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Originally published In Press as doi:10.1074/jbc.M505560200 on August 29, 2005

J. Biol. Chem., Vol. 280, Issue 44, 36824-36832, November 4, 2005
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Dopamine D2-like Receptors Are Expressed in Pancreatic Beta Cells and Mediate Inhibition of Insulin Secretion*

Blanca Rubí{ddagger}1, Sanda Ljubicic{ddagger}, Shirin Pournourmohammadi{ddagger}, Stefania Carobbio{ddagger}, Mathieu Armanet§, Clarissa Bartley{ddagger}, and Pierre Maechler{ddagger}2

From the {ddagger}Department of Cell Physiology and Metabolism, University Medical Center, and §Cell Isolation and Transplantation Center, Department of Surgery, Geneva University Hospitals, CH-1211 Geneva 4, Switzerland

Received for publication, May 20, 2005 , and in revised form, August 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dopamine signaling is mediated by five cloned receptors, grouped into D1-like (D1 and D5) and D2-like (D2, D3 and D4) families. We identified by reverse transcription-PCR the presence of dopamine receptors from both families in INS-1E insulin-secreting cells as well as in rodent and human isolated islets. D2 receptor expression was confirmed by immunodetection revealing localization on insulin secretory granules of INS-1E and primary rodent and human beta cells. We then tested potential effects mediated by the identified receptors on beta cell function. Dopamine (10 µM) and the D2-like receptor agonist quinpirole (5 µM) inhibited glucose-stimulated insulin secretion tested in several models, i.e. INS-1E beta cells, fluorescence-activated cell-sorted primary rat beta cells, and pancreatic islets of rat, mouse, and human origin. Insulin exocytosis is controlled by metabolism coupled to cytosolic calcium changes. Measurements of glucose-induced mitochondrial hyperpolarization and ATP generation showed that dopamine and D2-like agonists did not inhibit glucose metabolism. On the other hand, dopamine decreased cell membrane depolarization as well as cytosolic calcium increases evoked by glucose stimulation in INS-1E beta cells. These results show for the first time that dopamine receptors are expressed in pancreatic beta cells. Dopamine inhibited glucose-stimulated insulin secretion, an effect that could be ascribed to D2-like receptors. Regarding the molecular mechanisms implicated in dopamine-mediated inhibition of insulin release, our results point to distal steps in metabolism-secretion coupling. Thus, the role played by dopamine in glucose homeostasis might involve dopamine receptors, expressed in pancreatic beta cells, modulating insulin release.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dopamine is a neurotransmitter that plays a critical role in neurological and psychiatric disorders, such as schizophrenia, Parkinson disease, and drug addiction (1). Increasing evidence also shows implication of dopamine in various physiological functions such as cell proliferation (2), gastrointestinal protection (3), and inhibition of prolactin secretion (4). Effects of dopamine on insulin secretion in general and on pancreatic beta cell function in particular have been poorly studied. Insulin exocytosis from the beta cell is primarily controlled by metabolism-secretion coupling. First, glucose equilibrates across the plasma membrane and is phosphorylated by glucokinase, initiating glycolysis (5). Subsequently, mitochondrial metabolism generates ATP, which promotes the closure of ATP-sensitive potassium channels and, as a consequence, depolarization of the plasma membrane (6). This leads to calcium influx through voltage-gated calcium channels and a rise in cytosolic calcium, triggering insulin exocytosis (6, 7). Additional signals participating in the amplifying pathway (8) are necessary to reproduce the sustained secretion elicited by glucose. Insulin secretion evoked by glucose metabolism can be further modulated by parasympathetic and sympathetic neurotransmitters (9).

Treatment with dopamine precursor L-dopa in humans suffering from Parkinson disease reduces insulin secretion upon oral glucose tolerance test (10). In rodents, a single injection with L-dopa results in the accumulation of dopamine in beta cells and inhibition of the insulin secretory responses (11, 12). In isolated islets, analogues of dopamine inhibit glucose-stimulated insulin release (13), whereas one study reports potentiation of insulin secretion upon acute dopamine accumulation (14). Taken as a whole, these previous studies suggest that beta cells might be directly responsive to dopamine. Here, we investigated the molecular mechanisms implicated in beta cell responses to dopamine action. In particular, the present data demonstrate the presence of dopamine receptors in beta cells. Moreover, the inhibitory effects of dopamine are predominantly ascribed to activation of the D2-like receptor family members.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
INS-1E Cells and Pancreatic Islets—INS-1E cells, used as a well differentiated beta cell clone (15), were cultured in a humidified atmosphere containing 5% CO2 in a medium composed of RPMI 1640 supplemented with 10 mM Hepes, 5% (v/v) heat-inactivated fetal calf serum, 2mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 50 µM 2-mercaptoethanol. For rodent islets, Wistar rats or BALB/c mice weighing 200–250 g and 25–30 g, respectively, were obtained from in-house breeding (CMU-Zootechnie, Geneva, Switzerland). We followed the principles of laboratory animal care, and the study was approved by the responsible ethics committee. Pancreatic islets were isolated by collagenase digestion and handpicking from male Wistar rats or BALB/c mice as described previously (16). Isolated islets were cultured free-floating in RPMI 1640 medium before experiments. For human islets, pancreata were harvested from braindead donors and islets isolated after enzymatic ductal perfusion and cultured in non-adherent dishes.

Expression Analysis of Dopamine Receptors in INS-1E Cells and Rat Islets—Total RNA was extracted for RT3-PCR from INS-1E cells and rat islets by the use of Trizol (Invitrogen). Reverse transcription (see TABLES ONE and TWO for the list of oligonucleotides) was performed using 1 µg of total RNA by reverse transcriptase SuperScript II (Invitrogen). The PCR program for cDNA was as follows; initial denaturation step at 94 °C for 2 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at specific temperatures for 30 s, and elongation for 30–45 s at 72 °C. A final elongation step of 7 min at 72 °C allowed extension of truncated product to full-length. PCR reactions were performed using Taq DNA polymerase (Amersham Biosciences) with 1 µM concentrations of each primer. Control experiments demonstrated that amplification products were absent after reactions in the absence of reverse transcriptase.


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  		TABLE ONE
Oligonucleotides used for expression analysis by RT-PCR in INS1-E cells and rat islets

 


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  		TABLE TWO
Oligonucleotides used for expression analysis by RT-PCR in human islets

 
Immunostaining—INS-1E cells were grown on polyornithine-treated glass coverslips for 3 days. For immunofluorescence on rat, mouse, and human islets, islets were isolated and cultured for 24–48 h. The islet cells were then dispersed by trypsinization and plated onto polyornithine-treated glass coverslips (17). Monolayers of INS-1E or islet cells were rinsed twice with phosphate-buffered saline, fixed for 30 min in a 4% paraformaldehyde, and permeabilized for 10 min in 0.1% Triton. Cells were then incubated with primary antibodies, either rabbit anti-dopamine receptor antibody D2 recognizing the third intracellular loop of both short and long isoforms (18) D2L and D2S (1:150, AB5084P, Chemicon, Temecula, CA) or mouse monoclonal anti-human insulin (1:600, I-2018, Sigma-Aldrich) overnight at 4 °C. After rinsing with phosphate-buffered saline, the cells were exposed to the secondary antibodies goat anti-rabbit IgG fluorescein isothiocyanate and goat antimouse rhodamine, respectively, for 1 h at room temperature. Coverslips were mounted on glass slides with Dako, and samples were analyzed using a Zeiss laser confocal microscope (60x, LSM 510, Zurich, Switzerland).

Cell Fractionation and Immunoblotting—INS-1E beta cells were cultured for 4–5 days before cell fractionation. Cells were first washed with phosphate-buffered saline on ice, scraped in 0.32 M sucrose (10 mM Tris-HCl, pH 7.5), and homogenized in a Teflon-glass potter. The resulting suspension was centrifuged for 15 min at 800 g to pull down nuclei, and supernatant was centrifuged for 30 min at 18,000 x g, thereby giving the cytosolic fraction in the supernatant. The corresponding pellet was washed by a second run of suspension and centrifugation. The resulting pellet was resuspended in 2 ml of 0.32 M sucrose before incorporation onto a continuous sucrose gradient made of 5 ml of 1 M sucrose and 5 ml of 2.2 M sucrose at pH 7.5 (10 mM Tris-HCl) before centrifugation for 90 min at 100,000 x g. The visible cell membrane fraction was collected in the supernatant, and the pellet, corresponding to the granule fraction, was subjected to three freeze-thaw cycles using liquid nitrogen. After the Bradford assay for measurements of protein concentrations, fractions (2 µg of proteins per lane) were subjected to SDS/PAGE (10%) before transfer onto nitrocellulose membranes. The membranes were blocked for 1 h with Tris-buffered saline containing 0.1% Tween 20 and 3% (w/v) gelatin protein (Top-Block, Juro, Switzerland). The membranes were washed five times in Trisbuffered saline/Tween at room temperature. Membranes were probed with the rabbit anti-dopamine receptor antibody D2 (L/S) (1:300, AB5084P, Chemicon) overnight at 4 °C before incubation for 2 h at room temperature with anti-rabbit IgG conjugated to horseradish peroxidase (1:5000). The signal was detected by chemiluminescence. As controls, rat brain homogenates were used for detection of D2 receptors, and GLUT2 antibody served in parallel immunoblotting for assessment of cell membrane fraction.

Insulin Secretion—INS-1E cells cultured in 24-well plates (15) were washed twice and preincubated for 30 min at 37 °C in glucose-free Krebs-Ringer bicarbonate HEPES buffer (KRBH) of the following composition: 135 mM NaCl, 3.6 mM KCl, 5 mM NaHCO3, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 1.5 mM CaCl2, and 10 mM HEPES at pH 7.4 containing 0.1% bovine serum albumin. Next, cells were washed once with glucose-free KRBH and then incubated for 30 min in KRBH and test compounds as indicated. At the end of the incubation, supernatants were collected for measurement of insulin release and cellular insulin contents using rat insulin radioimmunoassay (15). Handpicked islets (10 per tube) were incubated over a period of 60 min in KRBH containing secretagogues as indicated, and insulin release was measured by radioimmunoassay (17).

Mitochondrial Membrane Potential and ATP Levels—Mitochondrial membrane potential was measured in INS-1E cells preincubated with 10 µg/ml rhodamine-123 for 20 min at 37 °C in KRBH. Mitochondrial membrane potential was monitored at 37 °C in a plate-reader fluorimeter (Fluostar Optima, BMG Labtechnologies, Offenburg, Germany) with excitation and emission filters set at 485 and 520 nm, respectively. ATP levels were monitored in INS-1E cells expressing the ATP-sensitive bioluminescent probe luciferase after infection with the specific viral constructs (19, 20). Briefly, cells were transduced with AdCA-cLuc, (driving the expression of cytosolic luciferase) and used 20 h later for experiments. Cells were washed with 1 ml of KRBH and incubated for 30 min at 37°C at 2.5 mM Glc. Then, KRBH containing 100 µM luciferin was added, and the luminescence was monitored in the plate-reader luminometer (Fluostar Optima) (15).

Calcium Concentration Measurements—Cytosolic calcium changes were monitored in INS-1E cells preincubated for 90 min with 2 µM Fura-2AM (Teflab, Austin, TX) in KRBH at 37 °C and then washed before the experiment. Ratiometric measurements of Fura-2 fluorescence were performed in a plate-reader fluorimeter (Fluostar Optima) with filters set at 510 nm for excitation and 340/380 nm for emission. For mitochondrial calcium measurements, INS-1E cells cultured in 24-well plates for 3–4 days were transduced with AdCA-mAeq adenovirus, enabling expression of the Ca2+-sensitive photoprotein aequorin targeted to the mitochondria. Twenty hours after viral treatment cells were loaded with the aequorin prosthetic group coelenterazine (2.5 µM) in KRBH for 1 h, and the luminescence was monitored in the plate reader luminometer (15).

Cell Membrane Potential and cAMP Levels—Cell membrane potential was monitored using 100 nM concentrations of the fluorescent probe bis-oxonol (bis-(1,3-diethylthiobarbituric acid) trimethine oxonol) in a thermostatted (37 °C) plate reader fluorimeter. Filters used for excitation and emission had wavelength optima at 544 and 590 nm, respectively (15). For cAMP measurements, INS-1E cells cultured in 24-well plates were exposed to 2.5 or 15 mM glucose for 30 min in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (1 mM) and tested compounds as indicated. At the end of incubation cells were lysed following the manufacturer's instructions, and cAMP quantification was determined by a commercially available enzyme immunoassay from Amersham Biosciences.

Statistical Analyses—Data are presented as the means ± S.E. as indicated. Differences between groups were assessed by Student's t test, and a p < 0.05 was considered as reflecting significant variance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression Analysis of Dopamine Receptors in INS1-E Beta Cells and Rat Islets—RT-PCR revealed the presence of dopamine receptors and dopamine decarboxylase in INS-1E cells and rat islets (Fig. 1). Specifically, clonal INS-1E beta cells expressed D1, D2 short and long isoforms, and D3 and D5 dopamine receptors, whereas rat islets expressed all known dopamine receptors: D1, D2 short and long isoforms, D3, D4, and D5. Amplification of the housekeeping gene (GAPDH) was performed to guarantee integrity of the cDNAs used. PCR reactions in which reverse transcriptase was omitted, as well as PCR reactions without cDNAs, were run as negative controls and gave no amplification product. The oligonucleotides used are listed in TABLE ONE. The sizes (bp) of the amplification products were as expected. Moreover, the specific fragments were cut from the gel and sequenced, confirming the identity of the amplification products.



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  		FIGURE 1.
Expression of dopamine receptors and L-dopa-decarboxylase in INS-1E cells and rat islets. RT-PCR analysis of dopamine receptors D1, D2L, D2S, D3, D4, D5, and L-dopa-decarboxylase (DDC). Lane 1, INS-1E cDNAs; lane 2, INS-1E cDNAs minus RT; lane 3, rat islet cDNAs; lane 4, rat islet cDNAs minus RT; lane 5, rat brain cDNAs; lane 6, rat brain cDNAs minus RT; M, molecular weight markers, 50 bp. GAPDH was amplified to determine integrity of the cDNAs used. The amplification products are indicated by arrows.

 
Immunodetection of Dopamine Receptor D2 in INS-1E Cells and Rodent Islets—We next assessed the presence of D2 receptors at the protein level in beta cells as well as its subcellular localization. INS-1E cells were immunostained with anti-D2 receptor antibody and visualized by fluorescent confocal microscopy. Double labeling experiments showed that dopamine receptor 2 colocalized with insulin-containing secretory granules without significant labeling on the plasma membrane (Fig. 2, A–C). Cell fractionation followed by immunoblotting using INS-1E cells was performed to further question this unexpected localization of the dopamine receptor 2. As shown in Fig. 2D, neither the cytoplasmic nor the plasma membrane fractions contained detectable levels of dopamine receptor 2. On the contrary, a band of ~64 kDa was observed on the granule fraction, corresponding to the upper band of the doublet exhibited by the rat brain extract used here as positive control. Therefore, only the D2 long isoform of the receptor was detected by immunoblotting in INS-1E beta cells. Nonetheless, according to relatively low amounts of proteins loaded on gels, the presence of other isoforms expressed at lower levels cannot be excluded. Quality of the plasma membrane fraction was assessed in parallel immunoblotting by GLUT2 detection (not shown). Double labeling experiments performed on islets of mouse and rat origin confirmed the colocalization of dopamine receptor 2 with insulin-containing secretory granules in primary beta cells (Fig. 3).

Effect of Dopamine on Insulin Secretion in INS-1E Beta Cells—Insulin secretion was tested over a 30-min stimulation period at basal 2.5 mM and stimulatory 15 mM glucose in INS-1E cells in the absence or in the presence of increasing concentrations of dopamine (1, 10, or 100 µM). Results are the means of three experiments performed in quadruplicate. Secretory responses in INS-1E cells stimulated with 15 mM glucose were 3.6-fold versus basal release (p < 0.05). As shown on Fig. 4A, dopamine dose-dependently inhibited glucose-stimulated insulin secretion at 1 µM (-33%), 10 µM (-56%, p < 0.05), and 100 µM (-76%, p < 0.05). Dopamine treatment over the 30-min period tested did not change cellular insulin contents (not shown).

Effect of D2/D3 and D1/D5 Agonists on Glucose-stimulated Insulin Secretion—We next tested if the effect of dopamine on insulin secretion could be mimicked by selected activation of D2 and D5 receptors expressed in the cells. To this end, glucose-stimulated insulin secretion experiments in INS1-E cells were performed in the absence or the presence of D2/D3 agonist quinpirole (21) and D1/D5 agonist SKF38393 (22). Incubation at 15 mM glucose stimulated insulin secretion in control cells 5.4-fold (p < 0.01). Fig. 4B shows that quinpirole (5 µM) inhibited glucose-stimulated insulin secretion by 43% (p < 0.05). D1/D5 agonist SKF38393 (10 µM) also inhibited the secretory response to glucose, although to a lesser extent (-26%, p < 0.01, Fig. 4C). The inhibitory effect of dopamine on insulin secretion was not affected when blocking GABA-A receptors and N-methyl-D-aspartate receptors by 10 µM AP-5 and 10 µM bicuculline (data not shown).

Effects of Dopamine and Quinpirole in Rodent Beta Cells—The following experiments compared the effects of dopamine and quinpirole on insulin secretion in pancreatic islets versus fluorescence-activated cell sorter-purified beta cells isolated from rats. Stimulation of isolated rat islets with 16.7 mM glucose for 30 min increased insulin secretion 4.5-fold (p < 0.0001) versus basal release at 2.8 mM glucose (Fig. 5A). Dopamine (10 µM) inhibited glucose-stimulated insulin secretion by 59% (p < 0.001) at stimulatory glucose concentrations. D2/D3 agonist quinpirole (10 µM) also inhibited glucose-stimulated insulin secretion (-50%, p < 0.05, Fig. 5B). Fluorescence-activated cell sorter-purified beta cells were responsive to glucose-stimulated insulin secretion (6.3-fold versus basal glucose, p < 0.05), and secretory responses were inhibited by the addition of 10 µM quinpirole (-41%, p < 0.05, Fig. 5C). The effects of dopamine and quinpirole were also tested in isolated mouse islets (Fig. 5D). Glucose (22.8 mM) stimulated insulin secretion in control islets (4.2-fold versus basal release, p < 0.01). The secretory responses to stimulatory glucose concentration were inhibited by dopamine (at 10 µM, -73%, p < 0.01) and quinpirole (at 5 µM, -58%, p < 0.05).

Effects of Dopamine and Quinpirole on Mitochondrial Activation— Because mitochondrial activation is mandatory for glucose-stimulated insulin secretion, we tested the effects of dopamine on mitochondrial membrane hyperpolarization induced by 15 mM glucose in INS-1E beta cells. Dopamine (10 µM) exhibited no effects on glucose-induced hyperpolarization of the mitochondrial membrane (Fig. 6A). Respiratory chain activation was not affected by quinpirole (up to 25 µM) as shown in Fig. 6B. Therefore, dopamine and quinpirole, in conditions inhibiting insulin secretion, do not affect glucose-induced mitochondrial hyperpolarization. Cellular ATP levels were monitored in cells expressing cytosolic luciferase. Glucose (15 mM) increased cytosolic ATP in INS-1E cells as expected (Fig. 6C). Dopamine and quinpirole did not inhibit glucose-induced ATP generation. On the contrary, dopamine (10 µM) increased ATP levels at 15 mM glucose (p < 0.05, Fig. 6C), and quinpirole (5 µM) exhibited similar effects (p < 0.05, Fig. 6D).



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  		FIGURE 2.
Immunodetection of D2 receptors in INS-1E cells. A–C, immunostaining on INS-1E cells was performed using the following primary antibodies: monoclonal anti-insulin (red staining, A) and polyclonal anti D2 receptor (green staining, B). C, the overlay indicates the co-localization. D, immunoblotting performed on subcellular INS-1E fractions after sucrose gradient fractionation using polyclonal antibody recognizing both short and long isoforms of D2 receptor, control rat brain (Br), cytoplasmic fraction (Cy), plasma membrane fraction (Me), granule fraction (Gr). MW, molecular weight.

 



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  		FIGURE 3.
Immunostaining of D2 receptor in rat and mouse islets. Immunostaining performed on dispersed islet cells used the primary antibodies monoclonal anti-insulin (red staining) and polyclonal anti D2 receptor (green staining). A–C, rat beta cells. D–F, mouse beta cells. A and D, insulin staining. B and E, D2 receptor staining. C and F, overlay.

 
Effects of Dopamine on Cell Membrane Potential, Ca2+ Levels, and cAMP Generation in INS-1E Cells—As expected, raising glucose from 2.5 to 15 mM induced cell membrane depolarization, as revealed by bisoxonol fluorescence recordings (Fig. 7A). The means of 4 independent experiments indicated inhibition of glucose-induced cell membrane depolarization in the presence of 10 µM dopamine. Elevation of cytosolic Ca2+ concentrations is the consequence of cell membrane depolarization and is required for the secretory response to glucose. Cytosolic Ca2+ changes evoked by 15 mM glucose were monitored in INS-1E cells loaded with Fura2-AM. As shown in Fig. 7B, glucose-induced cytosolic Ca2+ augmentations were reduced in the presence of 10 µM dopamine. Glucose-induced mitochondrial Ca2+ augmentations were monitored in INS-1E cells expressing the Ca2+-sensitive photoprotein aequorin targeted to the mitochondria. Upon 15 mM glucose stimulation, cells exhibited a biphasic increase in mitochondrial Ca2+. First-phase glucose-induced mitochondrial Ca2+ rise, reflecting changes in cytosolic Ca2+, was reduced by 10 µM dopamine (Fig. 7C). Because cAMP renders beta cells more responsive to glucose (9), we also tested such a possible dopamine target by quantifying cAMP in INS-1E cells. As expected, upon a 30-min incubation period in the presence of 1 mM 3-isobutyl-1-methylxanthine, cAMP levels were similar at 2.5 and 15 mM glucose (27.9 ± 1.0 versus 30.3 ± 3.4 fmol/µg protein, respectively; n = 4 in one of three independent experiments). At 15 mM glucose, glucagon-like peptide-1 (10 nM), used as a positive control, raised cAMP levels 7.3-fold (221.5 ± 47.8 fmol/µg of protein, p < 0.001 versus control at 15 mM glucose). Neither dopamine (10 µM) nor D2 agonist quinpirole (5 µM) reduced cAMP levels (45.2 ± 8.5 and 48.7 ± 7.3 fmol/µg of protein, respectively), arguing against cAMP-mediated inhibition by dopamine in beta cells.



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  		FIGURE 4.
Effects of dopamine and D2/D3 or D1/D5 receptor agonists on insulin secretion in INS-1E cells. A, insulin secretion in response to basal 2.5 mM and stimulatory 15 mM glucose (Glc) in INS-1E cells in the absence or in the presence of increasing concentrations (1, 10, or 100 µM) of dopamine (DA) tested over a 30-min stimulation period. Results are the means of three experiments performed in quadruplicate. B, insulin secretion in response to 2.5 and 15 mM glucose in INS-1E cells in the absence or in the presence of 5 µM quinpirole (D2/D3 receptor agonist (Q)) tested over a 30-min stimulation period. Results are the means of five experiments performed in quadruplicate. C, effects of D1/D5 receptor agonist SKF38393 (10 µM (SKF)) on glucose-stimulated insulin secretion. Results are the means of five independent experiments performed in quadruplicate. p < 0.05 (*) and p < 0.01 (**) versus basal Glc; p < 0.05 ({dagger}) and p < 0.01 ({dagger}{dagger}) versus Glc-matched control.

 
Dopamine Receptors in Human Beta Cells—Immunostaining studies revealed the presence of D2 receptors in human beta cells. Dopamine receptor 2 co-localized with insulin (Fig. 8A), in good agreement with observations in rodent beta cells (Fig. 3), showing that dopamine receptor 2 is present on insulin granules. RT-PCR analyses revealed the presence of D1, D2, D4, and D5 receptors as well as dopamine decarboxylase in human islets (Fig. 8B). Amplification of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was performed to guarantee integrity of the cDNAs used. PCR reactions without reverse transcriptase or without cDNAs were run as negative controls. The amplification products were found at the expected size, according to the oligonucleotides used (TABLE TWO). Insulin secretion was stimulated 3.1-fold (p < 0.05) in isolated human islets exposed to 16.7 mM glucose versus basal 2.8 mM glucose. The D2 receptor agonist quinpirole (5 µM) inhibited glucose-stimulated insulin secretion by 46% (p < 0.05, n = 5).



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  		FIGURE 5.
Effects of dopamine and quinpirole (D2/D3 receptor agonist) on insulin secretion in rodent islets. A, effects of 10 µM dopamine on glucose-induced insulin secretion in rat islets. Results are the means of five experiments performed in quadruplicate. B, effects of 10 µM quinpirole on glucose-induced insulin secretion in rat islets. Results are the means of five experiments performed in quadruplicate. C, effects of 10 µM quinpirole on glucose-induced insulin secretion in fluorescence-activated cell sorterpurified rat beta cells. Results are the means of four experiments performed in triplicate. D, effect of 10 µM dopamine and 5 µM quinpirole on glucose-induced insulin secretion in mouse islets. Results are the means of four experiments performed in quadruplicate. p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***) versus basal Glc; p < 0.05 ({dagger}),p < 0.01 ({dagger}{dagger}), and p < 0.001 ({dagger}{dagger}{dagger}) versus Glc-matched control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study provides the basis of molecular mechanisms mediating dopamine inhibition of glucose-stimulated insulin secretion in pancreatic beta cells. Dopamine receptors were present in INS-1E beta cells as well as rat, mouse, and human islets. Dopamine inhibited glucose-stimulated insulin secretion, an effect reproduced by activation of D2-like receptors using the dopamine 2/3 receptor agonist quinpirole. Regarding other dopamine receptors, expression of D1 and D5 correlated with only modest inhibition of secretory responses in the presence of D1/D5 receptor agonist SKF38393. Accordingly, the present study was based primarily on D2-like receptors.



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  		FIGURE 6.
Effects of dopamine and quinpirole on glucose-induced mitochondrial membrane potential and ATP generation. A and B, mitochondrial membrane potential was monitored as rhodamine-123 (Rh-123) fluorescence. A, INS-1E cells were either kept at 2.5 mM glucose (Basal), stimulated with 15 mM glucose (Glc), or Glc with 10 µM dopamine (DA). FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. B, glucose stimulation in the absence or presence of 25 µM quinpirole (Q). After 10 min of glucose stimulation complete depolarization of the mitochondrial membrane was induced by the addition of the protonophore FCCP (1 µM). Traces representative of three experiments were done in triplicate. C, effect of 10 µM dopamine on cellular ATP levels in INS-1E cells stimulated with 15 mM glucose compared with cells maintained at 2.5 mM glucose (Basal). Results are the means of four experiments performed in triplicate (*, p < 0.05 versus Glc-matched control). D, effect of 5 µM quinpirole on glucose-induced ATP increases in INS-1E cells. Results are the means of three experiments performed in triplicate (p < 0.05 (*) and p < 0.01 (**) versus Glc matched control).

 
Immunostaining revealed colocalization of D2 receptors with insulin. This subcellular localization on insulin granules is in accordance with studies performed on other cell types, where D2 receptor was found to be retained intracellularly upon overexpression (23, 24) or localized in secretory vesicles of retinal cells (25). The presence of D2 receptor in insulin granules of primary beta cells might also explain previous observations lacking interpretation. Indeed, dopamine has been shown to accumulate in mouse beta cells upon injection of its precursor L-dopa (11). Our immunoblotting after cell fractionation confirmed the presence of D2 receptors (specifically D2 long isoform) in the granule fraction which were undetectable in the plasma membrane fraction.



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  		FIGURE 7.
Effects of dopamine and quinpirole on cell membrane potential and calcium levels in INS-1E cells. A, cell membrane potential was monitored as bis-oxonol fluorescence in cells at 2.5 mM glucose (Basal), 15 mM glucose (Glc stimul.), and 15 mM glucose plus 10 µM dopamine (Glc+DA, *, p < 0.05 versus Glc stimulation). B, cytosolic calcium concentrations in cells loaded with Fura-2. After 5 min at basal (2.5 mM glucose), cells were challenged with 15 mM glucose alone or with 10 µM dopamine (Glc+DA). Traces are representative of four independent experiments done in triplicate. C, effects of 10 µM dopamine on mitochondrial calcium increases evoked by 15 mM glucose in INS-1E cells expressing the calcium-sensitive photoprotein aequorin targeted to mitochondria. Traces are representative of four independent experiments.

 
What could be the rationale of D2 receptor expression on the secretory granules? Recent dynamic studies on cargo release from secretory vesicles have shown that the rate of emptying of insulin granules is particularly slow after fusion of the membranes, i.e. in the range of seconds (26–28). It is also believed that the fusion pore might be open only transiently, resulting in incomplete release of the cargo, with possible regulatory functions (28). In this context D2 receptors expressed in insulin granules would be activated by extracellular dopamine only at the time as transient pore formation. Such a hypothetical model could possibly provide a new regulatory mechanism for negative feedback, while insulin exocytosis proceeds.

The observed inhibition of insulin secretion by dopamine or quinpirole was not the consequence of impaired metabolism, as glucose-induced mitochondrial membrane hyperpolarization was not affected by these ligands. Upon glucose stimulation, cellular ATP levels were slightly but significantly increased in the presence of dopamine or quinpirole. Because mitochondrial activation was not changed by dopamine, the increase in ATP levels might be the consequence of reduced ATP consumption rather than augmented production. One can hypothesize that the observed reduction of cytosolic Ca2+ concentrations upon glucose stimulation might result in lower ATP consumption by reticulum ATP-dependent Ca2+ pumps. In addition, hyperpolarizing effects of dopamine in beta cells could diminish the activation of the sodium/potassium ATP pump, as demonstrated in renal cells (29). All these results indicate that the increase in glucose-induced ATP generation induced by dopamine could be the consequence of reduced ATP consumption.



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  		FIGURE 8.
Dopamine receptors and L-dopa-decarboxylase expression in human islets. A, D2 receptor immunolocalization in human (h-) islets. Immunostaining was performed using the following primary antibodies: monoclonal anti insulin (red staining) and polyclonal anti D2 receptor (green staining). B, RT-PCR analysis of dopamine receptors (D1, hD1; D2, hD2; D3, hD3; D4, hD4; D5, hD5), L-dopa-decarboxylase (hDDC), and GAPDH in human islets (GAPDH). Lane 1, human islets cDNA; lane 2, human islets cDNA minus RT; lane 3, human brain cDNA; lane 4, human brain cDNA minus RT; C, absence of cDNA; M, molecular weight markers, 50 bp.

 
D2-like receptor activation inhibited glucose-evoked cell membrane depolarization. In neurons, dopamine hyperpolarizes the cell membrane and induces inward rectifier potassium current in voltage-clamp mode, an effect mimicked by the D2 receptor agonist quinpirole (30). Human dopamine D3 and D2L receptors activate G-protein-coupled inward rectifier potassium channels (GIRKs) in mammalian cells (31) and, specifically, dopamine receptor 2 forms stable complexes with Kir3 channels (32). Several GIRK isoforms have been found in human pancreatic islets (33). Moreover, KCNJ9 (GIRK3, member of the GIRK family) is present in the pancreas and was proposed as a candidate gene for type II diabetes mellitus in the Pima Indian population (34). Therefore, one can hypothesize that in beta cells, opening of GIRK3 channels by the action of dopamine on D2-like receptors would abolish glucose-induced cell membrane depolarization.

In beta cells the inhibition of insulin secretion by another catecholamine, namely epinephrine, has been extensively studied over the last two decades, although the precise mechanisms are still not fully understood. Epinephrine is known to inhibit insulin exocytosis at distal steps (35), including G-protein-mediated activation of potassium currents (36), repolarization of the cell membrane (37), and inhibition of Ca2+ influx (38) (see Ref. 39 and references therein). In the present study the inhibitory effects of dopamine and quinpirole on glucose-induced cell Ca2+ increases might be the consequence of the observed reduction in cell membrane depolarization. D2-like receptor activation in beta cells might also directly inhibit L-type voltage-gated Ca2+ channels, as shown in neurons (40, 41). In addition, it was previously reported that dopamine could also modulate hormone secretion independently of changes in cytosolic Ca2+ (42). Accordingly, regulation of insulin secretion by a mechanism downstream of Ca2+ signaling is not excluded and is possibly mediated by direct inhibition of granule exocytosis in beta cells, as dopamine 2 and 3 receptors interact with cytoskeletal proteins (43, 44). Because cAMP reinforces glucose secretory response at a distal step (9), such a parameter was measured, although we observed no reduction in cAMP levels upon dopamine exposure, rendering unlikely such a mechanism for dopamine action in beta cells. Further studies should investigate distal steps in the control of insulin exocytosis as potential targets for dopamine action in beta cells.

Where does dopamine, acting on pancreatic beta cells, come from? The exocrine pancreas is an important source of dopamine, proposed to be implicated in gastrointestinal mucosa protection (3). In the vicinity of beta cells, dopamine might be released from neurons innervating pancreatic islets (14). Dopamine could also be generated in pancreatic islets from its precursor L-dopa. Accordingly, we show here expression of the enzyme controlling dopamine production, namely L-dopa decarboxylase, in INS-1E beta cells and rat and human islets. This supports previously published data showing immunostaining of L-dopa-decarboxylase in pancreatic cells (45, 46) and accumulation of dopamine in beta cells of rodents injected with the substrate for L-dopa-decarboxylase (11, 14).

Modulation of insulin secretion by dopamine might depend on specific receptor-receptor interactions. Indeed, dopamine receptors are able to homodimerize (47, 48) and heterodimerize (49) with members of the same family or with another type of receptors. For instance, somatostatin type 5 receptors, which contribute to the regulation of glucose homeostasis and insulin sensitivity (50), can heterodimerize with D2 receptors, thereby enhancing functional activity (51). Therefore, in the context of insulin secretion, dopamine receptors could interact with somatostatin receptors known to be present in beta cells.

Both dopamine (52, 53) and insulin (54–56) actions in the brain modulate appetite and feeding behaviors. In this work we show for the first time that pancreatic beta cells express dopamine receptors mediating inhibition of glucose-stimulated insulin secretion. One can speculate that changes in dopamine levels might generate multiple responses, allowing coordination of feeding behaviors together with regulation of pancreatic endocrine and exocrine functions. Interestingly, treatment with L-dopa alters insulin secretion in patients with Parkinson disease (10, 57, 58). Moreover, antipsychotic (neuroleptic) drugs blocking dopamine receptors may cause hyperinsulinemia (59), hypoglycemia (60), increase appetite, and obesity (61, 62) and are associated with diabetes (61, 63, 64). Therefore, dopamine action on beta cells might have relevant implications for the study of obesity and diabetes, in particular in situations where dopamine transmission is altered.


   FOOTNOTES
 
* This work was supported by Swiss National Science Foundation Grants 3100-067023 and 3200B0-102134, the Max Cloetta Foundation (Zurich), and the Fondation Endocrinologie (Geneva). This study was part of the Geneva Programme for Metabolic Disorders. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence may be addressed: Dept. of Cell Physiology and Metabolism, University Medical Centre, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. E-mail: rubiweiss{at}yahoo.com. 2 To whom correspondence may be addressed: Dept. of Cell Physiology and Metabolism, University Medical Centre, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. Tel.: 41-22-379-55-54; E-mail: Pierre.Maechler{at}medecine.unige.ch.

3 The abbreviations used are: RT, reverse transcription; KRBH buffer, Krebs-Ringer bicarbonate HEPES buffer; GIRK, G-protein-coupled inward rectifier potassium channel; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Back


   ACKNOWLEDGMENTS
 
We thank Gaelle Chaffard for expert technical assistance, Domenico Bosco, Thierry Berney, and Philippe Halban for a generous supply of beta cell related materials, and Michelangelo Foti for valuable advice on cell fractionation as well as Paolo Meda for gift of antibodies.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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