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Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie and Interdisziplinäres Stoffwechsel-Centrum (E.G., M.Z.S., C.G., S.M., B.W., U.P.), Endokrinologie, Diabetes, und Stoffwechsel, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, 13353 Berlin, Germany; Department of Animal Physiology and Biochemistry (K.W.N., P.K., M.S.), August Cieszkowski University of Agriculture, 60–637 Poznan, Poland; Klinik für Unfall-, Wiederherstellungs-, und Handchirurgie (B.F.E.-Z.), Philipps-Universität Marburg, D-35032 Marburg, Germany; and Department of Endocrinology (M.T., G.K.S.), Max-Planck-Institut für Psychiatrie, 80804 München, Germany
Address all correspondence and requests for reprints to: Ursula Plöckinger, M.D., Interdisziplinäres Stoffwechsel-Centrum: Endokrinologie, Diabetes, und Stoffwechsel Medizinische Klinik mit Schwerpunkt Hepatologie und Gastroenterologie, Charité-Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. E-mail: ursula.ploeckinger{at}charite.de.
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Introduction |
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Recently evidence has been provided that changes in blood glucose concentration are reflected by OXA plasma concentration (8) as well as the activity of orexinergic neurons (9). In vivo studies in rats revealed that serum concentration of OXA increased during fasting and decreased in the hyperglycemic state (8). Furthermore, it has been demonstrated that administration of OXA increased plasma glucagon concentration in fasted rats (8).
Patients with narcolepsy have a reduced hypothalamic expression of orexin and significantly lower plasma orexin concentration, compared with healthy controls (10). It is known that narcolepsy is associated with metabolic abnormalities, including impaired glucose tolerance (11, 12, 13, 14). These observations in humans and rodents suggest that OXA may play a role in regulating glucose homeostasis and metabolism.
Recently immunohistochemical studies indicated the presence of OXA and OXR1 on pancreatic A and B cells, suggesting that OXA might affect insulin and/or glucagon secretion through a direct receptor-dependent interaction (8). To date, however, no further studies have been published characterizing the interaction of OXA with pancreatic A cells. We therefore investigated the role of OXA in the regulation of pancreatic A cell function and characterized the underlying signal transduction mechanisms.
Using three different biological systems (perfused rat pancreas, isolated pancreatic islets, and a clonal glucagon-producing cell line), we report that OXA modulates glucagon secretion and gene expression via the phosphatidyl-inositol 3-kinase (PI3-kinase)/adenylate-cyclase-kinase (AKT)-dependent pathway, involving the transcription factor forkhead box O-1 (Foxo1).
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Materials and Methods |
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Diagnostics (San Antonio, TX). Cy3-conjugated antigoat secondary antibody was purchased from Dianova (Hamburg, Germany). Antibodies against total AKT, pAkt-Ser, total phosphoinositide-dependent kinase (PDK)-1, phosphorylated (p) PDK-1, total Foxo1, and pFoxo1 as well as antirabbit/horseradish peroxidase-linked antibodies were obtained from Cell Signaling Technology (Beverly, MA). The specific inhibitor of PI3-kinase (LY294002) was from Calbiochem (San Diego, CA). Foxo1 small interfering RNAs (siRNAs) were purchased from QIAGEN Inc. (HP GenomeWide siRNA-SI01005179; official gene name: forkhead box O1, Entrez Gene ID: 56458, target transcript: NM 019739, Hilden, Germany). Rat glucagon RIA was from DPC Biermann (Bad Nauheim, Germany), Insulin ELISA was from Alpco (Windham, NH). The specificity for glucagon was 95%, the limit of sensitivity was 13 pg/ml, and intra- and interassay coefficient of variations were less than 6.5 and 12%, respectively.
Cell culture
InR1-G9 (hamster glucagonoma), INS-1 (rat insulinoma), and CHO-K1 cell lines were obtained from American Type Culture Collection (Rockville, MD) and maintained in RPMI 1640 medium supplemented with 10% (wt/vol) fetal bovine serum, L-glutamine, penicillin (50 µg/ml), and streptomycin (100 µg/ml) (all from Gibco, Karlsruhe, Germany). The cells were grown in a humidified atmosphere containing 5% CO2 at 37 C.
Animals
All animal experiments were approved by the Ethics Committees of the Philipps-University of Marburg, Germany, and the August Cieszkowski University of Poznan, Poland, respectively.
Adult male Wistar rats (180–200 g body weight) and C57BLmice (30–35 g body weight) were obtained from Charles River (Sulzfeld, Germany). The animals were fed chow diet and had water available ad libitum.
In situ perfusion of the rat pancreas
The rats were fasted for approximately 20 h before the experiment. Rats were anesthetized with pentobarbital sodium (60 mg/kg, ip), and the pancreas was dissected and perfused in situ at 37 C, as previously described (15). The perfusion medium consisted of 115 mM NaCl, 4.7 mM KCl, 2.6 mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4 · 7H2O, 24.9 mM NaHCO3, and 20 mM or 2.8 mM D-glucose. The solution was supplemented with 0.5% (wt/vol) Cohn fraction V bovine albumin (Sigma-Aldrich, Munich, Germany) and gassed for 30 min with 95% O2 and 5% CO2 at 37 C immediately before starting the perfusion experiment. Flow rate was maintained at 5 ml/min. After a 35-min equilibration period (perfusion solution with 10 mM D-glucose), glucagon secretion was induced by decreasing glucose concentration in the perfusate from 10–2.8 mM with or without 10 nM OXA. The temperature was kept constant at 37 C. In separate experiments, changes of glucagon and insulin secretion in response to different glucose concentrations (with or without 10 nM OXA) in the perfusate were characterized to monitor the quality of the perfusion experiments. Pancreata were perfused with 2.8 mM glucose followed by 10 mM glucose. In separate experiments, pancreata were perfused continuously with either 10 or 2.8 mM glucose. The stimulation of insulin secretion was induced by switching glucose concentration from 2.8 to 10 mM in the perfusate. The effluent samples were collected from the portal vein, without recycling, at 3-min intervals and frozen at –20 C until the time of assay. The concentration of glucagon and insulin was measured by RIA or ELISA according to the manufacturer’s instructions.
Static incubations of isolated rat pancreatic islets
Pancreatic islets were isolated from adult male rats by injecting liberase RI solution into the main pancreatic duct and processed as described previously (16). Briefly, islets were cultured for 24 h in RPMI 1640 containing 10% fetal calf serum (FCS) and antibiotics. Islets were preincubated for 2 h in serum-free Krebs Ringer buffer (pH 7.4) (10 islets/ml) at 37 C, containing 10 mM glucose. Afterward, islets were washed three times with glucose-free Krebs Ringer buffer. Glucagon secretion was stimulated by incubating the islets with 20 mM [SCAP];l-arginine and 2.8 mM D-glucose (the resulting glucagon concentration was defined as maximal secretion and set to 100%) for 1 h at 37 C. Orexin was present in the incubation medium at the indicated concentrations. The basal secretion was determined from supernatants derived from islets that were only incubated with 10 mM glucose. All experiments were performed in quadruplicates and repeated three times. At the end of the incubations, medium was aspirated and analyzed for the concentration of secreted glucagon.
Glucagon secretion from InR1-G9 cells
The cells were plated in 24-well plates and grown for 3 d under normal conditions. (as described in Cell culture). Before the experiment, cells were kept in serum-free medium for 12 h, followed by preincubation with fetal bovine serum-free medium containing 0.5% BSA and 2 mM glucose for 30 min before incubation with OXA (1 µM to 1 pM) in medium supplemented with 0.5% BSA and 10 mM glucose for various time periods in a total volume of 1 ml. Incubation medium was aspirated at the indicated time points and stored at –80 C for glucagon determination.
Protein preparations and Western blots
For protein isolation, 400 rat and murine pancreatic islets were purified individually by hand picking under a stereomicroscope and extensively washed with PBS. The islets were allowed to recover for 24 h in RPMI 1640 medium with additives. The next day islets were washed extensively again with PBS and processed in the same way as InR1-G9 cells to obtain total protein lysates. InR1-G9 cells were grown in petri dishes in a serum-free medium for 24 h. For total cell lysate, the cells were harvested by scraping in ice-cold lysis buffer [25 mM Tris-HCL (pH 6.8), 1.25% mercaptoethanol, 1% sodium dodecyl sulfate, 5% glycerol, 0.0125% bromophenol blue, and protease inhibitor cocktail (Roche, Mannheim, Germany)]. Proteins were separated on 12% Tris-HCl SDS-PAGE and blotted onto a nitrocellulose membrane (Amersham Biosciences, Freiburg, Germany). Proteins were detected by Ponceau S counterstaining of the membranes. Afterward the membrane was blocked with 5% nonfat dry milk in PBS and 0.2% Tween 20 for 60 min and then incubated with the primary antibody overnight at 4 C. After extensive washing with Tween 20 2% in PBS), the incubation with the secondary antibody followed for 90 min. at room temperature The antibody binding was detected by enhanced chemiluminescence kit (Amersham Biosciences). The antigen-antibody complex was visualized by exposure to a light-sensitive film for 1–10 min. The specificity of the signals detected by OXR1 antibody was confirmed by competing the primary antibody with the blocking peptide (at 10-fold molar excess) against OXR1 before incubating the membranes.
Immunocytochemistry
The cells were plated on coverslips and grown until reaching 80% confluence. They were fixed in 4% buffered paraformaldehyde for 20 min at room temperature and then extensively washed with PBS. Unspecific binding was blocked by incubation in PBS containing 2% nonfat dry milk (Bio-Rad Laboratories, Hercules, CA) for 30 min. Afterward cells were incubated with the primary antibodies overnight at 4 C. For detection, an indocarbocyanine Cy3-coupled secondary antibody (1:400) was added for 45 min followed by extensive washing. After preservation in 96% ethanol, coverslips were mounted with Evanol. Representative staining was captured using an AxioCam camera attached to an Axiosphot microscope (Carl Zeiss Inc., Jena, Germany). The images were merged using Photoshop software (Adobe, San Jose, CA).
Proglucagon gene expression
Cells were plated in 6-well plates and preincubated as described previously (described in Cell culture). On the day of the experiment, the cells were treated with FCS-free medium containing 2 mM glucose and OXA (1 µM to 1 pM) for various time periods in a total volume of 1 ml. Cells incubated without vehicle were used as controls. At the indicated time points, the medium was aspirated and total RNA was isolated using RNeasy minikit (QIAGEN), and then contaminating DNA was removed by using the RNase-Free DNase set (QIAGEN). RNA was quantified by measuring OD at 260 nm.
Quantitative RNA determinations
Total RNA was reversely transcribed to cDNA with oligo-dT primers and SuperScript II (Invitrogen, Karlsruhe, Germany). Real-time quantification of the target gene was performed with a FAM-490 real-time PCR assay using an ICycler PCR machine from Bio-Rad. Forward and reverse primers as well as FAM (6-carboxyfluorescein)-Tamra (6-carboxytetramethylrhodamine) probes for proglucagon were synthesized by Tibmolbiol (Berlin, Germany) (all sequences are given in Table 1
). A total reaction volume of 25 µl was set up, containing 12.5 µl TaqMan Universal PCR master mix (Applied Biosystems, Roche, Branchburg, NJ); 0.75–1.0 µl forward and reverse primers; and 0.2–0.5 µl FAM-labeled probe.
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CT method normalizing cycle threshold (CT) values and normalized against the expression level of β-actin (sequences are given in Table 1
). All samples were amplified in triplicate and the experiment was repeated twice. For negative control, the reaction was performed without reverse transcriptase and/or without cDNA.
Intracellular cAMP accumulation assay
The cells were plated in 96-well plates, grown until reaching 80% confluence, and serum starved overnight before the experiment. After washing steps with serum-free medium containing 1% BSA, the cells were preincubated with 0.5 M 3-isobutyl-1-methylxanthine for 30 min at 37 C. Then the cells were treated with OXA (1 µM to 1 pM) for various time periods and washed with PBS. cAMP measurement was carried out using cAMP Biotrak enzyme immunoassay system (Amersham Biosciences) according to the manufacturer’s instructions.
Determination of intracellular free Ca2+ by fura-2
Changes of cytosolic free Ca2+ were monitored by measuring fura-2 fluorescence. The cells were preincubated in supplement-free culture medium containing 1 µM fura-2-AM for 30 min. Thereafter the cells were washed in a sodium and potassium-free bath solution. Fluorescence measurements were performed at room temperature with a digital imaging system (T.I.L.L. Photonics, Munich, Germany). Fura-2 fluorescence was excited at 340 and 380 nm wavelength and changes in intracellular free Ca2+ concentrations were estimated based on the fluorescence emission ratio at the two excitation wavelengths according to Grynkiewicz et al. (17).
Design of Foxo1 siRNA and transfection of InR1-G9 cells
The Gene Globe design tool (QIAGEN) was used to identify target siRNAs for Foxo1 gene. InR1-G9 cells were transfected with Foxo1 siRNA (target script: NM_019739) using a HiPerFect transfection reagent (QIAGEN) according to the manufacturer’s instructions. Transfection with nonsilencing siRNA was performed as a negative control. The efficiency of gene silencing was detected by real-time RT-PCR and confirmed by Western blot analysis.
Statistical methods
Statistical analysis was performed by the GraphPad Prism software (version 4.0, San Diego, CA) using Student’s unpaired t test. Data are expressed as the mean ± SD. Differences were considered significant at P < 0.05.
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). Perfusion with OXA (10 nM) at 2.8 mM glucose resulted in an inhibition of low glucose-stimulated glucagon secretion (Fig. 1A). Similar effects were observed in the presence of OXB (10 nM; data not shown); however, OXB was a less potent inhibitor of glucagon secretion, compared with OXA. To assure that the pancreas responds to changes from low to high glucose concentration in the perfusion buffer, we perfused the pancreas first with low- and then high-glucose concentration of the perfusate. As expected, glucagon secretion decreased after increasing the glucose concentration in the perfusate from 2.8 to 10 mM, indicating a physiological response of pancreatic A cells to low- and high-glucose concentrations as well as an appropriate technical procedure (Fig. 1B).
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). As expected, insulin secretion increased after increasing the concentration of glucose in the perfusate from 2.8 to 10 mM (Fig. 1C). In contrast to the glucagonostatic activity of OXA, we observed a potentiation of 10 mM glucose-stimulated insulin secretion by OXA (Fig. 1C). In a separate control experiment, OXA failed to induce insulin secretion in the presence of 2.8 mM (data not shown).
The pancreas shows a complex architecture comprising the endocrine and exocrine compartments. To rule out an influence of exocrine pancreatic components on OXA-dependent regulation of A cell secretion, we evaluated the effects of OXA on glucagon secretion from isolated rat pancreatic islets (Fig. 1D). Isolated islets were statically incubated with 20 mM [SCAP];l-arginine at 1 mM glucose to induce glucagon secretion. OXA dose-dependently reduced 1 mM glucose/20 mM arginine-induced glucagon secretion, supporting our observations made in the perfused pancreas model.
Because isolated islets comprise several hormonally active cells that could potentially influence the effects of OXA on glucagon secretion we characterized the direct effects of OXA on glucagon secretion at the cellular level, using the glucagon-producing clonal cell line InR1-G9. In InR1-G9 cells, low (2 mM) or high (10 mM) glucose concentration had only a slight (P > 0.05) but not statistically significant stimulatory effect on glucagon secretion, compared with basal (5 mM glucose) (data not shown). These results indicate that, in contrast to the tight regulation of glucagon secretion from a perfused rat pancreas or isolated islets by glucose, glucagon secretion from InR1-G9 cells is not strictly controlled by glucose. Because glucagon secretion was slightly higher at 10 mM glucose, compared with 2 mM, we investigated the effects of OXA on glucagon secretion at 10 mM glucose. OXA reduced glucagon secretion from InR-G9 cells in a dose- and time-dependent fashion (IC50 value of 8.1 nM) in these cells (Fig. 1, E and F).
Taken together, these results indicate that OXA reduces glucagon secretion and increases insulin secretion. Moreover, we demonstrate that OXA reduces glucagon secretion from InR1-G9 cells as a result of the direct interaction of OXA with these cells.
Expression of OXR1 in pancreatic A cells
Because OXA influenced glucagon secretion, we investigated whether OXR1 is expressed on pancreatic A cells. We identified a single band of 52 kDa in protein lysates prepared from hand-picked and highly purified pancreatic islets of rats and mice by Western blot analysis (Fig. 2A). OXR1 expression was absent in protein lysates prepared from CHO-K1 cells that were used as negative control for Western blots. To exactly characterize the distribution of OXR1, we performed a double immunofluorescence on rat pancreatic sections (Fig. 2B). Consistent with the literature, glucagon immunopositivity (Fig. 2B, green) was located predominantly in the A cell-rich periphery of the pancreatic islets (Fig. 2B, a and c). We detected OXR1 expression in the endocrine as well as the exocrine pancreas (Fig. 2B, b and c red). Within the endocrine pancreas, OXR1 was expressed in both the A cell-rich periphery and the B cell-rich center (Fig. 2Bc). OXR1 colocalized with glucagon-immunoreactive cells (Fig. 2B, c and d). Negative controls, omitting the primary antibody, are given in Fig. 2B, e and f. We did not detect OXR2 expression in the rat endocrine pancreas using both islet cell preparations and frozen rat pancreas sections (data not shown). Using the same antibody as above, we tested the expression of OXR1 in InR1-G9 cells. A single band of the predicted molecular mass of 52 kDa was detected by Western blot (Fig. 2C, upper panel, lane 2). The specificity is demonstrated by competing the band by the immunizing OXR1 peptide antigen (Fig. 2 C, lower panel, lane 2). Protein lysates from the rat pituitary (Fig. 2C, upper panel, lane 1) and INS-1 cells (clonal rat insulin producing cells, lane 3) were used as positive controls, whereas CHO-K1 served as negative controls (Fig. 2C, upper panel, lane 4). The OXR1 signal detected by Western blots using protein lysates prepared from isolated rat pituitaries and INS-1 cells could be competed by the addition of immunizing peptide against OXR1, thereby confirming the specificity of the antibody (Fig. 2C, lower panel, lanes 1 and 3).
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). Negative controls omitting the primary antibody are given in Fig. 2D, a and b. OXR1-immunopositivity was detected predominantly at the plasma membrane compartment; however, the signal was also present intracellular (Fig. 2D). In summary, our data demonstrate the expression of OXR1 in A cells of the endocrine pancreas and the clonal glucagon-producing InR1-G9 cell line.
Effects of OXA on proglucagon gene expression in InR1-G9 cells
Hormone secretion is the net result of hormonal biosynthesis, storage and regulated hormonal release from storage vesicles. Any inhibition of glucagon secretion may thus be due to either the modification of secretory events and/or down-regulation of hormone synthesis, i.e. proglucagon gene expression. To clarify the responsible mechanism we measured proglucagon expression by real-time PCR in InR1-G9 cells, treated with OXA (10 nM) for various time periods. We observed a time-dependent inhibition of proglucagon gene expression in response to OXA treatment (Fig. 3), with the maximal effect observed after 24 h.
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Characterization of signal transduction pathways regulated by OXA
We next studied the intracellular effector molecules regulated by OXA in InR1-G9 cells.
Increased influx of calcium (e.g. due to depolarization) into cells increases intracellular calcium, which can stimulate glucagon gene transcription, biosynthesis, and secretion. These effects are modulated by calcium/calmodulin-dependent protein kinase II, which can phosphorylate the transcription factor cAMP response element-binding protein (CREB) to the glucagon cAMP response element (CRE) of the 5'flanking region of the proglucagon gene promoter. The result is an induction of proglucagon gene transcription. Some of the effects are mediated by calcium response elements or conferred by other transcription factors (e.g. nuclear factor of activated T cells). Enhanced glucagon gene expression is attributed to increased intracellular accumulation of cAMP, which can also act via CREB. OXA significantly reduced intracellular Ca2+ concentration in InR1-G9 cells (Fig. 4A). In addition, OXA dose-dependently decreased the production of cAMP in InR1-G9 cells (Fig. 4B). In agreement with these findings OXA decreased the phosphorylation of CREB (Fig. 4C).
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Because AKT and PDK-1 are two downstream target molecules of the PI3-kinase, we additionally investigated the phosphorylation of AKT and PDK-1 proteins in response to treatment with OXA in InR1-G9 cells. OXA (10 nM) increased the phosphorylation of both AKT and PDK-1 (Fig. 4E). These results suggest an inhibitory effect of OXA on proglucagon gene expression through activation of the PDK-1/AKT signal transduction pathway. The same mechanism has been described for insulin-induced inhibition of glucagon gene expression.
Role of the forkhead homeodomain transcription factor in mediating the effects of OXA on proglucagon gene transcription
One of the downstream targets of the PI3-kinase/AKT pathway is Foxo1, which becomes phosphorylated (inactivated) by AKT. There is increasing evidence for an important role of Foxo1 in glucose metabolism. To investigate whether Foxo1 is involved in the OXA-dependent reduction of proglucagon gene expression in InR1-G9 cells, RNA interference was used. To detect the effectiveness of gene silencing, we performed Western blot analysis for Foxo1 using protein lysate from transfected InR1-G9 cells (Fig. 5A). Silencing of Foxo1 resulted in an increased proglucagon mRNA after exposure to OXA, whereas basal proglucagon expression was not affected (Fig. 5B), as detected by real-time PCR. Furthermore, we provide direct evidence that OXA-induced suppression of the proglucagon gene is mediated via Foxo1 by demonstrating OXA-induced phosphorylation of Foxo1 (Fig. 4E). These data indicate that Foxo1 plays a role in conferring the inhibitory effects of OXA on proglucagon gene expression.
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Discussion |
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OXA has been proposed to act as a satiety factor and as a regulator of energy homeostasis (1, 2, 4). The increased incidence of impaired glucose tolerance or type 2 diabetes in patients with narcolepsy suggests that OXA may play a role in regulating glucose homeostasis and, potentially, the pathophysiology of type 2 diabetes (14). Patients with narcolepsy have a low plasma orexin concentration and decreased expression of hypothalamic orexin (10, 12). Glucose concentration is increased in a substantial number of patients with narcolepsy. The difference in glucose concentration is not related to higher body weight in narcoleptic patients because this is comparable with healthy controls. The differences in the blood glucose levels may be related to the low plasma orexin concentration and the resulting dysregulation of glucagon secretion.
Recent studies demonstrated that orexin receptors are expressed in the endocrine pancreas in humans and rats (18).
Administration of exogenous orexin into rodents influenced insulin secretion; however, inconsistent observations were reported regarding the effects of OXA on insulin secretion (19, 20). In animals with insulin-deficient diabetes, OXA lowered blood glucose levels, but this effect was not due to alterations of plasma insulin levels. We recently demonstrated that OXA stimulated insulin secretion from perfused rat pancreas (19).
To date, there is only a single study available demonstrating the reduction of plasma glucagon after administration of OXA (20). However, it is unknown whether these effects are due to a direct interaction with A cells or result from the interaction with other target tissues of OXA.
Despite the expression of OXRs in the endocrine pancreas, a direct interaction of OXA with glucagon-producing A cells has yet to be demonstrated. Our current data indicate that OXA decreases glucagon secretion. The expression of OXR1 in these cells suggests that the effects are mediated through a direct interaction with this receptor. Three independent experimental settings identified the glucagonostatic activity of OXA in our study: perfused pancreas, statically incubated isolated rat pancreatic islets, and the clonal glucagon-producing cell line InR1-G9.
OXA potently reduced glucagon secretion at low glucose concentration in a model of perfused rat pancreas. Several control experiments, undertaken to evaluate the response of the perfused pancreas to glucose (1 and 10 mM), demonstrated the expected suppression of glucagon secretion by higher glucose concentration in the perfusion buffer. Moreover, insulin secretion increased at 10 mM glucose and decreased at 1 mM glucose, confirming the physiological response of the pancreas to high and low glucose concentration. Importantly, we reproduced our previous observations in perfused rat pancreas (19), showing that OXA potentiates high glucose-stimulated insulin secretion, whereas no effects were observed on nonstimulated insulin secretion. The physiological response of the pancreas to glucose as well as differential response to OXA, as judged by the measurement of insulin and glucagon secretion, indicates a well-preserved condition of the perfused pancreas. These quality controls (physiological response to glucose) allowed us to confirm optimal experimental conditions of the perfusion technique.
The effects of OXA on glucagon secretion were basically reproduced using InR1-G9 cells. However, in contrast to the secretion results obtained with tissues (whole pancreas, isolated pancreatic islets), InR1-G9 cells did not show the typical response of inhibition or stimulation of glucagon secretion at high- or low-glucose concentration, respectively. Similar results have been demonstrated recently when InR1-G9 cells responded paradoxically to changes of the glucose concentration in the incubation medium (21). The highest increase of glucagon secretion was observed at glucose concentrations ranging from 12 to 20 mM. Noteworthy, glucagon secretion at these glucose concentrations was higher, compared with the secretion measured at very low-glucose concentrations. A similar paradox increase of glucagon secretion was also reported for the isolated mouse pancreatic islets; however, the increase was observed only at higher (20 mM) glucose concentration. At 10 mM glucose, the secretion of glucagon was well suppressed. Thus, there obviously is an atypical response to glucose in InR1-G9 cells that was again confirmed in our experimental setting. Despite the lack of a significant physiological increase of the glucagon secretion in response to low-glucose concentration in InR1-G9 cells, OXA was able to reduce glucagon secretion. This glucagonostatic effect is consistent with the observations made on isolated islets and perfused pancreas.
The results of our study using a perfused rat pancreas, isolated pancreatic islets and clonal glucagonoma cells are supported by previous observations in rats. In these animals OXA reduces the glucagon concentration in vivo (20). In contrast to these results, it has been demonstrated that OXA, in high concentrations of 0.8 x 10–7 and 1.0 x 10–7 M, increased glucagon secretion from isolated rat pancreatic islets as well as in vivo (8). There is no clearcut explanation for these discrepant findings. In the study by Quedraogo et al. (8), glucagon immunoreactive material was measured by ELISA with a low sensitivity (detection limit of glucagon immunoreactive material 60–80 pg/ml). Despite the relative low sensitivity, the authors reported that plasma concentration of immunoreactive glucagon in rats increased from 33.25 to 53.5 pg/ml. In contrast, Ehrstrom et al. (20) used a high sensitive RIA (detection limit 3 pg/ml). The range of the glucagon RIA was between 4.5 and 143.6 pmol/liter. The sensitivity of our glucagon RIA (13 pg/ml) compares well with the RIA of Ehrstrom et al. (20). Further differences between the studies were single vs. various different doses of OXA (8, 20). Thus, the discrepant observations regarding the effects of OXA on glucagon secretion could result from different biological systems (in vivo vs. in vitro), methods of glucagon detection (RIA vs. ELISA) and different doses of OXA tested.
Several studies demonstrated the expression of OXR1 in the endocrine pancreas in humans and rodents, suggesting that the effects of OXA are mediated through a direct interaction with OXR1. However, to date the expression of OXR1 in pancreatic A cells has not been demonstrated. We identified the expression of OXR1 in A cells of the rat pancreas and murine islets. The specificity of the antibody OXR1 interaction was confirmed by competing the binding to the islets with an excess of immunizing peptide. Moreover, we demonstrate the expression of OXR1 on glucagon-immunoreactive cells. Consistent with the literature (22), A cells are located in the periphery of rodent pancreas. These cells were OXR1 immunopositive; however, the OXR1 expression was also detected in the B cell-rich center of the islets as well as in the exocrine part of the pancreas. Whereas the function of OXR1 in pancreatic B cells has already been demonstrated, the physiological relevance of OXR1 expression in the exocrine pancreas remains to be further investigated. Consistent with the OXR1 expression in rat pancreatic A cells, InR1-G9 cells, considered a valid surrogate model of glucagon-producing A cells, showed OXR1-expression at both mRNA and protein levels. The OXR1 immunoreactivity was predominantly located at the cell membranes, as expected for this GPCR. Together, these findings indicate that the effects of OXA may be mediated through a specific interaction with the corresponding GPCR.
OXA decreased proglucagon gene expression, as detected by real-time PCR and potently inhibited glucagon secretion from A cells. The cAMP/protein kinase A-dependent pathway has been demonstrated to play a role in regulating glucagon gene expression in InR1-G9 cells (23). OXA binding by OXR1 has been shown to reduce adenylyl-cyclase activity and concomitantly lower the intracellular cAMP concentration in different cell lines (24). In concordance with these data, we found that OXA concentration-dependently reduced cAMP concentration in InR1-G9 cells.
Glucagon secretion and proglucagon gene expression positively correlated with the intracellular Ca2+ concentration. Depolarization increases Ca2+ influx via voltage-dependent calcium channels, which can stimulate glucagon transcription, biosynthesis, and secretion via calmodulin-dependent protein kinase II (25). These effects are mediated via CRE, located within the glucagon promoter and dependent on the transcriptional activation of phosphorylated nuclear factor of activated T cells (26). Consistent with the previously described effects of OXA on intracellular Ca2+ in different cell types (27), we observed a decrease of intracellular Ca2+ in InR1-G9 cells after treatment with OXA. These data suggest that the inhibitory effects of OXA on glucagon secretion/proglucagon gene expression are mediated via a Ca2+-dependent mechanism.
Activation of the PI3-kinase signal transduction pathway, i.e. by insulin, results in decreased proglucagon gene transcription (28). AKT seems to be an important player in this process, as overexpression of protein kinase B (PKB) in InR1-G9 cells can mimic the inhibitory effects of insulin of proglucagon gene transcription (28). OXA administration induced the phosphorylation, i.e. activation of PKB and PDK-1, two downstream target molecules of PI3-kinase. In addition, inhibition of PI3-kinase reduced the OXA-mediated inhibition of proglucagon gene expression. Both results are consistent with the involvement of the PI3-kinase pathway in the reduction of proglucagon gene expression.
As a further novel finding, we identify Foxo1 as a transcription factor mediating OXA effects on proglucagon gene suppression. The involvement of Foxo1 in glucose metabolism has already been demonstrated by showing the Foxo1-dependent effects of insulin on glucose metabolism in response to alterations of the feeding state (29, 30). Foxo1 activity is reduced by insulin-induced phosphorylation of Foxo1 via the PKB/PDK-1 signal transduction pathway (31). Moreover, haplo-insufficiency of Foxo1 in mice increased insulin sensitivity, whereas overexpression of the constitutively active Foxo1 in the liver-induced hyperglycemia, presumably due to increased glucose production (32). These data indicate that Foxo1 is an important subcellular regulator of glucose metabolism. We demonstrated OXA-induced phosphorylation of Foxo1. After silencing of Foxo1 by siRNA, OXA not only failed to inhibit proglucagon gene expression, but it strongly increased proglucagon gene expression in our study. This novel observations suggest that the OXA-induced inhibition of proglucagon gene expression in A cells is mediated through an interaction with the transcription factor Foxo1. This observation is compatible with the results of a very recent study demonstrating the crucial role of Foxo1 in suppressing the proglucagon gene transcription by insulin (33).
Interestingly, after silencing of Foxo1, we observed not only a lack of inhibition of glucagon gene expression but also a rather strong induction. This result was consistently observed in all experiments. We currently have no plausible explanation for this phenomenon, which obviously indicates a switch from inhibitory to stimulatory activity of OXA. Further studies are required to provide the full explanation for this phenomenon.
In summary, our study demonstrate the role for OXA in regulating glucagon secretion from pancreatic A cells using perfused rat pancreas, isolated rat islets and clonal glucagon-producing InR1-G9 cells. In addition to insulin, somatostatin, and glucagon-like peptide-1, we identify with OXA for the first time an additional potent physiological regulator of glucagon secretion and gene expression. We demonstrate that pancreatic A-cells express OXR1 and identify cAMP and Ca2+ as intracellular effector molecules of OXA action. Furthermore, we describe Foxo1 as a transcription factor mediating OXA effects on proglucagon gene suppression.
Hyperglucagonemia along with concomitant hyperglycemia is a hallmark of type 2 diabetes (34, 35). Reasons for hyperglucagonemia are still not well understood. New therapies aiming at the reduction of glucagon secretion in type 2 diabetes could lead to improved glucose control. Thus, the glucagonostatic activity of OXA deserves a further evaluation as a potential principle toward reducing hyperglucagonemia and toward improving glucose control in type 2 diabetes.
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Acknowledgments |
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Footnotes |
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Parts of this paper were presented at the 42nd annual meeting of the European Association for the Study of Diabetes (EASD) 2006 in Copenhagen, Denmark.
Disclosure: E.G., M.Z.S., C.G., K.W.N., P.K., M.S., S.M., B.F.E.-Z., M.T., G.K.S., and B.W. have nothing to disclose. U.P. consults for and received lecture fees from Novartis.
First Published Online December 27, 2007
1 E.G. and M.Z.S. contributed equally to the work. 
Abbreviations: AKT, Adenylate-cyclase-kinase; CREB, cAMP response element-binding protein; FCS, fetal calf serum; Foxo1, forkhead box O-1; GPCR, G-protein coupled receptor; OXA, orexin-A; OXB, orexin-B; OXR1, orexin receptor 1; OXR2, orexin receptor 2; p, phosphorylated; PDK, phosphoinositide-dependent kinase; PI3-kinase, phosphatidyl-inositol 3-kinase; PKB, protein kinase B; siRNA, small interfering RNA.
Received September 14, 2007.
Accepted for publication December 14, 2007.
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