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Louis-Jeantet Research Laboratories (V.L., K.R., P.A.H., J.-C.I.) and Department of Physiology (A.M.), University Medical Center, 1211 Geneva 4, Switzerland; and Department of Biochemistry and Molecular Biology and the Howard Hughes Medical Institute, University of Chicago (G.W., D.F.S.), Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Dr. Valérie Lilla, Laboratoires de Recherche Louis-Jeantet, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland. E-mail: valerie.lilla{at}medecine.unige.ch.
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Introduction |
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The search for molecules implicated in regulated insulin secretion has until now depended largely on classical biochemical or pharmacological approaches. Identification of glucokinase as the key glucose sensor of the ß-cells is a good example of the successful application of this approach (1). Contemporary techniques now allow for alternative approaches not dependent on previous knowledge or understanding of the events under study, including gene profiling and analysis of differential gene expression. These approaches depend upon access to cells with discrete phenotypic differences. Identification of many glucose-responsive genes in MIN6 cells by using high density oligonucleotide microarrays demonstrates the usefulness of this approach (2). MIN6 cells (3, 4) are a rare example of a transformed mouse ß-cell line that has retained many aspects of the differentiated state of a ß-cell, including glucose-induced insulin secretion. In addition, and in common with most ß-cell lines, MIN6 cells display functional heterogeneity with increasing passage number. With a view to identifying novel genes involved in regulated insulin secretion, the aim of the present study was to generate subclones of MIN6 cells with different insulin secretion profiles and to compare them using suppression subtractive hybridization (SSH) (5) and Affymetrix oligonucleotide microarrays (6).
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Materials and Methods |
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Insulin secretion assay and insulin content
Cells (2 x 105 cells/well) were seeded in 24-well plates 2 d before use. Cells were washed three times with a modified Krebs-Ringer bicarbonate HEPES buffer [KRBH; 125 mM NaCl, 4.74 mM KCl, 1 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 25 mM HEPES (pH 7.4), and 0.1% BSA] supplemented with 2.8 mM glucose and preincubated with this same buffer for 2 h at 37 C. Cells were then incubated for 1 h at 37 C with KRBH containing 2.8 mM glucose, followed by 1 h at 37 C with KRBH containing various concentrations of glucose or secretagogues of interest (stimulated secretion). The only exception was for stimulation by KCl, for which the stimulation period was limited to 10 min. The incubation buffer was recovered, and the cells were extracted with acid-ethanol. Insulin content is expressed as the sum of insulin extracted at the end of the stimulation test and insulin secreted during the first and second incubation periods. For the cell density assay, the same protocol was used, but the cells were seeded at three different densities (0.5 x 105, 2 x 105, and 5 x 105 cells/well). For estimation of insulin content per cell, cells were trypsinized, counted, and extracted with 1 M acid acetic/0.1% BSA. The amount of insulin in the incubation buffer and cellular extracts was measured by RIA using the charcoal separation technique previously described (7). Rat insulin was used as the standard with a guinea pig antiporcine insulin antibody and [125I]insulin as tracer.
Immunofluorescence
For insulin immunofluorescence, cells were fixed in Bouin’s solution for 12 h at room temperature, rinsed in PBS, and permeabilized using graded concentrations of ethanol. For neurofilament light polypeptide (NF-L) and neurofilament medium polypeptide (NF-M) immunofluorescence, cells were fixed with 100% ethanol for 3 min at -20 C. For both fixation methods, cells were rinsed in PBS and incubated 30 min in PBS containing 0.5% BSA. Cells were then exposed for 2 h at room temperature to a guinea pig serum against insulin (produced in our laboratory) diluted 1:400 or to a mouse antibody against NF-L (NCL-NF68, Novocastra Laboratories, Burlington, Canada) or NF-M (NN 18, Roche, Mannheim, Germany), both diluted 1:50. Cells were rinsed extensively in PBS and incubated for 1 h at room temperature in the presence of fluorescein-conjugated antibodies against guinea pig (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) or mouse IgG (Antibodies, Inc., Davis, CA) diluted 1:400 and 1:1000, respectively. After rinsing, cells were covered with a solution of PBS/glycerol (1:2) containing 0.02% paraphenylendiamine and finally sealed with nail varnish.
Measurement of intracellular calcium concentration
Cells were grown on glass coverslips for 72 h. Then, cells were preincubated 2 h at 37 C in KRBH containing 2.8 mM glucose and subsequently loaded in the same buffer for 30 min at room temperature in the presence of 1.5 µM fura-2/acetoxymethyl ester (Molecular Probes, Inc., Eugene, OR). The coverslips were washed, placed in a thermostatic chamber at 37 C, and incubated in KRBH containing first 2.8 mM glucose, then 16.7 mM glucose, and finally 16.7 mM glucose supplemented with 20 mM KCl. Cells were illuminated alternatively at 340 and 380 nm using an Axiovert S100TV microscope (Carl Zeiss AG, Feldbach, Switzerland). Fluorescence emission at 510 nm was captured every 10 sec using a cooled, back-illuminated, 16-bit, charge-coupled device, frame transfer camera (Princeton, Roper Scientific, Trenton, NJ) controlled by Metamorph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). Using established procedures (8), changes in the emission intensity of fura-2, expressed as a ratio of dual excitation, were used as indicators of changes in the intracellular free Ca2+ concentration ([Ca2+]i). Three independent experiments were performed on each subline, with 30 cells examined for each experiment.
SDS-PAGE and Western blotting
The DC (detergent-compatible) protein assay (Bio-Rad Laboratories, Inc., Reinach, Switzerland) was used to quantify the amount of total protein in samples according to the manufacturer’s instructions. Equal amounts of protein were loaded and separated on 8% polyacrylamide gels under reducing conditions according to Laemmli’s procedure (9). After SDS-PAGE, proteins were transferred onto nitrocellulose (Schleicher \|[amp ]\| Schuell, Inc., Dassel, Germany). NF-L and NF-M were detected using the monoclonal antibodies NCL-NF68 and NN 18 (both diluted 1:1000), an antimouse horseradish-peroxidase-conjugated second antibody (diluted 1:1000), and the enhanced chemiluminescence detection system (Amersham Pharmacia Biotech, Uppsala, Sweden).
RNA isolation
Cells (107) grown to approximately 70–80% confluence were lysed for preparation of mRNA or total RNA. mRNA was isolated using the Fast Track 2.0 Kit (Invitrogen, Groningen, The Netherlands), and total RNA was isolated using the RNeasy Midi Kit (QIAGEN, Basel, Switzerland) according to the manufacturer’s instructions.
Generation of cDNA libraries using SSH
mRNA (2 µg) from the B1 and C3 cells was used to generate cDNA libraries by SSH using the PCR select cDNA subtraction kit (CLONTECH Laboratories, Inc., Basel, Switzerland). Two subtractions were performed: forward SSH (subtraction of C3 from B1 cells) and reverse SSH (subtraction of B1 from C3 cells). Mirror orientation selection (MOS), a modification to eliminate false positive clones, was used as described by Rebrikov et al. (10). PCR products from SSH/MOS were cloned into plasmid vector pGEM-T Easy and transformed into JM109 competent cells (Promega Corp., Zurich, Switzerland).
Screening of the subtracted libraries
The subtracted libraries were differentially screened with the 32P-labeled tester cDNA as positive and with the 32P-labeled driver cDNA as negative. The differential clones were picked and confirmed by real-time PCR. Screening of the subtracted libraries was performed using the PCR-Select differential screening kit (CLONTECH Laboratories, Inc.). Briefly, cDNA inserts were amplified by PCR, NaOH-denatured, blotted on Hybond-N+ nylon membranes (Amersham Pharmacia Biotech), and UV cross-linked in a Stratalinker (Stratagene, Heidelberg, Germany). Subtracted B1 cell cDNA and subtracted C3 cell cDNA were digested with RsaI to remove the adaptors, purified (Concert Rapid PCR Purification System kit, Invitrogen), and 32P-labeled by random priming (Random Primed DNA Labeling Kit, Roche). Unincorporated radionucleotides were removed using Microcon YM-30 (Centrifugal Filter Devices, Millipore Corp., Bedford, MA). Membranes were hybridized overnight at 72 C in ExpressHyb hybridization solution and specific blocking solution (CLONTECH Laboratories, Inc.). Membranes were washed as recommended at 68 C (four times for 20 min each time in 2x standard saline citrate/0.5% sodium dodecyl sulfate and twice for 20 min each time in 0.2x standard saline citrate/0.5% sodium dodecyl sulfate) and exposed to Kodak X-OMAT AR film (Eastman Kodak Co., Rochester, NY). Candidate positive clones were sequenced, and the sequences were compared using the National Center for Biotechnology Information for homology search. Differential gene expression was confirmed by real-time PCR.
Preparation of labeled targets for oligonucleotide microarray hybridization
Double-stranded cDNA was synthesized from 1.5 µg polyadenylated RNA using the Superscript Choice System (Invitrogen) with an HPLC-purified oligo(deoxythymidine) primer containing a T7 RNA polymerase promoter (5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24–3'; GENSET, La Jolla, CA). After second strand synthesis, reactions were extracted with phenol/chloroform/isoamyl alcohol, cDNA-precipitated with ethanol, and resuspended in 3 µl diethyl pyrocarbonate-treated water. In vitro transcription was carried out on 1.5 µl cDNA using Bioarray High Yield RNA transcript labeling reagents (Enzo Diagnostics, Farmingdale, NY) following the manufacturer’s instructions and incorporating biotinylated CTP and UTP. In vitro transcription reactions yielded 50–70 µg biotin-labeled cRNA. Biotin-labeled cRNA was purified on RNeasy affinity columns (QIAGEN) and fragmented at 94 C for 35 min in fragmentation buffer [40 mM/Tris-acetate (pH 8.1), 100 mM KOAc, and 30 mM MgOAc].
Microarray hybridization
Hybridization solution [1 M NaCl, 20 mM EDTA, 100 mM 2-(N-morpholino)ethanesulfonic acid, and 0.01% Tween 20] was used to prehybridize Affymetrix MG-U74 oligonucleotide microarrays for 30 min at 40 C. The prehybridization solution was removed and replaced with 200 µl hybridization solution containing 0.05 µg/µl fragmented cRNA. The arrays were hybridized for 16 h at 40 C. After hybridization, arrays were washed on an Affymetrix fluidics station and stained with streptavidin-phycoerythrin (hybridization solution, 2 mg/ml acetylated BSA, and 5 µg/µl streptavidin R-phycoerythrin; Molecular Probes, Inc., Eugene, OR). After staining, arrays were washed extensively in fresh hybridization buffer. Arrays were scanned on a GeneArray Scanner (Hewlett-Packard Co., Palo Alto, CA), and the data obtained were analyzed using Affymetrix Microarray Suite 5.0 and Affymetrix Data Mining Tool 2.0.
Criteria for selecting induced/suppressed genes and functional assignment
The following criteria were set for determining which genes are differentially expressed in the two sublines. Genes were considered up- or down-regulated if the fold change was at least 1.5 in individual experiments and the averaged fold change was 2 or greater in triplicate experiments. These limits are in general agreement with array experiments conducted in other mammalian systems. It was noted empirically that relaxing the criteria led to identification of large numbers of genes that were not functionally related and/or not present in pancreatic ß-cells. Genes were assigned to functional groups by database searches on PubMed and Affymetrix websites.
Real-time PCR
cDNA was synthesized with Superscript II (Invitrogen) using 1 µg total RNA in a 20-µl reaction volume. For real-time PCR, the cDNA was amplified using an ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems, Foster City, CA). For this purpose, primers were designed according to the Primer Express software and the dsDNA-specific dye SYBR Green I incorporated into the PCR reaction buffer (PE Applied Biosystems) to allow for quantitative detection of the PCR product. The temperature profile of the reaction was 95 C for 10 min, 40 cycles of denaturation at 95 C for 15 sec, and annealing/extension at 60 C for 1 min. An internal housekeeping gene control, ß-actin, was used to normalize differences in RNA isolation, RNA degradation, and the efficiencies of the RT.
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Results |
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). Immunofluorescence labeling using antiinsulin serum was comparable, however (Fig. 2A), suggesting similar insulin contents. This was confirmed by RIA (Fig. 2B). Basal insulin secretion (2.8 mM glucose) was also comparable for B1 and C3 cells (1.2% and 0.8% cell content/h, respectively; P = 0.3). However, the two clones showed quite different secretory responses to glucose (Fig. 3). Insulin secretion from B1 cells was stimulated by glucose in a concentration-dependent fashion, reaching 12-fold after 1 h of stimulation with 16.7 vs. 2.8 mM glucose. C3 cells were insensitive to glucose, with insulin secretion remaining at basal levels even at 16.7 mM glucose.
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). B1 cells responded to varying degrees to all secretagogues tested [20 mM leucine, 20 mM arginine, 1 mM isobutylmethylxanthine (IBMX), 100 nM phorbol 12-myristate 13-acetate (PMA), and 30 mM KCl], with the most marked stimulation elicited by PMA. C3 cells failed to respond to either leucine or arginine and showed only modest stimulation to IBMX, PMA, or KCl compared with B1 cells. To confirm that mitochondrial fuel-mediated insulin secretion is defective in C3 cells and that the response to phospholipase C is attenuated, we tested a mitochondrial substrate other than leucine (methyl succinate) and a phospholipase C agonist (carbachol), respectively. Similar to leucine, 10 mM methyl succinate increased insulin secretion from B1 cells (4.4 ± 0.6-fold stimulation vs. basal condition), whereas it did not stimulate secretion from C3 cells. Carbachol (50 µM) increased insulin secretion markedly from B1 cells (12.9 ± 1.6-fold stimulation), but only weakly from C3 cells (2.1 ± 0.3-fold). This confirmed results obtained with PMA.
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). Finally, mastoparan (10 µM), which is a classical G protein agonist, stimulated insulin secretion 4.6 ± 0.7- and 3.3 ± 0.4-fold from B1 and C3 cells, respectively. These results are not significantly different. It is understood that mastoparan-stimulated insulin release is due to activation of the exocytotic machinery itself (11, 12). These results would thus support the hypothesis that the secretory defect of C3 cells arises at the late step in the stimulus-secretion pathway, but upstream of the steps stimulated by mastoparan.
Effect of cell density on insulin secretion from B1 and C3 cells
Cell to cell contact has been shown to improve insulin secretion from both primary ß-cells (13) and MIN6 cells (14). To study whether cell to cell contact affects insulin release from B1 and C3 cells, glucose stimulation was performed on cells seeded at three different densities. At low density (seeded at 0.5 x 105 cells/well), insulin release from B1 cells amounted to 7% of content/h at 2.8 mM glucose and did not significantly increase at 16.7 mM glucose (Fig. 5). At higher densities, basal insulin secretion decreased to reach 2%/h at a seeding density of 5 x 105 cells/well. Conversely, glucose-stimulated secretion increased from 10% to 20%/h as cell density was increased from 0.5 to 2 x 105 cells/well, staying at this level even at 5 x 105 cells/well. In C3 cells there was no effect of glucose on insulin secretion at any cell density tested (data not shown).
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for representative traces). B1 cells showed a rise in [Ca2+]i in response to glucose, whereas no change in [Ca2+]i was observed in C3 cells. On the other hand, B1 and C3 cells showed similar Ca2+ responses to KCl despite the markedly different responses in terms of insulin secretion (Fig. 4A).
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). Gene expression of glucose transporters, GLUT-1 and GLUT-2, and of glucokinase, which is considered the glucose sensor of ß-cells, was not significantly different between B1 and C3 cells. Expression of hexokinase was not detectable in either of the sublines. In addition, mRNA levels of the ATP-sensitive K+ channel subunits, Kir6.2 and SUR1, were similar in the two sublines. On the other hand, gene coding for the transcription factor pancreatic-duodenum homeobox-1 (PDX-1), which regulates genes associated with pancreatic cell differentiation and maturation, was 2.5-fold more highly expressed in C3 vs. B1 cells. The fact that the less well differentiated C3 cells display higher levels of expression of PDX1, argues against PDX1 being a major player underlying the phenotypic differences between B1 and C3 cells. Because cell density affects insulin secretion by B1 cells, we examined the E-cadherin gene, which is involved in cell to cell adhesion. This gene was, on the average, 3.5-fold more highly expressed in B1 cells than in C3 cells.
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) and a further 5 in C3 (Table 3) cells, whereas the remaining 9 genes were revealed as false positive.
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lists genes for which a mean 2-fold or greater change in transcript levels was demonstrated in three independent experiments. Genes were clustered according to known cellular function. For several genes of interest, the relative expression levels were confirmed by real-time PCR (Rab3D, NF-M, NF-L, DNA-binding protein A, and tau) and found to be comparable to the values shown in Table 4.
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) and Western blotting (Fig. 7B). Most B1 cells were labeled for NF-L and NF-M, whereas only occasional C3 cells were labeled. This strong difference was confirmed by Western blotting (Fig. 7B).
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Discussion |
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Measurements of [Ca2+]i in B1 and C3 cells show that both sublines increased their [Ca2+]i in response to KCl in a similar manner. Unlike B1 cells, in C3 cells this rise in [Ca2+]i consequent to depolarization by KCl did not lead to an equally impressive increase in insulin secretion, suggesting either a defect in coupling calcium signaling to exocytosis or an altered calcium set-point, as seen in other nonglucose-responsive MIN6 cells (17). The failure of C3 cells to respond to glucose with a rise in [Ca2+]i suggests a metabolic defect or a defect in KATP channel function that could perhaps again be the consequence of or associated with possibly abnormally elevated [Ca2+]i (17). There was, however, no apparent difference in mRNA levels for either the KATP channel itself (Kir6.2) or its associated sulfonylurea receptor subunit (SUR1), but whether protein levels for these two KATP channel subunits or channel function differ between the two sublines remains to be investigated. Regardless, and as discussed below, the unresponsive phenotype of C3 cells reflects not only potential problems at the level of ion channel function and accounting for altered calcium handling, but also defects in other proximal or distal events important for stimulus-secretion coupling and the exocytotic process itself.
To identify genes important for the differentiated function of ß-cells, and most notably for normal regulation of insulin secretion, we compared gene expression in B1 vs. C3 cells using three complementary approaches: analysis of candidate gene expression, SSH, and high density microarrays.
Some of the most obvious candidate genes, the low affinity and high capacity glucose transporter GLUT-2 and glucokinase, the enzyme that is considered to be the glucose sensor of ß-cells (18, 19), showed comparable expression in the two sublines. Minami et al. (20) have previously described two sublines (m9 and m14) derived from MIN6 cells that also show different secretory behaviors. Their glucose-responsive m9 cells express higher levels of glucokinase than their nonglucose-responsive m14 cells, and GLUT-2 mRNA was not detected in either subline (20). Although m14 and our own nonresponsive C3 cells both failed to respond to metabolic stimuli such as leucine (or 
-ketoisocaproic acid), the former responded as well as their responsive counterpart m9 cells to IBMX, whereas C3 cells were much less responsive to IBMX than B1 cells. Both m14 and C3 cells have defects in calcium signaling, with a recent study from Minami et al. (17) suggesting that for m14 cells this can be reversed by lowering [Ca2+]i.
Another candidate gene, E-cadherin, was found to be overexpressed in B1 compared with C3 cells. It has been shown that E-cadherin is involved in aggregation and insulin secretion of both MIN6 and pancreatic ß-cells (14, 21, 22, 23). This is in keeping with observed differences in phenotype. In particular, the presence of higher levels of E-cadherin on B1 cells might serve to promote cell-cell aggregation and, in turn, underlie the cell confluence dependence of glucose-stimulated insulin secretion from these cells.
The two complementary approaches, SSH and Affymetrix high density arrays, revealed a number of interesting genes directly involved in insulin secretion [Rab3D (24) and calcium/calmodulin-dependent protein kinase II (25, 26)] or involved in cell adhesion and cytoarchitecture. Nectin-3 has the ability to recruit the E-cadherin/ß-catenin complex at the cell membrane (27). Overexpression of nectin-3 in B1 cells together with that of E-cadherin could be involved in the elevated glucose-stimulated secretion that we observed only at high cell density. Reelin and protocadherins, both involved in cell-cell aggregation in neurons (28), are overexpressed in B1 compared with C3 cells. Overexpression in B1 cells of integrin 6 and the laminin 
2 chain, specific to laminin 5 (29), could provide cell-matrix interactions important in ß-cell secretion (30). The elevated expression of the microtubule-associated tau gene in C3 vs. B1 cells could be implied in the distal defect of C3 cells. Indeed, it has been shown that overexpression of tau inhibits kinesin-dependent trafficking of vesicles along microtubules (31, 32). All of these proteins are particularly interesting because the cytoskeleton is pivotal for membrane vesicle transport and therefore in exocytosis (23, 33, 34, 35). The higher levels of expression of the neurofilament subunits NF-L and NF-M in B1 cells could contribute to secretion in a similar fashion.
Taken together, our results indicate that C3 cells suffer from a complex constellation of defects some, but probably not all, of which combined lead to the altered phenotype compared with B1 cells. On the one hand, the absent or attenuated secretory response to a variety of stimuli in these cells suggests a defect in the availability of granules for exocytosis or in a late common step in exocytosis itself; on the other hand, [Ca2+]i experiments show that there is also a problem at the level of intermediary metabolism and/or KATP channel function. All three approaches used to identify genes possibly responsible for the phenotypic differences revealed differential expression of genes that could be involved at these different levels of insulin secretion and its regulation. It appears that there is a central role for elements of the cytoskeleton, cell-cell communication, and cell-matrix adhesion in the maintenance of normal differentiated function of the pancreatic ß-cell. It will now be necessary to elucidate the precise roles played by the various genes shown to be differentially expressed in one or the other of the MIN6 sublines to form a more complete picture of the integrated protein circuits implicated in regulated insulin secretion.
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Acknowledgments |
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Footnotes |
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Abbreviations: [Ca2+]i, Intracellular Ca2+; IBMX, isobutylmethylxanthine; KATP, ATP-sensitive K+; KRBH, Krebs-Ringer bicarbonate HEPES buffer; MOS, mirror orientation selection; NF-L, neurofilament light polypeptide; NF-M, neurofilament medium polypeptide; PDX-1, pancreatic-duodenum homeobox-1; PMA, phorbol 12-myristate 13-acetate; SSH, suppression subtractive hybridization.
Received September 3, 2002.
Accepted for publication December 30, 2002.
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References |
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in pancreatic ß-cells causes abnormal islet architecture with decreased expression of E-cadherin, reduced ß-cell proliferation, and diabetes. Diabetes 51:114–1236ß1 integrin. Diabetes 49:233–243[Abstract]
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 K. Hamaguchi, N. Utsunomiya, R. Takaki, H. Yoshimatsu, and T. Sakata Cellular Interaction Between Mouse Pancreatic {alpha}-Cell and {beta}-Cell Lines: Possible Contact-Dependent Inhibition of Insulin Secretion Experimental Biology and Medicine, November 1, 2003; 228(10): 1227 - 1233. [Abstract] [Full Text] [PDF]
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) was not stimulated by glucose, whereas secretion of B1 cells (
) significantly increased (P = < 0.006 for all incremental steps) in a concentration-dependent fashion. Data are the mean ± SEM of three independent experiments.
