3BGo). These results clearly showed the presence of the {delta}2 isoform in MIN6 cells. In rat islets, an immunoreactive band of 55 kDa corresponding to the {delta}2 isoform was barely detected (Fig. 3BGo), whereas an immunoreactive band of 55 kDa was observed in HIT-T15 cells (Fig. 3BGo). To detect the ß'e isoform in MIN6 cells, we first used a mAb to ß-subunit. Unexpectedly, no immunoreactive bands were observed in MIN6 cells (data not shown). Therefore, we examined whether the ß-subunit mAb detected the overexpressed ß'e isoform. For this purpose, we transfected NG108–15 cells with the isoforms, because the levels of overexpression of proteins in NG108–15 cells were higher than those in MIN6 cells. The monoclonal antibody reacted with the ß isoform, but not with the ß'e isoform (data not shown). Next, we used anti-CaM kinase IIß{gamma} antibody and found that the antibody reacted with the ß'e isoform as well as the ß and {gamma}A isoforms (data not shown). The antibody did not react with the {delta}2 isoform overexpressed in MIN6 cells (Fig. 3CGo). When anti-CaM kinase IIß{gamma} antibody was used, a clear single immunoreactive band of 55 kDa was observed in mock-transfected MIN6 cells, rat islets, and HIT-T15 cells. A 55-kDa protein comigrated with the overexpressed ß'e isoform in MIN6 cells. The {alpha}-subunit mAb detected the overexpressed {alpha} isoform, but no immunoreactive bands were observed in mock-transfected cells (data not shown). These results strongly suggested that the identified isoforms of CaM kinase II in MIN6 cells and rat islets were ß'e and {delta}2, and ß'e, respectively, at the protein level.



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  		Figure 3. Immunoblot analysis of CaM kinase II. Extracts of MIN6 cells nontransfected (15 µg) or transfected with {delta}6 (15 µg), {delta}2, or ß'e isoform (15 µg); rat islets (15 µg); and HIT-T15 cells (15 µg) were subjected to immunoblot analysis. Anti-brain CaM kinase II antibody (A), anti-CaM kinase II{delta}1-{delta}4 antibody (B), and anti-CaM kinase IIß{gamma} antibody (C) were used at dilutions of 1:500, 1:1000, and 1:200, respectively.

 
Activation of the ß'e and {delta}2 isoforms overexpressed in MIN6 cells
To examine the involvement of ß'e and {delta}2 isoforms in secretagogues-induced insulin secretion, the isoforms were overexpressed in MIN6 cells. To estimate the transfection efficiency of MIN6 cells, we transfected the cells with the {delta}3 isoform of rat brain, which was not observed in MIN6 cells and was exclusively localized at the nucleus in cerebellar granule cells (28). When we immunostained the cells with anti-CaM kinase II {delta}1-{delta}4 antibody, 35.9 ± 3.2% of the nuclei of the cells were immunostained among those of the cells examined. These results indicated that the transfection efficiency of MIN6 cells was about 36%. When we prepared the cell extracts in the absence of protein phosphatase inhibitors (15 mM Na4P2O7 and 25 mM NaF), the activities of CaM kinase II in extracts of cells transfected with both ß'e and {delta}2 isoforms were about 2.5-fold higher than those of mock-transfected cells and were dependent on Ca2+/calmodulin (data not shown). In the next step, we examined whether autophosphorylation of overexpressed isoforms occurred in a manner similar to that of endogenous CaM kinase II. Figure 4AGo shows that treatment of mock-transfected cells with tolbutamide enhanced autophosphorylation of endogenous CaM kinase II, as previously reported (7). Autophosphorylation of overexpressed {delta}2 and ß'e was higher than that in mock-transfected cells and increased by treatment with tolbutamide (Fig. 4AGo). The autophosphorylated ß'e isoform migrated at a higher position than the autophosphorylated {delta}2 isoform (Fig. 4AGo). Autophosphorylation of the ß'e isoform may change the migration position by SDS-PAGE. The autophosphorylated bands in mock-transfected cells were relatively broad compared with the overexpressed ß'e or {delta}2 isoform and may contain autophosphorylated ß'e and {delta}2 isoforms. Furthermore, we examined the generation of the Ca2+/CaM-independent form of CaM kinase II by treatment with tolbutamide (Fig. 4BGo). Without tolbutamide treatment, Ca2+/CaM-independent activity of CaM kinase II in cells transfected with the ß'e or {delta}2 isoform was significantly higher than that in mock-transfected cells. Treatment with tolbutamide increased Ca2+/CaM-independent activity to 1.39 ± 0.01-, 1.45 ± 0.02-, and 1.43 ± 0.06-fold in mock-, {delta}2-, and ß'e-transfected cells, respectively (n = 6; Fig. 4BGo). These results indicate that the overexpressed isoforms are activated by tolbutamide through autophosphorylation and generation of Ca2+/CaM-independent forms.



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  		Figure 4. CaM kinase II activity of isoform-transfected cells. A, Autophosphorylation of CaM kinase II in MIN6 cells transfected with ß'e or {delta}2 isoform. MIN6 cells were labeled with [32P]orthophosphate, as described in Materials and Methods. Cells were then incubated in KRH without (Control) or with 0.37 mM tolbutamide for 3 min. CaM kinase II in the cell extract was immunoprecipitated with anti-brain CaM kinase II antibody and subjected to SDS-PAGE, followed by autoradiography. B, Ca2+/calmodulin-independent activity of CaM kinase II in MIN6 cells transfected with ß'e or {delta}2 isoform. MIN6 cells transfected with ß'e or {delta}2 isoform were treated with or without 0.37 mM tolbutamide for 3 min. The cell extract was prepared in the presence of 15 mM Na4P2O7 and 25 mM NaF, and the Ca2+/calmodulin-independent activity of CaM kinase II was measured. Values are the mean ± SEM (n = 6).

 
Potentiation of phosphorylation of synapsin I by overexpression of ß'e and {delta}2 isoforms
Previous studies from our laboratories showed that synapsin I was associated with insulin secretory granules (24) and that phosphorylation correlated with activation of CaM kinase II (7). Therefore, we next examined whether overexpression of the ß'e and {delta}2 isoforms increased phosphorylation of synapsin I. For this purpose, we prepared the antibodies specific to the synapsin I phosphorylated at Ser566 and Ser603, which are recognized as phosphorylation sites of CaM kinase II. Figure 5AGo shows the immunoreactivities of the antibodies with various synthesized phosphopeptide as antigens. pS566-Syn I-Ab and pS603-Syn I-Ab showed high titers against P-Ser566 peptide and P-Ser603 peptide, respectively, but did not cross-react with other phosphopeptides and purified synapsin I. As these phosphopeptides corresponded to the phosphorylation sites of other protein kinases, the antibodies seemed to be specific for synapsin I phosphorylated by CaM kinase II. Immunoblot analysis was performed using pS566-Syn I-Ab, purified synapsin I, and particulate fractions from MIN6 cells and rat brains (Fig. 5BGo). pS566-Syn I-Ab reacted with purified synapsin I phosphorylated by CaM kinase II and with synapsin I in MIN6 cells and rat brains only after phosphorylation by CaM kinase II (Fig. 5BGo). No other proteins were detected with the antibody. Pretreatment of the antibody with the P-Ser566 peptide completely abolished the immunoreactive bands (Fig. 5BGo). We confirmed the phosphorylation of synapsin I by MAP kinase with [{gamma}-32P]ATP (Fig. 5CGo), but the antibody did not react with synapsin I phosphorylated by MAP kinase (Fig. 5BGo). The similar results were obtained with pS603-Syn I-Ab (data not shown).



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  		Figure 5. Characterization of pS566-Syn I-Ab and pS603-Syn I-Ab. A, The specificity of each antibody was determined by ELISA. B, Purified synapsin I (3 µg), and extracts of MIN6 cells (5 µg) and rat brains (3.6 µg) were phosphorylated without or with MAP kinase or CaM kinase II, as indicated, for 10 min. Immunoblot analysis was performed with pS566-Syn I-Ab at a dilution of 1:1000. C, Phosphorylation of synapsin I with [{gamma}-32P]ATP was detected by SDS-PAGE, followed by autoradiography. MAP kinase-phosphorylated synapsin I migrated higher than CaM kinase II under experimental conditions.

 
In the next series of studies we investigated the time course of phosphorylation of synapsin I using pS566-Syn I-Ab (Fig. 6Go). Stimulation of MIN6 cells with 25 mM glucose increased phosphorylation of synapsin I, with a peak occurring after 5 min of incubation, but it gradually decreased to the basal level in 15 min (Fig. 6AGo). Furthermore, stimulation with 0.37 mM tolbutamide induced a rapid increase in phosphorylation of synapsin I, with a peak level noted after 3 min, which decreased to the basal level within 15 min (Fig. 6BGo).



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  		Figure 6. Serial changes in phosphorylation of synapsin I by CaM kinase II. After MIN6 cells were treated with KRH containing 25 mM glucose (A) and 0.37 mM tolbutamide (B) for the indicated time intervals, the medium was removed, and cells were frozen in liquid N2. The cell extract (6.4 µg) was used for immunoblot analysis with pS566-Syn I-Ab at a dilution of 1:1000. The phosphorylation of synapsin I with glucose or tolbutamide treatment for 5 or 3 min, respectively, was taken as 100%, and from this value, other values were calculated. Values are the mean ± SEM (n = 4).

 
Figure 7Go shows phosphorylation of synapsin I by overexpression of the ß'e and {delta}2 isoforms. In control MIN6 cells that were not treated with glucose or tolbutamide, synapsin I was barely phosphorylated regardless of overexpression of isoforms (Fig. 7Go). When the phosphorylation of synapsin I at Ser566 without any stimulants was taken as 100%, stimulation of mock-transfected cells with glucose and tolbutamide augmented phosphorylation of synapsin I to 943 ± 16% and 729 ± 56%, respectively (Fig. 7Go, A and B). Stimulation of cells with glucose significantly increased phosphorylation of synapsin I by overexpression of the {delta}2 and ß'e isoforms to 371.6 ± 11.1% and 651.8 ± 34.3%, respectively, compared with the mock-transfected cells (Fig. 7AGo). Tolbutamide also significantly stimulated phosphorylation of synapsin I by overexpression of {delta}2 and ß'e isoforms to 190.2 ± 10.3% and 164.5 ± 3.7%, respectively (Fig. 7BGo). When the phosphorylation of synapsin I at Ser603 without any stimulants was taken as 100%, stimulation of mock-transfected cells with glucose and tolbutamide augmented phosphorylation of Ser603 to 354% and 1000%, respectively. Overexpression of the {delta}2 and ß'e isoforms significantly potentiated phosphorylation of Ser603 by treatment with tolbutamide to 272% and 165%, respectively, compared with the mock-transfected cells (Fig. 7Go, C and D).



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  		Figure 7. Phosphorylation of synapsin I by CaM kinase II in MIN6 cells transfected with the ß'e or {delta}2 isoform. MIN6 cells transfected with pCAGGSneo plasmid alone (Mock) or with the ß'e or {delta}2 isoform were treated with KRH containing 25 mM glucose for 5 min (A and C) or with 0.37 mM tolbutamide for 3 min (B and D). The cell extract (15 µg) was used for immunoblot analysis with pS566-Syn I-Ab (A and B) or pS603-Syn I-Ab (C and D) at a dilution of 1:1000 or 1:2000, respectively. After the antibody was stripped, immunoblot analysis with antisynapsin I antibody was carried out at a dilution of 1:500. The phosphorylation of synapsin I of the mock-transfected cells treated with glucose (A and C) or tolbutamide (B and D) was taken as 100%, and from this value, other values were calculated. Values are the mean ± SEM (A and B, n = 4; C and D, n = 2)

 
Potentiation of insulin secretion by overexpression of ß'e and {delta}2 isoforms
In previous work we demonstrated that activation of CaM kinase II correlated with insulin secretion (8). In the next series of experiments we examined whether overexpression of the ß'e and {delta}2 isoforms increased insulin secretion (Fig. 8Go). Without any stimulants, overexpression of ß'e or {delta}2 isoform did not increase insulin secretion compared with that in mock-transfected cells. Treatment of mock-transfected cells with glucose and tolbutamide significantly increased insulin secretion from 141.4 ± 8.0 to 169.8 ± 3.0 and 178.4 ± 4.3 U/mg, respectively. Stimulation of cells with glucose significantly increased insulin secretion by overexpression of {delta}2 and ß'e isoforms to 190.7 ± 8.3 and 192.6 ± 9.4 U/mg, respectively, compared with that of mock-transfected cells. Tolbutamide also significantly stimulated insulin secretion to 241.5 ± 17.0 and 225.4 ± 7.8 U/mg, respectively.



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  		Figure 8. Effects of transfection of MIN6 cells with ß'e or {delta}2 isoform on insulin secretion. MIN6 cells transfected with pCAGGSneo plasmid alone (Mock) or with the ß'e or {delta}2 isoform were preincubated with 3 mM glucose in KRH for 30 min and then incubated with 3 or 25 mM glucose or 0.37 mM tolbutamide for 10 min as described in Materials and Methods. Insulin content in the medium was measured by RIA. Values are the mean ± SEM (six wells per condition in a single experiment). The experiments were repeated at least three times with reproducible results, and representative results were shown. *, P < 0.05; **, P < 0.01 (vs. control).

 
Overexpression of ß'e(K43A) and {delta}2(K43A) mutants, and their effects on insulin secretion
To confirm that the activity of CaM kinase II was necessary for potentiation of insulin secretion, we generated ß'e(K43A) and {delta}2(K43A) mutants by site-directed mutagenesis, which were overexpressed in MIN6 cells. Immunoblot analysis revealed that both mutants and ß'e and {delta}2 isoforms migrated at the same position, and expression levels were comparable (Fig. 9AGo). The activities of CaM kinase II in cell extracts of ß'e(K43A) mutant and {delta}2(K43A) mutant-transfected cells were similar to those in mock-transfected cells (Fig. 9BGo). We also measured insulin secretion from the cells and found that overexpression of the ß'e(K43A) mutant or the {delta}2(K43A) mutant did not increase insulin secretion in the presence or absence of tolbutamide compared with that in mock-transfected cells (Fig. 9CGo). In these experiments, overexpression of the wild-type ß'e and {delta}2 isoforms potentiated insulin secretion by tolbutamide from 142.0 ± 3.1 to 166.1 ± 5.1 U/mg and from 164.7 ± 3.2 to 220.9 ± 7.0 U/mg, respectively (Fig. 9CGo).



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  		Figure 9. Overexpression of ß'e(K43A) mutant and {delta}2(K43A) mutant and their effects on insulin secretion. A, Extracts (10 µg) of MIN6 cells transfected with pCAGGSneo plasmid alone (Mock), {delta}2(K43A) mutant, {delta}2 isoform, ß'e (K43A) mutant, or ß'e isoform was subjected to immunoblot analysis using anti-brain CaM kinase II antibody at a dilution of 1:500. B, The activity of CaM kinase II was measured in the presence or absence of Ca2+/calmodulin (Ca2+/CaM) with extracts of transfected MIN6 cells. Values are the mean ± SEM (n = 4). C, Effects of overexpression of {delta}2(K43A) mutant, {delta}2 isoform, ß'e(K43A) mutant, and ß'e isoform on insulin secretion from MIN6 cells by treatment without (Cont.) or with 0.37 mM tolbutamide (Tolbut.) for 10 min. Values are the mean ± SEM (four wells per condition in a single experiment). The experiments were repeated at least three times with reproducible results, and representative results were shown. **, P < 0.01 (vs. control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is growing evidence that CaM kinase II is involved in Ca2+-induced insulin secretion (6, 7). We reported that activation of CaM kinase II and concomitant phosphorylation of synapsin I correlate with secretagogue-induced insulin secretion (7). Furthermore, inhibition of dephosphorylation of synapsin I enhances insulin secretion induced by secretagogues (9). In the present study we examined whether overexpression of CaM kinase II enhanced the phosphorylation of synapsin I and increased insulin secretion. We considered that the major isoforms of CaM kinase II in pancreatic ß-cells should be overexpressed in MIN6 cells for this purpose.

Therefore, in the first step we identified the isoforms of CaM kinase II in MIN6 cells and rat islets. We found that all subunits of CaM kinase II were present in MIN6 cells and identified seven isoforms at the mRNA level. Among the isoforms, the ß'e and {delta}2 isoforms were suggested to be the major isoforms in MIN6 cells at the protein level, as follows. 1) Immunoblot analysis with anti-brain CaM kinase II antibody showed that the major isoforms of CaM kinase II in MIN6 cells had an apparent molecular mass of 55 kDa. 2) Immunoblot analysis with antibrain CaM kinase IIß{gamma} antibody indicated the occurrence of the ß'e isoform, which had the apparent molecular mass of 55 kDa. 3) Anti-CaM kinase II{delta}1-{delta}4 antibody detected the {delta}2 isoform in MIN6 cells, which had the apparent molecular mass of 55 kDa. The overexpressed {delta}6 isoform migrated at the lower position than 55 kDa. 4) The monoclonal antibody to the {alpha}-subunit did not react with the 55-kDa enzyme. These results suggested that the {alpha}-subunit was not abundant in MIN6 cells at the protein level. The mRNA of the {gamma}-subunit was found in MIN6 cells (Fig. 1Go). However, it is not clear at present whether the {gamma}-subunit was present in MIN6 cells at the protein level, because the specific antibody to the {gamma}-subunit was not available. Only the ß'e isoform was detected in rat islets at both mRNA and protein levels. Therefore, we decided to overexpress the ß'e and {delta}2 isoforms in MIN6 cells. The ß'e isoform was originally found in rat brains (43), and to our knowledge, our finding is apparently the first report on the occurrence of the ß'e isoform in insulinoma cells and islets. We found that the ß'e isoform comigrated with the {delta}2 isoform on SDS-PAGE and that the ß'e isoform was not detected using a commercially available monoclonal antibody to the ß-subunit. These characteristics of the ß'e isoform may explain why the ß'e isoform has not been reported previously in insulinoma cells and islets. The ß3 isoform has been reported to be present in MIN6 cells as well as neonatal rat islets and human islets at the mRNA level (12, 44). However, we could not detect the isoform under our experimental conditions. Differences between the experimental results are not clear at present.

Overexpression of the isoforms potentiated phosphorylation of synapsin I by CaM kinase II after treatment with tolbutamide and glucose. Synapsin I has at least six phosphorylation sites, and three of them are phosphorylated by MAP kinase (45). Site 2 (Ser566) and site 3 (Ser603) are phosphorylated by CaM kinase II (18), and sites 4, 5, and 6 (Ser62, Ser67, and Ser549, respectively) are phosphorylated by MAP kinase (45). MAP kinase kinase and MAP kinase are present in both rat islets and MIN6 cells (46). MAP kinase was partially active in nonstimulated conditions and was activated by stimulation with glucose (46). Therefore, MAP kinase may phosphorylate synapsin I at sites different from site 2 in the presence or absence of secretagogues. Immunoblot analysis with pS566-Syn I-Ab clearly showed that the antibody did not cross-react with synapsin I phosphorylated by MAP kinase and that overexpression of CaM kinase II potentiated the secretagogue-induced phosphorylation of synapsin I at site 2. Immunoblot analysis with pS603-Syn I Ab showed the potentiation of phosphorylation of synapsin I at site 3 by overexpression of CaM kinase II.

We confirmed that both sites of Ser566 and Ser603 of synapsin I were phosphorylated in MIN6 cells in response to glucose and tolbutamide, using the specific antibody to each site. These are phosphorylation sites by CaM kinase II. However, in the present study we could not distinguish the role of each site phosphorylation in insulin secretion.

It was unexpected that overexpressed CaM kinase II was partially activated under basal, unstimulated conditions. However, overexpression of the isoforms did not induce phosphorylation of synapsin I or insulin secretion without stimulation with any secretagogues. As synapsin I is readily phosphorylated by CaM kinase II in vitro, it may be relevant that phosphorylation of synapsin I by overexpressed CaM kinase II occurred even under basal conditions. At present, the reasons for this are not clear. One explanation is that a small activity of control under basal conditions is not high enough to phosphorylate synapsin I by spatial distance in situ between CaM kinase II and synapsin I in the cells and therefore does not stimulate insulin secretion. Furthermore, it cannot be ruled out that additional conditions to increase autonomous CaM kinase II activity, such as Ca2+ influx, are needed to stimulate exocytosis of insulin. The SNARE complex and synaptotagmin have important roles in neurotransmitter release (47). Synaptotagmin is an integral Ca2+-binding protein of synaptic vesicle membranes, and binding of Ca2+ to synaptotagmin is necessary for fusion of the membranes of synaptic vesicles and the presynaptic terminal (48). As synaptotagmin was reported to exist in MIN6 cells and pancreatic ß-cells (49), similar mechanisms may be involved in insulin secretion. An elevation of Ca2+ beneath the plasma membrane by secretagogues may be necessary for fusion of insulin secretory granules to plasma membrane.

Received November 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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