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Journal of Molecular Endocrinology (2008) 41 1-11    DOI: 10.1677/JME-07-0133
© 2008 Society for Endocrinology
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CaV1.2 rather than CaV1.3 is coupled to glucose-stimulated insulin secretion in INS-1 832/13 cells

 Marloes Dekker Nitert1,3, Cecilia L F Nagorny1,3, Anna Wendt2,3, Lena Eliasson2,3 and Hindrik Mulder1,3

1 Molecular Metabolism2 Islet Cell Exocytosis, Department of Clinical Science, Clinical Research Center, SE-205 02 Malmö, Sweden3 Lund University Diabetes Center, Malmö, Sweden

(Correspondence should be addressed to M D Nitert who is now at Unit of Molecular Metabolism, Department of Clinical Science, CRC Floor 12, SE-205 02 Malmö, Sweden; Email: marloes.dekker_nitert{at}med.lu.se)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
In clonal β-cell lines and islets from different species, a variety of calcium channels are coupled to glucose-stimulated insulin secretion. The aim of this study was to identify the voltage-gated calcium channels that control insulin secretion in insulinoma (INS)-1 832/13 cells. The mRNA level of CaV1.2 exceeded that of CaV1.3 and CaV2.3 two-fold. Insulin secretion, which rose tenfold in response to 16.7 mM glucose, was completely abolished by 5 µM isradipine that blocks CaV1.2 and CaV1.3. Similarly, the increase in intracellular calcium in response to 15 mM glucose was decreased in the presence of 5 µM isradipine, and the frequency of calcium spikes was decreased to the level seen at 2.8 mM glucose. By contrast, inhibition of CaV2.3 with 100 nM SNX-482 did not significantly affect insulin secretion or intracellular calcium. Using RNA interference, CaV1.2 mRNA and protein levels were knocked down by ~65% and ~34% respectively, which reduced insulin secretion in response to 16.7 mM glucose by 50%. Similar reductions in calcium currents and cell capacitance were seen in standard whole-cell patch-clamp experiments. The remaining secretion of insulin could be reduced to the basal level by 5 µM isradipine. Calcium influx underlying this residual insulin secretion could result from persisting CaV1.2 expression in transfected cells since knock-down of CaV1.3 did not affect glucose-stimulated insulin secretion. In summary, our results suggest that CaV1.2 is critical for insulin secretion in INS-1 832/13 cells.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The β-cells of pancreatic islets secrete insulin in response to an increase in glucose. Glucose enters the cells and is metabolised, provoking a rise in the ATP:ADP ratio, which leads to closure of ATP-sensitive K+ (KATP) channels. This sets off a depolarisation of the plasma membrane, which opens the voltage-gated calcium channels. The subsequent increase in intracellular calcium [Ca2+]i initiates the secretion of insulin granules from the β-cells. The voltage-gated calcium channels are part of a family of multi-subunit proteins, consisting of {alpha}1-, {alpha}2-, {delta}- and β-subunits, and additionally in some cell types a {gamma}-subunit (Jay et al. 1990). These have different primary structures and electrophysiological and pharmacological properties (for a review see (Yang & Berggren 2005). The {alpha}1-subunits form the ion-conducting pore of the calcium channel. Several pore-forming calcium channel {alpha}1-subunits of voltage-sensitive calcium channels have been identified in rat pancreatic islets and β-cell lines: CaV1.2, CaV1.3, CaV2.1, CaV2.2, CaV2.3 and CaV3.1 (Plant 1988, Sala & Matteson 1990, Safayhi et al. 1997, Ligon et al. 1998, Grabsch et al. 1999, Ertel et al. 2000, Zhuang et al. 2000, Scholze et al. 2001). The contribution of the different calcium channels to the increase in [Ca2+]i has been extensively studied. Approximately, 60–70% of the rat whole-cell Ca2+ current in β-cells is sensitive to dihydropyridines (Schulla et al. 2003, Huang et al. 2004, Taylor et al. 2005). The major dihydropyridine-sensitive calcium channels in rat β-cells are CaV1.2 or CaV1.3, with different studies emphasising the role of either one (Iwashima et al. 1993, Liu et al. 2004, Taylor et al. 2005). Ca2+ currents that can be inhibited by {omega}-conotoxin GVIA, which is known to block CaV2.2, have been reported in rat β-cells, but their role in insulin secretion is controversial (Satin et al. 1995, Sher et al. 2003). CaV2.3, which is inhibited by SNX-482, accounts for another 18% of the Ca2+ current in mouse β-cells (Jing et al. 2005). The role of CaV2.3 in rat β-cells is not clear since Vajna et al. (2001) reported that 25% of insulin secretion in rat insulinoma (INS)-1 β-cells was inhibited by SNX-482, whereas others did not find an effect of SNX-482 in the same cells (MacDonald et al. 2005).

Clonal cells derived from the rat insulinoma cell line INS-1, 832/13, have previously been shown to exhibit strong responses to stimulation with glucose (Hohmeier et al. 2000). A robust increase in insulin secretion is demonstrated by 832/13 cells when stimulated with glucose as well as a number of other secretagogues (Hohmeier et al. 2000). The identity of voltage-gated calcium channels in these cells is not known and neither is the relative importance of CaV1.2 versus CaV1.3 for insulin secretion. The distribution pattern of voltage-gated calcium channels could also give rise to differences in insulin secretion between 832/13 and 832/2 cells, the latter being weakly responsive to glucose (Hohmeier et al. 2000). However, 832/2 cells do respond to high potassium with an increase in insulin secretion, although not to the same extent as 832/13 cells (Yang et al. 2004). In both studies, it was shown that the insulin content of 832/13 and 832/2 cells is similar, and the blunting of insulin secretion can therefore not be attributed to differences in insulin content. Yang et al. (2004) showed that increased signalling in the cAMP/protein kinase A system accounts for part of the exaggerated glucose responsiveness of 832/13 cells. However, additional differences must exist. For one, the secretory differences between the two cell types could be explained by the fact that different calcium channels are expressed in the two cell types, resulting in different sensitivities to metabolic coupling signals in the two clones. Also, metabolic differences between the glucose-responsive and -unresponsive lines exist: anaplerosis manifested as pyruvate cycling is much more prominent in 832/13 than 832/2 cells (Lu et al. 2002).

To resolve possible differences in Ca2+ dynamics between the different 832 lines, we studied the expression levels of three voltage-gated calcium channels (CaV1.2, CaV1.3 and CaV2.3), and the calcium influx in response to changes in glucose concentrations in the presence and absence of calcium channel blockers. The effects of calcium blockers and RNA silencing of calcium channels on insulin secretion, changes in [Ca2+]i, voltage-gated Ca2+ currents and cell capacitance ({Delta}Cm) were also studied. We found that CaV1.2 is the major voltage-gated calcium channel coupled to insulin secretion in 832/13 cells. However, differences in glucose responsiveness of the two clonal β-cell lines could not be attributed to the distribution of voltage-gated calcium channels, since the 832 clones expressed similar levels of calcium channels.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials

All chemicals were obtained from Sigma–Aldrich, unless otherwise indicated.

Cell culture

Insulinoma-derived β-cell clones INS 832/13 and 832/2 were cultured in RPMI 1640 medium containing 11.1 mM glucose supplemented with 10% foetal calf serum, 1 mM sodium pyruvate, 10 mM HEPES, 50 µM β-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere containing 95% air and 5% CO2.

Insulin secretion assays

Cells were cultured in 24-well plates until confluence. After an overnight incubation in full medium containing 5 mM glucose, the cells were first washed and then pre-incubated in HEPES balanced salt solution (HBSS) containing 114 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.16 mM MgSO4, 20 mM HEPES, 25.5 mM NaHCO3, 2.5 mM CaCl2 at pH 7.2 with 0.575 mM BSA and 2.8 mM glucose for 2 h at 37 °C. The buffer was then replaced by HBSS containing glucose, the calcium channel inhibitors isradipine and SNX-482, and KCl in combination with diazoxide at the indicated concentrations for 1 h at 37 °C. The buffer was carefully removed and the amount of insulin was determined with the Coat-A-Count insulin RIA (DPC, Los Angeles, CA, USA), which recognises human insulin and cross-reacts by ~20% with rat insulin. The cells were washed with PBS and stored at –20 °C for determination of protein content. Protein content was measured with the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL, USA).

Measurements of [Ca2+]i

Dual-wavelength microfluorimetry was performed to measure changes in [Ca2+]i in individual cells. The cells were cultured overnight on 22 mm microscope cover plates. The cells were loaded with 2.5 µM fura-2-acetoxymethyl ester (fura-2 AM) in the presence of 0.0007% w/v pluronic acid (Molecular Probes, Leiden, The Netherlands) for 30–40 min at 37 °C. Experiments were performed in a perfusion chamber; the perfusion solution consisted of 138 mM NaCl, 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 5 mM HEPES at pH 7.4, held at a temperature of 32 °C. Glucose, the calcium channel inhibitors isradipine (which blocks Cav1.2 and Cav1.3) and SNX-482 (which blocks Cav2.3), and KCl were added at this solution at the indicated concentrations. The experiments were conducted on an IonOptix fluorescence imaging system (Milton, MA, USA). Excitation of fura-2 AM at 340 and 380 nm was alternately performed, and the emitted light was collected at 510 nm. The fluorescence (F) ratio F340/F380 was determined and equation 5 of (Grynkiewicz et al. 1985) with a Kd of 224 nM was then used to estimate {Delta}[Ca2+]i. The maximum ratio (Rmax) in the equation was established by the addition of 100 µM ionomycin in the presence of 10 mM CaCl2 at the end of each experiment.

Real-time PCR

Total RNA was isolated from 832/13 and 832/2 cells grown to confluence with the RNeasy Plus Mini kit (Qiagen GmbH), which removes genomic DNA from the sample. One microgram of RNA was transcribed into single-stranded cDNA with 100 U M-MuLV Reverse Transcriptase RNase H– (Finnzymes Oy, Espoo, Finland). TaqMan gene expression assays were purchased from Applied Biosystems (ABI; Stockholm, Sweden) for the various target genes: Rn00709287, directed against rat calcium channel Cav1.2; Rn00568820, directed against rat calcium channel CaV1.3; Rn00494444, directed against rat calcium channel Cav2.3 and Rn0138745, directed against hypoxanthine-guanine phosphoribosyl transferase, which served as endogenous control gene. The reaction consisted of 50 ng cDNA, 10 µl TaqMan Universal PCR mix (ABI), primers at a final concentration of 300 nM, a final concentration of the probe of 100 nM and RNase-free water for a volume of 20 µl per well. The PCR was performed in an ABI 7900HT Fast Real-Time PCR system. The reactions were performed in duplicate. The relative quantity of calcium channel Cav1.2, CaV1.3 and Cav2.3 mRNA was calculated using the comparative CT method after initial experiments showed similar quantitative PCR efficiency rates for all assays.

RNA interference

Two different siRNA sequences for CaV1.2 were purchased with siRNA identification numbers 199812, with an antisense sequence of 5'-AUUUCAAUGGUGAAAAUGG-3' and a sense sequence of 5'-CCAUUUUCACCAUUGAAAU-3', and 47874, with an antisense sequence of 5'-AAAUACAGACAUACAUCCC-3' and a sense sequence of 5'-GGGAUGUUAGUCUGUAUUU-3'. For CaV1.3, one siRNA sequence was obtained with identification number 198492 and with an antisense sequence of 5'-UCCUUGCAUAGUUUGCUG-3' and a sense sequence of 5'-CGAGGCAAACUAUGCAAGA-3' (Ambion, Huntingdon, UK). Negative control siRNA coupled to the fluorophore Alexa488 were used (1027284; Qiagen). Cells were reverse transfected with 30 nM siRNA, using HiPerFect transfection medium for siRNA (Qiagen). The transfection efficiency amounted to 70%. The medium was changed after 24 h and the cells were assayed for knock-down at the mRNA level after yet another 24 h. Alternatively, the cells were cultured for 48 h post-transfection and then insulin secretion was subsequently performed. For patch-clamp measurements, the cells were co-transfected with CaV1.2 siRNA and negative control siRNA or negative control siRNA; the presence of control siRNA allowed us to identify cells transfected with siRNA.

Western blot

Cells transfected with siRNA for CaV1.2 or CaV1.3 were cultured for 48 h and homogenised in a homogenisation buffer containing 50 mM Tris, 5 mM MgCl2, 1 mM CaCl2, 1 mM EDTA, 0.1x10–3 mM EGTA and 1:100 protease inhibitor cocktail. Protein content was determined by the BCA method and 20 and 35 µg protein was run onto a 7.5% SDS-PAGE gel for CaV1.2 and CaV1.3 respectively, and subsequently blotted onto polyvinylidene difluoride (PVDF) membranes. Protein for CaV1.2 was detected with primary antibody rabbit anti-Ca2+ channel (CaV1.2 ({alpha}-1C)) polyclonal antibody in a dilution of 1:1000 (BD Biosciences, Stockholm, Sweden) and for CaV1.3 with primary rabbit anti-CaV1.3 polyclonal antibody in a dilution of 1: 350 (Alomone Labs, Jerusalem, Israel). Horseradish peroxidase-coupled anti-rabbit IgG antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used as a secondary antibody (1:5000). Enhanced chemiluminescence was used to detect protein on the blots. The protein levels were determined by densitometry, and normalised to the levels of {alpha}-tubulin (Santa Cruz Biotechnology), which corrected for variations in protein loading. The results are expressed as proportion of the density for samples treated with negative control siRNA.

Electrophysiology

Whole-cell currents and exocytosis were recorded on single 832/13 cells 48 h after transfection using an EPC-9 patch-clamp recorder and the software Pulse (Heka Elektronik, Lambrecht/Pfalz, Germany). Measurements were only performed on transfected cells that were identified by Alexa488. All measurements were conducted at 32–34 °C. Patch electrodes were pulled from borosilicate glass (tip resistance 3–6 M{Omega} when filled with intracellular solution). Experiments were conducted using the standard whole-cell configuration, which results in electrical contact with the whole cell and allows the pipette solution to diffuse into the cell and exchange the cytosol. The pipette solution contained 125 mM Cs-Glut, 10 mM NaCl, 10 mM CsCl, 1 mM MgCl2, 0.05 mM EGTA, 3 mM Mg-ATP, 10 mM HEPES (pH 7.15 using CsOH) and 0.1 mM cAMP. The standard extracellular solution consisted of 118 mM NaCl, 20 mM tetraethylammonium chloride (to block voltage-gated K+-currents), 5.6 mM KCl, 2.6 mM CaCl2, 1.2 mM MgCl2, 5 mM glucose and 5 mM HEPES (pH 7.4 using NaOH). Exocytosis was detected as changes in membrane capacitance using the software-based lock-in application (which add a sine wave with a frequency of 500 Hz to the holding potential) of the amplifier. Exocytosis was elicited by a train of 500 ms voltage-clamp depolarisations from –70 to 0 mV applied at 1 Hz. The steady-state inactivation of the current was investigated using a conventional two-pulse protocol in which a 5 ms test depolarisation to zero was preceded by a 50 ms conditioning pulse to voltages between –150 and 0 mV. The current(I)–voltage(V) relationship was studied using a protocol in which a series of 50 ms depolarisations from –70 mV to increasing voltages between –50 mV to 20 mV is applied to the cell. Both the inactivation properties and the I–V relationship were analysed using different features of the PulseFit function of the Heka software.

Statistical analysis

Values are expressed as mean±S.E.M. Differences between the two clones or the groups were performed by Student's t-test for unpaired data, unless otherwise indicated, and P<0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Insulin secretion is reduced in the presence of isradipine in 832/13 but not 832/2 cells

The two clones of the INS-1 cell line differed in their basal secretion of insulin in the presence of 2.8 mM glucose. 832/2 cells displayed basal insulin secretion, which was three- to fourfold higher than basal insulin secretion in 832/13 cells (Fig. 1A). However, whereas 832/13 cells responded to stimulation by 16.7 mM glucose with a tenfold increase in insulin secretion, 832/2 cells did not significantly increase insulin secretion from the basal level. The KATP-independent pathway of glucose-stimulated insulin secretion was tested by incubating the cells with 2.8 or 16.7 mM glucose in the presence of 35 mM KCl and 250 µM diazoxide. In 832/13 cells, this resulted in increased insulin secretion both at basal (by 10-fold) and high glucose concentrations (by 25-fold over basal glucose concentrations and by 2.6-fold over 16.7 mM glucose). In 832/2 cells, an increase in insulin secretion could also be observed, although not to the same extent as in 832/13 cells (a 4.1-fold increase over basal glucose concentrations). There was, however, no stimulatory effect of 16.7 mM glucose in 832/2 cells under KATP-independent conditions.


Figure 1
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  		Figure 1 Insulin secretion in 832 INS-1 cells. (A) Insulin secretion in response to stimulation with 2.8 and 16.7 mM glucose with and without 35 mM KCl and 250 µM diazoxide; 832/13 cells (black bars), 832/2 cells (white bars). (B) Insulin secretion in 832/13 cells after stimulation with 2.8 or 16.7 mM glucose in combination with 5 µM isradipine or 100 nM SNX-482. (C) Effect of 5 µM isradipine on insulin secretion elicited by 35 mM KCl and 250 µM diazoxide and 16.7 mM glucose. Number 2 denotes 832/2 cells. n=3 for each condition (*P<0.05, **P<0.01).

 
Since we hypothesised that the disparity in glucose responsiveness could be attributed to differences in the expression of the various calcium channels, we incubated the cells with inhibitors of three different calcium channels: Cav1.2, CaV1.3 and Cav2.3. Isradipine, which blocks Cav1.2 and CaV1.3, was added at 5 µM to basal and high glucose concentrations. In 832/13 cells, isradipine did not significantly affect insulin secretion at 2.8 mM glucose. However, insulin secretion in response to 16.7 mM glucose was reduced by 90% in the presence of isradipine (Fig. 1B). In 832/2 cells, no effect of isradipine could be detected irrespective of the glucose concentration (results not shown). When the inhibitor of Cav2.3, SNX-482 (100 nM), was used, there was no significant effect on insulin secretion in either 832/13 or 832/2 cells. Under KATP channel-independent conditions (in the presence of KCl and diazoxide), addition of isradipine resulted in a significant decrease in insulin secretion at 2.8 and 16.7 mM glucose in both 832/13 and 832/2 cells (Fig. 1C).

Dynamics of [Ca2+]i in 832/13 cells

The differences in insulin secretion between the two clones were also reflected in the measurements of {Delta}[Ca2+]i. Figure 2A shows the pattern of changes in [Ca2+]i in 832/13 cells. Here, an increase in glucose concentration of the perfusion medium from 3 to 15 mM resulted in a rise in [Ca2+]i. This rise in calcium was not constant but rather of a more oscillatory nature. When expressed as number of peaks per minute, it increased from 0.44±0.04 at 3 mM glucose to 0.74±0.06 (P<0.001) at 15 mM glucose. In 832/2 cells (Fig. 2B), there was no significant increase in the number of [Ca2+]i peaks per minute when the glucose concentration was raised (0.46±0.07 vs 0.37±0.06 peaks per minute at basal and high glucose respectively). Comparison of the two clones revealed that 832/13 cells displayed significantly more calcium peaks per minute than 832/2 cells at 15 mM glucose (0.74±0.06 vs 0.37±0.06, P<0.001), while there was no difference in the frequency of peaks at 3 mM glucose. The change in [Ca2+]i after application of 15 mM glucose was also significantly different between 832/13 and 832/2 cells. In 832/13 cells, the mean increase in [Ca2+]i was 61±15 nM, whereas in 832/2 cells, it amounted to 7.4±3.4 nM (P<0.001).


Figure 2
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  		Figure 2 Changes in [Ca2+]i in 832 INS-1 cells. Response to 3 mM (3G) and 15 mM glucose (15G) on {Delta}[Ca2+]i in an (A) 832/13 cell and (B) 832/2 cell. (C) Same cell as in (A) but the response on {Delta}[Ca2+]i to 70 mM KCl (KCl). (D) The same cell as in (B) stimulated as in (C). (E) Changes in {Delta}[Ca2+]i in response to 15 mM glucose and 5 µM isradipine in 832/13 (black traces) and 832/2 cells (grey traces). (F) The response on {Delta}[Ca2+]i in an 832/13 cell stimulated with 15 mM glucose and after the addition of 100 nM SNX-482 in the continued presence of glucose. The traces are representative examples of respective traces.

 
Both clones displayed a rise in [Ca2+]i after the addition of 75 mM KCl to the perfusion solution (Fig. 2C and D). In fact, the increase in [Ca2+]i was similar in 832/2 and 832/13 cells, averaging ~1 µM. This increase in 832/2 cells shows that the cells do contain voltage-gated calcium channels that permit the entry of Ca2+ into the cell in response to depolarisation of the cell membrane. These results are consistent with the results from the insulin secretion experiments performed with KCl (Fig. 1).

Next, measurements of [Ca2+]i in both cell types were performed in the presence of inhibitors of the voltage-gated calcium channel. The CaV1.2 and CaV1.3 channels were blocked by the addition of isradipine (5 µM) to the perfusion medium containing 15 mM glucose. In 832/13 cells, this led to a decrease in the frequency of [Ca2+]i peaks to that seen at 3 mM glucose (0.40±0.05, P<0.001; Fig. 2E). The effect of isradipine was also observed as a decrease in [Ca2+]i by 21.23±11.53 nM (P<0.01.). In 832/2 cells, no effect of isradipine on the number of peaks per minute, nor on the [Ca2+]i, could be seen. In separate experiments, SNX-482, which is a blocker of CaV2.3, was co-perfused with 15 mM glucose. Here, no effect on the number of peaks per minute was noted; only a slight decrease in [Ca2+]i was seen in 832/13 cells (Fig. 2F), whereas no effect on 832/2 cells was observed (results not shown).

Real-time PCR reveals the presence of several voltage-dependent Ca2+ channel subtypes

The relative levels of RNA for CaV1.2, CaV1.3 and CaV2.3 were determined by real-time PCR. The expression of all three channels was readily detected; however, no differences between the 832 lines with respect to the mRNA expression levels were found (Fig. 3). However, the expression levels of the individual calcium channels varied widely. CaV1.2 mRNA expression levels were two orders of magnitude higher than those for CaV1.3 and CaV2.3. The expression levels for CaV1.3 and CaV2.3 were low but did not differ from each other.


Figure 3
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  		Figure 3 mRNA expression levels for CaV1.2, CaV1.3 and CaV2.3 in 832/13 and 832/2 cells. Expression levels in 832/13 cells (black bars; n=4 for CaV2.3 or n=5 for CaV1.2 and CaV1.3), and 832/2 cells (white bars; n=3). The results were evaluated with a one-way ANOVA and the post hoc Bonferroni test (*P<0.05, ***P<0.001).

 
RNA interference of CaV1.2 reduces insulin secretion and exocytosis in 832/13 cells

Since isradipine is a pharmacological inhibitor of both CaV1.2 and CaV1.3, our results could not discern the importance of CaV1.2 versus CaV1.3 in glucose-stimulated insulin secretion in 832/13 cells. Therefore, expression of CaV1.2 was knocked down, using RNA interference. Two different siRNAs were employed and found to be equally efficient; a knock-down by 65% at the mRNA level was achieved (Fig. 4A, results of one siRNA shown). Knock-down of CaV1.2 did not influence mRNA expression levels of CaV1.3 (Fig. 4A), which concurs with the fact that the siRNA sequences were found to be specific for CaV1.2 upon blasting of the sequences in the NCBI database. The signal of the protein level on western blot for CaV1.2 was found to be reduced (Fig. 4C) by 34% upon RNA interference, when the density of the protein band of siRNA-treated cells was compared with the density of the protein band of negative control-treated cells (Fig. 4D). As a consequence of CaV1.2 knock-down, insulin secretion in response to 16.7 mM glucose was decreased by 50% (Fig. 5A). Co-incubation of 5 µM isradipine and 16.7 mM glucose further reduced insulin secretion by 55 and 75% respectively, of that provoked by 16.7 mM glucose alone in CaV1.2 siRNA-treated and negative control-treated cells. Expression of CaV1.3 was knocked down to a similar extent as CaV1.2 (by 70%), whereas the expression levels of CaV1.2 were not affected (Fig. 4B) and CaV1.3 protein signals were decreased by 31% (Fig. 4C and D). The siRNA sequences were specific for CaV1.3 in the analysis of the sequence in the NCBI database. Nevertheless, the knock-down of CaV1.3 did not impact on glucose-stimulated insulin secretion (Fig. 5B).


Figure 4
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  		Figure 4 CaV1.2 and CaV1.3 downregulated by RNA interference in 832/13 cells. (A) CaV1.2 or CaV1.3 mRNA levels in cells treated with CaV1.2 siRNA (black bars) or negative control siRNA (white bars) respectively (n=3, *P<0.05). (B) CaV1.3 or CaV1.2 mRNA levels in cells treated with CaV1.3 siRNA (black bars) or negative control siRNA (white bars) respectively. Negative control expression levels for each voltage-gated calcium channel were set to 1 (n=4). Protein expression signals for CaV1.2 after siRNA treatment (C, upper panel) or for CaV1.3 (C, lower panel). (D) Protein expression was normalised to expression levels of {alpha}-tubulin and expressed as proportion of negative control protein signal. The results were evaluated with a paired t-test: CaV1.2 (n=4, P=0.0135), CaV1.3 (n=3, P=0.0536).

 

Figure 5
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  		Figure 5 Influence of CaV RNA knock-down on insulin secretion. (A) Insulin secretion after RNA interference for CaV1.2 (black bars). Cells were stimulated with 2.8 and 16.7 mM glucose or 16.7 mM glucose with 5 µM isradipine. Results were evaluated with a paired t-test due to variation in absolute values of secreted insulin between different experiments (n=3, *P<0.05). (B) Insulin secretion after siRNA for CaV1.3 (black bars). Cells were stimulated with 2.8 mM glucose or 16.7 mM glucose (n=3).

 
To investigate whether the 50% reduction in insulin secretion was due to effects on the exocytotic machinery, standard whole-cell patch-clamp recordings on single INS 832/13 cells transfected with negative control siRNA or siRNA against CaV1.2 were performed. First, we investigated the voltage-dependent currents in 832/13 cells by application of 50 ms depolarisations from –70 mV to increasing voltages between –50 mV to +20 mV. The voltage-dependent current contained both a transient Na+-dependent component and a Ca2+-dependent component (Fig. 6A), as has been observed in primary Sprague–Dawley rat β-cells (Hiriart & Matteson 1988). The peak of the Na+ component (Ip, Na) was maximal when the membrane potential was depolarised to 0 mV and was not significantly different between control cells and cells treated with CaV1.2 siRNA (data not shown). Thus, the RNA silencing had no effect on the Na+ current. The steady-state inactivation properties of the Na+ current measured using a two-pulse protocol with a 5 ms test pulse preceded by a 50 ms conditioning pulse was also the same in control and siRNA-treated cells; the half-maximal inactivation (Vhalf) was –55±4 mV (n=8) in the control cells and –58±3 (n=11) in the CaV1.2 siRNA-treated cells. This is slightly higher than that previously reported in primary rat β-cells, where the half-maximal inactivation of Na+ currents was –70 mV (Hiriart & Matteson 1988), but closer to that reported in human islets (Barnett et al. 1995).


Figure 6
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  		Figure 6 Ca2+ current and exocytosis upon RNA silencing of CaV1.2 in 832/13 cells. (A) Voltage-dependent inward current initiated by a depolarisation from –70 to 0 mV in a control cell and CaV1.2 siRNA-treated cell as indicated. Note that the rapid Na+ peak inactivated within a few milliseconds is followed by the more sustained Ca2+ current. (B) Current (I)–voltage (V) relationship of the peak Ca2+ current (Ip, Ca) in control cells (black) and siRNA CaV1.2 cells (grey). (C) Charge (Q)–voltage (V) relationship of the voltage-dependent current under same conditions as in (B). (D) Increase in membrane capacitance ({Delta}Cm, bottom) evoked by a train of membrane depolarisations (V, top) in a control cell. (E) The same as in (D) but the experiment was performed on a cell transfected with siRNA against CaV1.2. (F) Histogram summarising the total increase in membrane capacitance in the experiments performed in (D)–(E) (n=7–10, *P<0.05 and **P<0.01).

 
To analyse the voltage dependence of the Ca2+ current, the peak current (Ip,Ca) was measured 2.5 ms after the onset of the depolarization when the transient Na+ current was inactivated (Hiriart & Matteson 1988). The peak Ca2+ current was maximal at 0 mV and amounted to –163±36 pA (n=8) in the control cells (Fig. 6B). This maximal peak current was significantly smaller in the siRNA-treated cells, demonstrating that the knock-down of CaV1.2 by RNA silencing indeed reduced Ip,Ca by ~60% (P<0.05; n=8–10). This was also confirmed by analysis of the voltage dependence of the charge (Q; Fig. 6C), which mainly represents the amount of Ca2+ that is transferred into the cell through the voltage-dependent Ca2+ channels. The charge at 0 mV was estimated to be –6.1±1.8 (n=8) and –2.7±0.7 (n=10; P<0.05) in control and CaV1.2 siRNA-treated cells respectively.

Next, we investigated the effects of RNA silencing of the CaV1.2 channel on exocytosis in the 832/13 cells, using capacitance measurement in combination with standard whole-cell patch-clamp recordings. Exocytosis was evoked by a train of ten 500 ms depolarisations from –70 to 0 mV (Fig. 6D and E). The total increase in membrane capacitance under control conditions was 182±38 fF (n=7). When 832/13 cells had been transfected with siRNA against Cav1.2, the increase in membrane capacitance was significantly reduced and amounted to 81±15 fF (n=9; P<0.01). The decrease in membrane capacitance was equal throughout the train and not linked to the first depolarisations, as in primary mouse β-cells (Schulla et al. 2003, Jing et al. 2005). The difference between control cells and siRNA-treated cells was sustained also during the application of a second train, 2 min after the first. Here, the increase in membrane capacitance was 209±38 (n=7) during control conditions and 121±13 (n=9; P<0.05) in the CaV1.2 siRNA-treated cells.


   Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
We found that CaV1.2 mRNA is expressed at a substantially higher level in 832 cells than either CaV1.3 or CaV2.3 mRNA. The expression levels for CaV1.2 and CaV1.3 have previously been reported for different types of rat β-cells (Safayhi et al. 1997, Taylor et al. 2005) and rat islets (Iwashima et al. 1993, Taylor et al. 2005). Whereas minor differences in expression of the two channels have been found before, differences of the extent that we found here have not been reported. However, the mRNA expression levels of the three calcium channels studied did not differ between the two INS-1 832 clones, indicating that the number of calcium channels is not the causal factor for the discrepant insulin secretion responses. By contrast, we have previously reported that pyruvate cycling (Lu et al. 2002) and signalling in the cAMP/protein kinase A system (Yang et al. 2004) are exaggerated in the highly glucose-responsive 832/13 cells compared with unresponsive 832 clones.

The 832/13 cells are a model for β-cells with a strong insulin secretory response to increases in glucose concentration (Hohmeier et al. 2000). This increase in insulin secretion is preceded by a rise in Ca2+ influx into the cytoplasm. This increase and its oscillatory nature was recently described in 832/13 cells (Baroukh et al. 2007). Ca2+ enters the cells via dihydropyridine-sensitive voltage-gated calcium channels. We found that isradipine blocked calcium influx and virtually abolished glucose-induced insulin secretion in the 832/13 cells. These findings are in accordance with the findings of others, who noted significant inhibition of insulin secretion with dihydropyridines (Satin et al. 1995, MacDonald et al. 2005, Taylor et al. 2005). This emphasises the importance of dihydropyridine-sensitive calcium channels in rat β-cells. Next, we silenced CaV1.2 mRNA levels in order to distinguish between the role of CaV1.2 and CaV1.3, since both these channels are inhibited by dihydropyridines. The cells treated with CaV1.2 siRNA exhibited a significant decrease in glucose-stimulated insulin secretion, a similar decrease in calcium currents and a decrease in capacitance upon a train of depolarisations. Knock-down of CaV1.3 with siRNA did not affect glucose-stimulated insulin secretion. Given the high expression level of CaV1.2, the decrease in insulin secretion after RNA interference, the effect on capacitance increases and calcium currents after RNA interference, and the lack of effect on insulin secretion of CaV1.3 downregulation, we conclude that CaV1.2 is the critical voltage-gated calcium channel in 832/13 cells. The residual insulin secretion that was inhibited by isradipine could be attributed to remaining CaV1.2 expression considering the relatively modest decreases in protein signal and/or persistent expression of CaV1.3. CaV1.3 mRNA expression levels were not significantly altered by CaV1.2 downregulation, in contrast to the reported compensatory upregulation of CaV1.2 in CaV1.3 knock-out mice (Namkung et al. 2001). Liu et al. have previously performed studies on the role of CaV1.2 versus CaV1.3 in INS-1 cells. They found that CaV1.3 was responsible for insulin secretion and calcium influx into the cells (Liu et al. 2003, 2004). In these studies, the pore-forming units of the channels were over-expressed on top of normal expression patterns without taking into account constitutive expression of CaV1.2 and CaV1.3. By contrast, our model does not rely on over-expression of calcium channels, and the discrepancy of the results could thus be explained by the differences in the methodology used.

The CaV2.3 calcium channel was blocked by SNX-482, which previously has been shown to inhibit 25% of insulin secretion in mouse β-cells as well as 20% in a knock-out mouse model (Jing et al. 2005). This study indicated the importance of this channel in the second phase of insulin secretion. In our cells, SNX-482 did not exert a pronounced effect on either insulin secretion or [Ca2+]i influx. This is in accordance with our previous results from the experiments performed in INS-1 cells, where no effect of SNX-482 on exocytosis could be detected (MacDonald et al. 2005). On the other hand, Pereverzev et al. (2002) studied the effect of a CaV2.3 antisense expression cassette and reported a reduction in insulin secretion. In this study, the transfection procedure lowered the insulin content of the cells significantly. Glucose-stimulated insulin secretion was expressed as percentage of basal insulin secretion, which in turn was expressed as percentage of insulin content. Since the insulin content was very different in the transfected and the untransfected cells, it is difficult to compare these results with those obtained by us.

About 832/2 cells were found to have functional dihydropyridine-sensitive calcium channels, since insulin secretion in response to high KCl concentrations could be inhibited by isradipine. The failure of 832/2 cells to respond to increased glucose concentrations can thus not be attributed to dysfunctional calcium channels. Most likely, a metabolic coupling signal is lacking in 832/2 cells. In fact, we have previously examined mitochondrial metabolism in different 832 clones derived from INS-1 cells. We found that pyruvate cycling, where pyruvate fed into the tricarboxylic acid (TCA) cycle from a distinct cellular pool of the triose, correlated strongly with glucose responsiveness (Lu et al. 2002). Thus, 832/2 cells exhibit a much less pronounced activity of this anaplerotic pathway than 832/13 cells. The high basal insulin secretion might also be a reflection of this disturbed metabolism.

Both CaV1.2 and CaV1.3 calcium channels and sodium channels have been reported in humans (Seino et al. 1992, Barnett et al. 1995). The sodium channel was not found to affect insulin secretion after stimulation with 10 mM glucose because of rapid inactivation (Barnett et al. 1995). This is likely to be the same in our study since the half-maximal inactivation of the sodium channel in 832/13 cells is similar to that reported in human islets (Barnett et al. 1995). In the same study, around 70% of the calcium channels in human β-cells were sensitive to dihydropyridines, indicating the importance of these channels in β-cells. Also, the rapid first phase of insulin secretion in mouse β-cells has been coupled to the close association between the secretory granules and the calcium channels (Barg et al. 2001). The relative contribution of CaV1.2 and CaV1.3 calcium channels to insulin secretion has not been fully elucidated in most species (Yang & Berggren 2006). Nevertheless, it has been established in mouse β-cells that CaV1.2 contributes to calcium influx coupled to insulin secretion (Barg et al. 2001). In fact, a missense mutation in the CaV1.2-subunit has been identified in humans (Splawski et al. 2004). This reduces the inactivation of the calcium channels and results in episodic hypoglycaemia in the patients, emphasizing the importance of CaV1.2 for insulin secretion. Moreover, both CaV1.2 and CaV1.3 mRNA levels are reduced in the Zucker diabetic fatty rat, which results in reduced calcium currents (Roe et al. 1996). In humans, insulin secretion is defective in the pre-diabetic stage of impaired glucose tolerance (Hosker et al. 1989, Tripathy et al. 2000, Abdul-Ghani et al. 2006), and an absolute decompensation of β-cell function characterises the transition to mild diabetes (Tripathy et al. 2000). Voltage-gated calcium channels could be involved in this transition. Therefore, the expression pattern of voltage-gated calcium channels is of interest to study in different animals and cell models.


   Acknowledgements
 
The studies were supported by the Swedish Research Council (grant 14196 to H M and 13147 to L E), the Crafoord, Thelma and Albert Påhlsson Foundations, the Swedish Diabetes Association, the Medical Faculty at Lund University, the European Union (BioSim LSHB-CT-2004-005137 (L E)), The Novo Nordisk Foundation and Lund University Diabetes Center (Linnaeus grant from Swedish Research Council). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


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 Discussion
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Received in final form 1 April 2008
Accepted 9 April 2008
Made available online as an Accepted Preprint 9 April 2008




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M. Hiriart and L. Aguilar-Bryan
Channel regulation of glucose sensing in the pancreatic {beta}-cell
Am J Physiol Endocrinol Metab, December 1, 2008; 295(6): E1298 - E1306.
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