Metabotropic Glutamate Receptor Type 4 Is Involved in Autoinhibitory Cascade for Glucagon Secretion by α-Cells of Islet of Langerhans

RESEARCH DESIGN AND METHODS

Preparation.

Islets of Langerhans were isolated from male Wistar rats at 7–8 postnatal weeks by the collagenase digestion method combined with discontinuous Ficoll gradient centrifugation (22). Islets were then handpicked and suspended in a bicarbonate-buffered Hank’s solution supplemented with 0.2% BSA. In some experiments, islets (500 pieces) were washed with 20 mmol/l MOPS-Tris (pH. 7.0) containing 0.3 mol/l sucrose, 5 mmol/l EDTA, 5 μg/ml leupeptin, 5 μg/ml chymostatin, 5 μg/ml antipain, and 5 μg/ml pepstatin A and then extensively homogenized. The homogenate was centrifuged at 800g for 10 min, and the resultant supernatant was centrifuged at 140.000g for 1 h.

RT-PCR.

Total RNA extracted from isolated islets (1 μg) was transcribed into cDNA in a final volume of 20 μl of a reaction buffer containing 0.2 mmol/l each deoxynucleotide triphosphate (dNTP), 10 mmol/l dithiothreitol, 25 pmol of random octamers, and 200 units of Moloney murine leukemia virus reverse transcriptase (Amersham). After 1 h incubation at 42°C, the reaction was terminated by heating at 90°C for 5 min. For PCR amplification, the 10-fold–diluted synthesized cDNA solution was added to the reaction buffer containing 0.6 mmol/l total dNTP (150 μmol/l each dNTP), 25 pmol of primers (see Fig. 1A), and 1.5 units of Ampli Taq-Gold DNA polymerase (Perkin Elmer). A total of 35 temperature cycles were conducted, as follows: denaturation at 94°C for 30 s, annealing at a specific temperature shown in Fig. 1A for 30 s, and extension at 72°C for 1 min. The amplification products were analyzed by polyacrylamide gel electrophoresis. The sequences of the oligonucleotides used as primers were based on the published sequences of various mGluRs. DNA sequencing was performed by the chain-termination method (23).

Expression of mGluR4.

Rat mGluR4 cDNA, kindly provided by Dr. S. Nakanishi (24), was subcloned into the EcoRI-XhoI site of expression vector pcDNA3.1 (Invitrogen, San Diego, CA). The resultant construct was used to transfect COS7 cells by lipofection using Trans It reagent (Mirus, Madison, WI). COS7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS as described previously (25). Membrane fraction was prepared as described above.

Antibodies.

Site-specific rabbit polyclonal antibodies against rat mGluR4 were produced as follows: DNA fragments encoding MSGKGGWAWWWARLPLCLLLSLYAPWVPSSLGKPKGHPHMNS (24) were amplified by PCR and cloned into the EcoR1 site of expression vector pGEX3X (Amersham Pharmacia Biotech) to form glutathione S-transferase (GST) fusion plasmids. DNA fragments encoding the NH₂-terminal region of mGluR8, MVCEGKRLASCPCFFLLTAKFYWILTMMQRTHSQEYAH (GenBank accession no. U63288) (26), were also amplified and cloned into pGEX3X as described above. After transformation, the GST fusion proteins were purified on a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech) and then injected into a rabbit with complete adjuvant two times with a 2-week interval. The DNA fragments encoding the above-mentioned polypeptides of the NH₂-terminal region of mGluR4 and -8 were also cloned into the XhoI and EcoRI site of expression vector pBAD/myc-His B (Invitrogen). After transformation of the resultant plasmids, the six His-tagged polypeptides were purified through a Ni-NTA column (Qiagen, Tokyo) according to the manufacturer’s manuals. The mouse monoclonal antibodies against glucagon were from Progen. The rat monoclonal antibodies against somatostatin, mGluR2/3, mGluR5, and mGluR8 were from Chemicon. The guinea pig polyclonal antibodies against insulin and rat pancreatic polypeptide were from Biogenesis and Linco Research, respectively. Alexa Fluor 568–labeled anti-mouse IgG and Alexa Fluor 488–labeled goat anti-rabbit IgG were from Molecular Probes.

Western blotting.

Membrane fraction was suspended in SDS sample buffer and then subjected to SDS gel electrophoresis. Immunoblotting was performed according to the published protocol, with visualization by enhanced chemiluminescence amplification (Amersham Pharmacia Biotech) (4).

Immunohistochemistry.

Indirect immunofluorescence microscopy was performed as described previously, using an Olympus FV-300 confocal laser microscope (25).

Assays.

Isolated islets (20 pieces per assay) or cultured cells (4.0 × 10⁶ cells/dish) were washed three times with DMEM and then incubated in Ringer’s solution containing 10 mmol/l HEPES (pH 7.4), 0.2% BSA, and glucose at the specified concentration for 1 h at 37°C. Then, the islets or cultured cells were transferred to 2 ml of the above Ringer’s solution containing glucose at the specified concentration. At the times indicated, samples (10 μl) were taken, and the amount of glucagon and cAMP was quantified with enzyme immunosorbent assay kits obtained from Amersham Pharmacia Biotech and Yanaihara (Shizuoka, Japan) according to the manufacturers’ manuals. For measurement of cAMP, islets were extensively homogenized with 20 mmol/l MOPS-Tris (pH 7.0) containing 0.3 mol/l sucrose, 5 mmol/l EDTA, 5 μg/ml of leupeptin, 5 μg/ml of chymostatin, 5 μg/ml of antipain, and 5 μg/ml of pepstatin A and then centrifuged at 140.000g for 1 h. The resultant supernatant was used for assay.

Statistics.

Statistical significance between sets of data were assessed using Student’s t test.

Materials.

Agonists and antagonists for GluRs listed in Table 1 were purchased from Tocris Cookson (Avonmouth, U.K.). Other chemicals were of the highest grade commercially available.

RESULTS

Pharmacological evidence for involvement of class III mGluR(s) in the inhibition of glucagon secretion.

As the first step of this study, we investigated whether mGluRs are involved in the regulation of glucagon secretion, using a pharmacological approach (Table 1). l-Glutamate at 1 mmol/l inhibited low-glucose–dependent glucagon secretion, as glucose secreted at 16.7 mmol/l subtracted from that secreted at 3.3 mmol/l glucose, by 87%. l-Glutamate at 100 μmol/l was inhibitory, with 55% less effectiveness than that at 1 mmol/l. l-Glutamate is also inhibitory to the arginine-evoked secretion of glucagon: arginine at 15 mmol/l enhanced secretion of glucagon 2.3-fold at 3.3 mmol/l glucose, and arginine-enhanced secretion of glucagon was inhibited by 79% upon treatment of l-glutamate at 1 mmol/l. PPG and DCPG (agonists for class III mGluRs) (27) at 100 μmol/l also inhibited glucagon secretion by 81 and 73%, respectively. The inhibition was dose dependent: at 40 nmol/l both agonists were not effective, the effectiveness being 50% at 40 μmol/l. At >100 μmol/l, no further inhibition was observed. Moreover, it was found that (1S,3R,4S)-1-aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I; another agonist for class III mGluR) (28) at 50 μmol/l inhibited glucagon secretion by 87%. The l-glutamate–, PPG-, DCPG-, and ACPT-I–evoked inhibition of glucagon secretion was blocked by (R,S)-cyclopropyl-4-phosphonophenylglycine (CPPG), an antagonist for class III mGluRs (29), at 100 μmol/l. Essentially the same glucagon secretion was observed when CPPG itself was included in the assay mixture. (2S,1′S,2′S)-2-(2-carboxycyclopropyl)-2-(9H-xanthen-9-yl)glycine (LY341495) is an antagonist with a K_i value of 57 nmol/l for mGluR8, which is a ∼1,000-fold higher affinity than that for mGluR4 (30). The compound moderately blocked the l-glutamate–evoked inhibition of glucagon secretion at 30 μmol/l, but it did not block inhibition at all at 300 nmol/l. (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG] and quisqualate (specific agonists for class I mGluR) (31,32) at 1 mmol/l or (2S,3S,4S)-2(carboxycyclopropyl)glycine (l-CCG-I) at 100 μmol/l and (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) at 1 mmol/l (agonists for class II mGluRs) (20,33,34) were only slightly effective (Table 1). AMPA and kainate (agonists for ionotropic glutamate receptors) at 0.5 mmol/l also slightly affected glucagon secretion (Table 1). These results suggest that class III mGluRs, most probably mGluR4, are involved in the inhibition of glucagon secretion.

Expression and localization of mGluR4 in islets.

Subsequently, we examined which types of mGluRs are expressed in islets. Among the known mGluRs examined, RT-PCR analysis indicated the amplification of RNA fragments of mGluR4 (Fig. 1). The amplified products covered all predicted sequences. The sequence of the amplified RNA fragment was identical to the corresponding sequence of mGluR4. On the other hand, no amplification of RNA fragments for other mGluRs was observed, although the same probes could amplify the predicted gene fragments from brain sources (Fig. 1). In particular, three different sets of primers for mGluR8 did not give any positive products for islet RNA, although these primers gave amplified products with the expected sizes and sequences for brain RNA (Fig. 1). Essentially the same results were obtained with the various amplification protocols used with an annealing temperature of 60 or 65°C (data not shown). These results led us to conclude that the mGluR4 gene is actually expressed in the islets, but expression of the genes of other mGluRs is negative or under the detection limit of our assay.

Similar RT-PCR analyses were performed using RNA prepared from exocrine pancreas. Fragments of mGluR3 and -5 with the expected sizes and sequences were amplified, whereas no amplification of the mGluR4 gene was observed (Fig. 1). Thus, in contrast with isolated islets, the exocrine part of pancreas shows different expression of mGluRs.

To detect the expression of mGluR4 at the protein level, we prepared site-specific antibodies against mGluR4. The mGluR4 antibodies recognized a 100-kDa polypeptide of authentic mGluR4 expressed in COS7 cells (Fig. 2A, left panel). The immunoreactivity disappeared when antigenic polypeptides (1 mg) were included during the immunoreaction. Dot blot analysis indicated that the mGluR4 antibodies recognized the NH₂-terminal region of mGluR4, but they did not recognize the counterpart of mGluR8 (Fig. 2A, right panel). These results gave credence to the immunological specificity of the mGluR4 antibodies used in the study. The mGluR4 antibodies recognized a single 100-kDa polypeptide in islet membranes as well as brain membrane (Fig. 2B). The immunoreactivity disappeared when antigenic polypeptides (1 mg) were included during the immunoreaction. These results demonstrated the presence of the mGluR4 protein in islets. Double-labeling indirect immunofluorescence microscopy revealed that mGluR4 immunoreactivity is confined to islets but not to exocrine parts of pancreas (Fig. 2C). mGluR4 immunoreactivity is colocalized with glucagon and with pancreatic polypeptide but not with insulin or somatostatin (Fig. 2C). Treatment with antigenic peptides caused the mGluR4 immunoreactivity to disappear (data not shown). These results indicate that mGluR4 is present in α- and F cells but not in β- and δ-cells.

Consistent with the absence of mRNA of other mGluRs in islets, neither antibodies against mGluR2/3 nor those against mGluR5 recognized any polypeptides of islets on immunoblotting, whereas these antibodies recognized counterparts of brain membranes (Fig. 3A). Consistently, anti-mGluR2/3 and -mGluR5 antibodies did not immunostain any islet cells, but they immunostained synaptic terminals and blood vessels in pancreas as well as brain sections (Fig. 3B). As for mGluR8, mGluR8 antibodies did not recognize any polypeptides of islets on immunoblotting (Fig. 3A). After immunohistochemistry, mGluR8 antibodies slightly immunostained islet cells (Fig. 3B), although the immunoreactivity did not disappear even in the presence of antigenic peptides (data not shown). These results indicated that the level of mGluR2/3, mGluR5, or mGluR8 in islets is, if anything, very low or under the detection limit of our assay.

Intracellular signaling pathway for the inhibition of glucagon secretion.

Because mGluR4 is a class III mGluR, its stimulation may inhibit adenylate cyclase through activation of G_i protein, followed by a decrease in a cAMP level, resulting in the inhibition of glucagon secretion. To obtain convincing evidence for functional coupling between mGluR4 and adenylate cyclase, the effects of dibutyryl cAMP (DBcAMP; a nonhydrolyzable cAMP analog) and pertussis toxin (PTX; a specific uncoupler of adenylate cyclase and G_i) were investigated. DBcAMP treatment abolished the glutamate- and PPG- or ACPT-I–evoked decreases in glucagon secretion (Fig. 4). PTX also blocked the glutamate- and PPG- or ACPT-I–dependent decreases in glucagon secretion and cAMP content (Figs. 4 and 5). Because it is known that islet α-cells contain PTX-sensitive G_i protein (35,36), these results strongly suggest that mGluR4 and adenylate cyclase are functionally coupled through G_i in α-cells.

DISCUSSION

In this study, we showed that l-glutamate inhibits glucagon secretion from isolated islets under low-glucose conditions. AMPA and kainate did not stimulate glucagon secretion under low-glucose conditions. These results support our proposal that glutamatergic signaling is involved in the inhibition of glucagon secretion (3,4). Furthermore, l-glutamate–evoked inhibition of glucagon secretion was blocked on treatment with CPPG, indicating possible involvement of class III mGluR in this inhibitory process. We then investigated which mGluR is responsible for the inhibition. We found that mGluR4 is the major mGluR in the islet of Langerhans, and it is involved in l-glutamate–evoked inhibition of glucagon secretion. Four lines of evidence support this conclusion. At first, pharmacological analysis suggested the functional involvement of class III mGluRs in the inhibition of low-glucose–stimulated glucagon secretion. Second, RT-PCR analysis demonstrated the presence of mRNA of mGluR4. Third, we prepared antibodies specific to mGluR4 and demonstrated the presence of the mGluR4 polypeptide in islets. Finally, immunohistochemical analysis indicated that mGluR4 is present in α- and F cells but not δ- and β-cells, supporting the results of our pharmacological analysis.

Our results are partly consistent with previous observations reported by two groups (6,21): no expression of mGluRs was detected in β-cells and clonal β-cells. Brice et al. (12) also detected amplification of a gene fragment of mGluR4 after RT-PCR, although they did not perform immunological identification. They also reported amplification of mRNA fragments of other mGluRs (mGluR3, -5, and -8), although no expression of these mGluRs was observed in our case. At present, we do not know the reason for the apparent discrepancy. It should be stressed, however, that the mGluR3 and -5 genes are expressed in the exocrine parts of pancreas (Figs. 1 and 3). Thus, the presence of acinar cells in an islet preparation may be expected to lead to amplification of the gene fragments of these mGluRs.

More importantly, we could not confirm the expression and localization of mGluR8 in islet α-cells. Even with the same DNA probes for RT-PCR analysis (Fig. 1A) (11), we could not detect any expression of the mGluR8 gene. Although the mGluR8 antibodies slightly immunostained islets, as judged by fluorescent microscopy, the immunoreactivity was observed throughout the islets, especially in β-cells (Fig. 3), and did not disappear when excess amounts of antigenic peptides were included during immunoreaction. Consistent with these observations, Western analysis with mGluR8 antibodies did not reveal any polypeptide from isolated islets (Fig. 3). These results strongly suggest that immunoreaction by mGluR8 antibodies is artificial in nature, and the concentration of mGluR8 protein in α-cells is under the detection limit or very low as compared with that of mGluR4.

In addition, Tong et al. (11) reported that DCPG and PPG at 20 and 40 nmol/l had the maximum inhibitory effect on glucagon secretion. The values are actually too low compared with the above-mentioned abilities of authentic mGluR8 or even authentic mGluR4 of rat (27,28), and they are far from the concentrations required for the inhibition of glucagon secretion (Table 1). We showed that the concentrations giving 50% effectiveness of DCPG and PPG were ∼28 and ∼19 μmol/l, respectively, which are around the K_d value of CPPG with respect to recombinant mGluR4 expressed in cultured cells or rat tissues (37). ACPT-I most potently inhibited glucagon secretion (Table 1). Moreover, LY341495 did not affect the l-glutamate–evoked inhibition of glucagon secretion at 300 nmol/l, a several-fold higher concentration of K_i value for mGluR8 (30). These results further support the involvement of mGluR4 but not mGluR8 in the inhibition of glucagon secretion. Thus, the observation of Tong et al. (11) is incorrect.

Stimulation of mGluR4 causes inhibition of glucagon secretion. mGluR4 is a class III mGluR and is thus believed to be negatively coupled with adenylate cyclase. As expected, the addition of l-glutamate, PPG, or ACPT-I decreased the cellular cAMP level, which was inhibited by CPPG and PTX (Fig. 5). The involvement of DBcAMP also led to the recovery of the PPG-evoked inhibition of secretion of glucagon (Fig. 4). As for the cellular cAMP level, essentially the same responses to l-glutamate and CPPG were observed in αTC6 cells, which are clonal α-cells (S.U., Y.M., unpublished observation). CPPG resulted in enhanced glucagon secretion and cAMP content (Table 1, Fig. 5), suggesting that endogenous l-glutamate released from isolated islets tonically acts through an inhibitory cascade. Although PTX is expected to show the same effect as CPPG, the compound did not enhance either glucagon secretion or cAMP content (Figs. 4 and 5). The reason for the apparent ineffectiveness of PTX is unknown at present but seems to be caused by decreased viable cells during incubation over a long period. Overall, we concluded that signaling pathways from mGluR4 to G_i and adenylate cyclase operate in α-cells. In vivo, the following signaling cascade can be expected to operate: under low-glucose conditions, l-glutamate is secreted by α- or F cells. Then, the released l-glutamate may bind to the mGluR4 on α-cells so as to inhibit glucagon secretion through an inhibitory cAMP cascade. The mGluR4-mediated signaling pathway will provide the molecular basis for chemotherapeutics for hyperglycemia, one of the symptoms for type 2 diabetes.

In conclusion, islet α-cells express functional mGluR4 as a major mGluR. l-Glutamate itself may function as an autocrine transmitter and inhibit glucagon secretion by way of the mGluR4-mediated inhibitory cascade. The glutamatergic signaling also triggers GABA secretion from β-cells, and the released GABA in turn inhibits glucagon secretion by way of the GABAA receptor. Thus, the l-glutamatergic system may inhibit glucagon secretion through two distinct signaling pathways, and it may constitute a novel regulatory mechanism of glucagon secretion in the islet of Langerhans.