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Departments of Cell and Molecular Biology (S.Y., U.F., L.F., L.S.H., H.M.), and Physiological Sciences (E.R.), Lund University, Lund SE-221 84, Sweden; and Sarah Stedman Center for Nutritional Studies and Duke Program in Diabetes Research (H.E.H.), Departments of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: Hindrik Mulder, M.D., Ph.D, Department of Cell and Molecular Biology, Section for Molecular Signaling, Lund University, Biomedical Center C11, SE-221 84 Lund, Sweden. E-mail: hindrik.mulder{at}medkem.lu.se.
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ABSTRACT |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Given the fact that the 832 cells arise from the same parental cell line (INS-1) (1, 4), it is likely that very distinct genetic and molecular differences may underlie the difference in glucose responsiveness, and as such, these differences may be possible to resolve. In our first screen of insulin secretion (1), we noted that cAMP-mediated insulin secretion was particularly strong, as evidenced by a 25-fold increase in insulin release compared with an unstimulatory glucose concentration. It has long been recognized that cAMP is essential for maintaining primary ß-cells in a glucose-competent state (6), and this may also hold true for clonal ß-cells. Furthermore, cAMP has again been in focus during the last couple of years, because incretin hormones released from the gut, such as glucagon-like peptide 1 (GLP-1), which potentiate GSIS, exert their effects via an increase in cellular cAMP (7). Given this background, we hypothesized that cAMP plays a critical role in the enhanced secretory performance of the 832/13 line. To explore this possibility, a glucose-responsive (832/13) and a glucose-unresponsive (832/2) ß-cell line were compared with regard to cAMP signaling. We found that glucose responsiveness was associated with increased expression of the catalytic subunit of protein kinase A (PKAcat). Pharmacological and molecular interference with cAMP/PKA signaling markedly impaired secretory performance in the glucose-responsive ß-cell line. Our findings emphasize the critical role of cAMP in nutrient- and hormone-stimulated insulin secretion.
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RESULTS |
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, 832/13 cells exhibited a gradual increment in insulin secretion, with a half-maximal response at 12 mM glucose, and attaining an 8-fold maximal response. In contrast, 832/2 cells responded less than 1.5-fold at most. Membrane depolarization, using 35 mM KCl, provoked a 3.5-fold greater response in 832/13 cells compared with 832/2 cells (Fig. 1B). Furthermore, under conditions when the KATP-channel was bypassed (250 µM diazoxide and 35 mM KCl), a rise in glucose from 3–15 mM failed to increase insulin secretion in 832/2 cells, whereas 832/13 cells exhibited a further potentiation of insulin release, indicating an operational KATP-independent pathway of insulin secretion in these cells. Despite this profound difference in GSIS, insulin content of both lines was similar (Fig. 1C), suggesting that the difference in secretory performance is due to differences in stimulus-secretion coupling.
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). Thus, patch-clamped single cells were voltage clamped at –70 mV; after establishing the standard whole-cell configuration at t = 0, exocytosis was evoked by intracellular dialysis with a Ca2+-, ATP-and cAMP-containing patch electrode solution. Although exocytosis was rapidly initiated and continued for several minutes, differences in exocytotic capacity were evident from the onset. Under the conditions employed, the 832/13 cells responded vigorously with an almost 2-fold greater rate of capacitance increase than that exhibited by 832/2 cells (Fig. 2A). Thus, our observations indicate that release of insulin granules is less efficient in 832/2 cells than in 832/13 cells.
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, forskolin potentiated GSIS in 832/13 cells 17-fold compared with secretion at unstimulatory glucose (3 mM); in contrast, forskolin was much less efficient in 832/2 cells, increasing GSIS only 3-fold compared with secretion at unstimulatory glucose (3 mM). Addition of isobutylmethylxanthine (IBMX), an inhibitor of phosphodiesterases that hydrolyze cAMP, was even more efficient than forskolin to potentiate GSIS in 832/13 cells (24-fold; Fig. 3B), emphasizing the role of cAMP in GSIS. Next, during our static incubations of the 832 cells, we included rp-8-bromo (Br)-cAMPS (8-bromoadenosine-3',5'-cyclic monophosphorothioate, rp-isomer), a stable pseudosubstrate for the regulatory subunit of PKA (PKAreg) that inhibits PKA activity. When 832/13 cells were stimulated by 15 mM glucose, rp-8-Br-cAMPS inhibited secretion by 21% (P < 0.01; Fig. 3C); when cells were stimulated by forskolin in addition to glucose, the high concentration of rp-8-Br-cAMPS completely blocked the potentiating effect of forskolin (P < 0.001; Fig. 3C). In 832/2 cells, neither concentration of rp-8-Br-cAMPS exerted a significant effect on insulin secretion in the presence or absence of forskolin (Fig. 3D).
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); when forskolin was added, cAMP levels were increased slightly, but significantly, by 0.7-fold (P < 0.01). 832/2 Cells exhibited moderately elevated basal levels of cAMP (Fig. 3E); when glucose was raised to 15 mM, cAMP levels did not change. In contrast, addition of forskolin to 832/2 cells provoked a dramatic increase in cAMP both at 3 and 15 mM glucose (3.3- and 4-fold, respectively; P < 0.001 for both).
Our findings thus far suggest that 832/13 cells are more sensitive to cAMP than 832/2 cells. To confirm this finding, we performed an additional series of single-cell experiments, again using patch-clamped cells in the whole-cell configuration (Fig. 4). This time, the cells were preloaded with biologically inert caged cAMP that was activated by a UV flash, producing an instantaneous and uniform increase in cytosolic cAMP. Exocytosis was assessed by capacitance measurements before and after release of cAMP. Before release of cAMP, i.e. when exocytosis was stimulated by Ca2+ only, basal rates of capacitance increases were similar in both cell types (16 ± 4 fF/sec vs. 21 ± 3 fF/sec in 832/2 and 832/13 cells, respectively). After photoliberation of cAMP, 832/2 cells did not increase their exocytotic capacity in response to cAMP. In contrast, 832/13 cells exhibited a robust greater than 60% acceleration of exocytosis (21 ± 3 fF/sec vs. 33 ± 4 fF/sec, before and after cAMP liberation, respectively). These observations further indicate that 832/13 cells are more sensitive to changes in cAMP. The greater increments in cAMP in 832/2 cells may represent attempts to adapt to this apparent insensitivity.
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- and ß-isoform of the catalytic subunit of PKA (PKAcat), an immunoreactive band of approximately 40 kDa was seen in the cytosolic fractions of both cell lines (Fig. 5). However, the immunoreactive band was considerably stronger in lysates from 832/13 cells. Accordingly, densitometric analysis showed a 2-fold higher level of PKA expression in 832/13 cells, indicating that the protein is expressed at a higher level in these cells.
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As shown in Fig. 6A, PKAreg expression was appropriately increased by transduction of 832/13 cells with a recombinant adenovirus housing the cDNA of PKAreg. To assess whether such overexpression affects PKA activity, cellular lysates of 832/13 cells were prepared and assayed for activity of the kinase. In a series of pilot experiments, we found that a rise in glucose from 3–15 mM did not increase PKA activity in cellular extracts (data not shown). We found that maximal activation of the kinase in 832/13 cells occurred 10 min after addition of 2.5 µM forskolin and 15 mM glucose. Therefore, we determined the effect of PKAreg overexpression on PKA activity under these conditions. We found that PKA activity was reduced by 22% (P < 0.01) compared with cells transduced with the control virus (AdCMV-ßGal; Fig. 6B). This indicates that the strategy was successful in terms of inhibiting PKA.
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, GSIS was inhibited by 32% (P < 0.01) in PKAreg-overexpressing cells. Moreover, forskolin-potentiated GSIS was blocked by 31% (P < 0.01) in 832/13 cells transduced with AdCMV-PKAreg, whereas basal secretion of insulin was not affected by overexpression of PKAreg. Thus, the molecular manipulation, using AdCMV-PKAreg, agrees with our findings using the PKA-inhibitor rp-8-Br-cAMPS. Clearly, there is a discrepancy between the extent of PKA inhibition in the in vitro experiments (Fig. 6B), and the inhibition of glucose- and forskolin-potentiated insulin secretion (Fig. 6C). This probably reflects that insulin secretion is determined in a functional cell, where compartmentation and targeting of PKAreg are intact, whereas PKA activity was determined in a cell lysate. Nevertheless, the results are in accordance and emphasize the important role of PKA in stimulus-secretion coupling in the glucose-responsive 832/13 line. |
DISCUSSION |
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Using 832/13 cells, we were able to demonstrate that robust KATP-dependent and -independent insulin secretion occurs even when the glucose-induced increase in malonyl-coenzyme A levels is blocked (9). This is a much debated mechanism in ß-cell stimulus-secretion coupling (11, 12), for which we were unable to find experimental support. Recently, we took advantage of the range of glucose responsiveness in our clonal ß-cell lines and compared fluxes through the tricarboxylic acid cycle in the different lines (10). We found that glucose responsiveness was highly correlated with pyruvate cycling, an anaplerotic pathway dependent on separate pools of pyruvate in the cell. This important finding agrees with the prevailing concept of metabolic coupling of stimulus to secretion in the pancreatic ß-cell and implies that a high anaplerotic capacity (i.e. pyruvate cycling) determines high glucose responsiveness in a ß-cell. These studies also indicate that the INS-1-derived 832 lines are appropriate models for the pancreatic ß-cell.
Whereas our previous work shows that metabolic differences between the ß-cell lines account for differences in glucose responsiveness (10), the current work highlights another aspect of stimulus-secretion coupling that determines a robust glucose response in the ß-cell, i.e. cAMP. The role of cAMP in nutrient-induced insulin secretion has long been controversial. Whereas increases in cellular cAMP in response to glucose have been observed in many studies (13, 14), it has been more difficult to link an activation of PKA, the classical effector of cAMP (8), to GSIS (reviewed in Ref. 15). Recently, however, using a different approach, we provided evidence for a critical role of cAMP in GSIS (16). Thus, cAMP levels were reduced in 832/13 cells or rat islets by adenoviral overexpression of the cAMP-hydrolyzing enzyme phosphodiesterase 3B (16, 17). Such reductions caused a significant impairment of GSIS, as well as secretion potentiated by GLP-1, an incretin hormone that increases cellular cAMP. In addition, it has been recognized for some time that a PKA-independent effect of cAMP exists in pancreatic ß-cells (18). This pathway has recently been described on a molecular level (19); it appears that cAMP binds to a guanidine exchange factor (cAMP-GEFII). This complex binds to a Rab3a-interacting molecule, which is a putative regulator of fusion of membrane vesicles to the plasma membrane. When this pathway is blocked, a significant reduction in GLP-1-induced insulin secretion is observed (20); the reduction is additive to that observed by H89, an inhibitor of PKA, suggesting that the effect on GLP-1-induced insulin secretion is PKA independent. At this point, we have yet to examine PKA-independent effects of cAMP in the 832 cells. Although it is likely that this exists also in these cells, we focused here on the role of PKA, based on our findings of an enhanced expression of the kinase.
In the current work, through comparisons of a poorly and robustly glucose-responsive ß-cell line, we identified an important role for cAMP in stimulus-secretion coupling. The poorly glucose-responsive 832/2 line exhibited a blunted exocytotic response despite a marked rise in cAMP upon stimulation by forskolin. In contrast, the robustly glucose-responsive 832/13 line responded vigorously in terms of exocytosis despite a marginal increase in cAMP. This difference could be accounted for by an increase in expression of PKAcat in 832/13 cells. Moreover, when PKA activation was blocked, using a pharmacological agent or a molecular approach, 832/13 cells lost their robust secretory response. Worthy of note is that characterization of glucose-responsive and unresponsive ß-cell lines derived from the MIN6 insulinoma line did not reveal differences in cAMP signaling (2, 3), because both lines were equally responsive to IBMX.
In our experiments, we observed only a marginal increase in cAMP levels in 832/13 cells in response to glucose alone, whereas the response to forskolin was more apparent. Our previous work (16), involving overexpression of phosphodiesterase 3B in these cells, showed that the exocytotic response, assessed by increases in cell capacitance, was decreased by this measure. This observation is most likely explained by a tight association of the esterase and the exocytotic machinery, because the overall cAMP concentration is unlikely to be affected because the cell was continuously dialyzed with cAMP. Another implication of this finding is compartmentation of cAMP signaling in ß-cells; this conceivably explains that a localized increase in cAMP activates PKA, an increase that we were unable to record in a whole-cell lysate. Nevertheless, although it is rather clear that cAMP levels rise in response to cAMP-raising agents, such as incretins, it remains to be shown how the nucleotide responds to changes in ß-cell metabolism. One possibility is that a rise in intracellular Ca2+, triggered by a rise in the ATP:ADP ratio (21), activates production of cAMP, thereby inducing a feed-forward system for glucose to promote the release of insulin.
It was shown recently that the clonal mouse ß-cell line ßTC6 expresses the -, ß-, and 
-isoforms of PKAcat (22). Interestingly, upon glucose- and GLP-1 stimulation, the ß-isoform translocates to the plasma membrane, suggesting that it participates in the exocytotic process. In contrast, the -isoform translocates to nucleus, suggesting a role primarily in regulation of gene transcription, whereas the -isoform of PKAcat does not respond. The antibodies used in our experiments do not recognize the -isoform of PKA and cannot distinguish between the - and ß-isoforms of the kinase. However, judging from the molecular size on the Western blots (
40 kDa), it appears to be the -, rather than the ß-isoform, of the kinase that is most highly expressed in the 832 cells. Whether this has any functional implications remains to be clarified.
Another remaining question is what determines the differential regulation of the cAMP/PKA system in the INS-1-derived ß-cell lines that we have created. Although transcriptional control of the PKAcat ß-gene is incompletely understood, it has been noted that the protooncogene product c-Myc activates transcription of this gene in a number of different cells (23). In fact, this early response gene is induced by glucose in rat islets and purified rat ß-cells (24). Chronic overexpression of c-Myc, however, in transgenic mice results in deranged islet architecture and subsequently diabetes (25). Whether c-Myc is induced in 832/13 cells has not been determined. To address this issue as well as other questions, global characterization of the transcriptome in the 832 lines is currently ongoing. Understanding the genetic machinery that determines glucose responsiveness will be of key importance in the development of novel therapies as well as cells for replacement therapy in diabetes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cell Culture
The clonal ß-cell lines, 832/2 and 832/13, were derived from the INS-1 insulinoma cell line by a transfection-selection strategy, as recently reported by us (1). In brief, parental INS-1 cells were electroporated with a plasmid containing cDNA for human insulin, under control of the cytomegalovirus (CMV) promoter, and the gene for neomycin resistance. After electroporation, the cells were cultured in the presence of G418, yielding 58 discrete colonies. These colonies were expanded, and insulin secretion was examined. Thirty-nine clones (67%) exhibited a foldresponse up to 2 to an increase in glucose from 3–15 mM, 10 clones (17%) responded 2- to 5-fold, and nine clones (16%) responded 5- to 13-fold (1). The cell lines used in this study were selected from the first, unresponsive, category (832/2 cells), and from the last, strongly responsive, category (832/13 cells). The chosen cell lines were cultured in RPMI-1640 containing 11.1 mM D-glucose and supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES, 2 mM glutamine, 1 mM sodium pyruvate, and 50 µM ß-mercaptoethanol, at 37 C in a humidified atmosphere containing 95% air and 5% CO2.
Generation of Recombinant Adenoviruses and Transduction of 832 Cells
The full-length cDNA of the rat type 1 PKAreg (a kind gift from Dr. Susan S. Taylor at the Howard Hughes Medical Institute and the Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA) was subcloned into the EcoRI site of the shuttle plasmid pACCMV.pLpA. A recombinant adenovirus was generated by cotransfection of pACCMV.pLpA-PKAreg and pJM17 in human embryonic kidney 293 cells, as described in detail elsewhere (26). A single-clone virus was isolated after plaque purification, and high-titer viral stocks were prepared by successive amplifications in human embryonic kidney 293 cells.
Confluent 832/13 cells in 12-well dishes were infected with adenovirus expressing PKAreg (AdCMV-PKAreg) in RPMI-1640 medium (1.5 ml) for 2 h, after which 3 ml fresh complete RPMI-1640 medium, containing 5 mM glucose, were added to the cells. The cells were analyzed 18–24 h after infection with respect to insulin secretion and PKA expression and activity. As control, cells were infected by a similar titer of a recombinant adenovirus expressing ß-galactosidase (AdCMV-ßGal).
Insulin Secretion Studies
For assay of insulin secretion, the cells were grown to confluence in 24-well dishes, and the glucose concentration in the culture medium was switched to 5 mM 18 h before assay. When assayed, the cells were washed in HEPES balanced salt solution (HBSS; 114 mM NaCl; 4.7 mM KCl; 1.2 mM KH2PO4; 1.16 mM MgSO4; 20 mM HEPES; 2.5 mM CaCl2; 25.5 mM NaHCO3; 0.2% BSA, pH 7.2) supplemented with 3 mM glucose for 2 h at 37 C. Insulin secretion was then measured by static incubation of the cells for 1 h in 0.8 ml HBSS containing the glucose concentration indicated in the figure legends. When KATP-independent glucose sensing was examined, the K+ concentration in the HBSS during the static incubation was increased to 35 mM, whereas the Na+ concentration was reduced to 89.8 mM, and 250 µM diazoxide was added. Insulin was measured by the Coat-a-Count kit (DPC, Los Angeles, CA), which recognizes human insulin and cross reacts approximately 20% with rat insulin. Insulin content of cells was determined after acid ethanol extraction of the hormone.
Western Blot Analysis
Cells were homogenized in 0.25 M sucrose; 1 mM EDTA, pH 7.0; 1 mM dithioerytritol (DTE); 20 µg/ml leupeptin; 20 µg/ml antipain; and 1 µg/ml pepstatin A. A cytosolic fraction was prepared by centrifugation at 110,000 x g for 45 min at 4 C. Proteins were resolved by SDS-PAGE and electroblotted to nitrocellulose membranes. Western blot analysis was performed by the enhanced chemiluminescence system (SuperSignal ULTRA, Pierce Chemical Co., Rockford, IL), using polyclonal rabbit antihuman PKAcat or PKAreg antibodies (sc-903 and sc 907, respectively; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). In the experiments for quantitation, 5 µg protein (determined by the BCA Protein Assay Kit; Pierce) from each cell line was loaded onto the gel. Expression was determined by densitometric analysis of the Western blots (n = 5 for each line).
Determination of cAMP
For determination of cAMP accumulation, cells were grown to confluence in 12-well dishes in complete RPMI-1640 medium, as described above. The cells were then kept in RPMI-1640 medium containing 5 mM glucose for 12 h, followed by a 2-h incubation in HBSS supplemented with 3 mM glucose. Two minutes after a switch to HBSS containing either 3 or 15 mM glucose with or without 2.5 µM forskolin, cAMP was extracted from cells by adding 0.5 ml 80% ethanol to the cells. cAMP levels were determined after centrifugation of the cellular extracts by RIA (Amersham Pharmacia Biotech, Braunschweig, Germany).
Capacitance Measurements and Flash Photolysis
For electrophysiology (Figs. 2, A and B, and 4), the cells were cultured in 35-mm Nunc petri dishes, and continuously perifused with a preheated (34 C) extracellular solution consisting of (in mM) 138 NaCl, 5.6 KCl, 1.2 MgCl2, 2.6 CaCl2, 3 D-glucose, and 5 HEPES (pH 7.4 with NaOH). Exocytosis was measured as increases in cell membrane capacitance, using an EPC9 amplifier in conjunction with the sine+dc mode of the lock-in amplifier, integrated in the Pulse software suite (v. 8.4 and later; HEKA Elektronik, Lambrecht/Pfalz, Germany). The standard whole-cell configuration of the patch clamp technique was used, and exocytosis was elicited by intracellular dialysis of a Ca2+-containing patch electrode solution composed of (in mM) 125 K-glutamate, 10 KCl, 10 NaCl, 1 MgCl2, 5 HEPES, 3 Mg-ATP, 10 EGTA, 9 CaCl2 (free cytosolic Ca2+2 µM), and 0.1 cAMP. In experiments shown in Fig. 4, cAMP was replaced by its photolabile inactive precursor, caged cAMP (0.2 mM; Molecular Probes, Leiden, The Netherlands), which was liberated during the recording by 2-msec UV flashes, using a XF-10 photolysis apparatus (HiTech Scientific, Salisbury, UK). The efficacy of release was determined to approximately 40% in response to a single UV flash.
PKA Activity
Confluent 832/13 cells in 12-well dishes kept in RPMI-1640 medium were infected with an equal titer of AdCMV-PKAreg or AdCMV-ßGal as described above. The medium was removed, and the cells were washed in HBSS-3 mM glucose and subsequently incubated for 10 min in HBSS containing 15 mM glucose and 2.5 µM forskolin. Then, the cells were washed once in ice-cold PBS and placed on ice, and 150 µl homogenization buffer [50 mM N-[Tris(hydroxymethyl)methyl]-2-aminoethane-sulfonic acid (TES), 1 mM EDTA, 0.1 mM EGTA, 250 µM sucrose; pH 7.4] containing protease inhibitors (1 µg/ml pepstatin, 10 µg/ml leupeptin, 1 µg/ml antipain), phosphatase inhibitor (0.1 µM okadaic acid), and phosphodiesterase inhibitor (1.67 mM IBMX) were added. Homogenates were prepared by aspirating cells through a syringe five times followed by centrifugation at 5000 rpm for 10 min at 4 C. PKA in 10-µl aliquots of the homogenates was allowed to phosphorylate a synthetic peptide substrate (13 µg Kemptide) in a mixture of 39 mM TES, 0.5 M sucrose, 0.1 M MgSO4, 10 mM dithioerytritol, 0.4 mM ATP (pH 7.4), and 15 µCi [32P]ATP; the reactions were carried out in duplicate at 30 C in the presence or absence of protein kinase inhibitor (17 µM). The reaction was terminated after 30 min by addition of 10 µl 1% BSA-1 mM ATP and 10 µl 12.5% trichloroacetic acid and subsequently put on ice for 10 min. The reaction mixture was centrifuged at 3000 rpm for 3 min, after which 10 µl homogenate were transferred to a Whatman P81 membrane. The membrane was washed three times in H3PO4 and once in acetone. Activity was determined by scintillation counting. PKA activity was calculated by subtracting the activity in reactions to which protein kinase inhibitor had been added. In each experiment, PKA activity was expressed as percent of that in controls.
Statistical Analysis
Data are given as mean ± SEM and were, unless otherwise stated, compared with a two-tailed Student’s t test. A probability level of P < 0.05 was considered significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AdCMV, Adenovirus cytomegalovirus; ßGal, ß-galactosidase; GLP-1, glucagon-like peptide 1; GSIS, glucose-stimulated insulin secretion; HBSS, HEPES balanced salt solution; IBMX, isobutylmethylxanthine; PKA, protein kinase A; PKAcat, catalytic subunit of PKA; PKAreg, regulatory subunit of PKA.
Received for publication April 13, 2004. Accepted for publication May 19, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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anomer. FEBS Lett 54:80–83[CrossRef][Medline]
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C), evoked by intracellular dialysis with a Ca2+- and cAMP-containing patch electrode solution, starting when the standard whole-cell configuration was established at the onset of the recording. B, Average exocytotic rates (
