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Am J Physiol Endocrinol Metab 290: E308-E316, 2006. First published September 27, 2005; doi:10.1152/ajpendo.00131.2005
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Cell type-specific activation of metabolism reveals that {beta}-cell secretion suppresses glucagon release from {alpha}-cells in rat pancreatic islets

Rui Takahashi,1 Hisamitsu Ishihara,1 Akira Tamura,1 Suguru Yamaguchi,1 Takahiro Yamada,1 Daisuke Takei,1 Hideki Katagiri,2 Hitoshi Endou,3 and Yoshitomo Oka1

Divisions of 1Molecular Metabolism and Diabetes and 2Advanced Therapeutics for Metabolic Diseases, Tohoku University Graduate School of Medicine, Sendai, Miyagi; and 3Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Mitaka, Tokyo, Japan

Submitted 22 March 2005 ; accepted in final form 19 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Abnormal glucagon secretion is often associated with diabetes mellitus. However, the mechanisms by which nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by {beta}- or {delta}-cells is among the postulated mechanisms. Herein we present further evidence of the paracrine mechanism. First, to activate cellular metabolism and thus hormone secretion in response to specific secretagogues, we engineered insulinoma INS-1E cells using an adenovirus-mediated expression system. Expression of the Na+-dependent dicarboxylate transporter (NaDC)-1 resulted in 2.5- to 4.6-fold (P < 0.01) increases in insulin secretion in response to various tricarboxylic acid cycle intermediates. Similarly, expression of glycerol kinase (GlyK) increased insulin secretion 3.8- or 4.2-fold (P < 0.01) in response to glycerol or dihydroxyacetone, respectively. This cell engineering method was then modified, using the Cre-loxP switching system, to activate {beta}-cells and non-{beta}-cells separately in rat islets. NaDC-1 expression only in non-{beta}-cells, among which {alpha}-cells are predominant, caused an increase (by 1.8-fold, P < 0.05) in glucagon secretion in response to malate or succinate. However, the increase in glucagon release was prevented when NaDC-1 was expressed in whole islets, i.e., both {beta}-cells and non-{beta}-cells. Similarly, an increase in glucagon release with glycerol was observed when GlyK was expressed only in non-{beta}-cells but not when it was expressed in whole islets. Furthermore, dicarboxylates suppressed basal glucagon secretion by 30% (P < 0.05) when NaDC-1 was expressed only in {beta}-cells. These data demonstrate that glucagon secretion from rat {alpha}-cells depends on {beta}-cell activation and provide insights into the coordinated mechanisms underlying hormone secretion from pancreatic islets.

pancreatic islet; paracrine regulation; glucagon secretion; cell activation

PANCREATIC ISLETS OF LANGERHANS play a central role in glucose homeostasis. In diabetic patients, not only insulin but also glucagon secretion is impaired. Basal levels of serum glucagon are elevated, and a rise in blood glucose fails to inhibit, and can paradoxically even stimulate, glucagon release in subjects with diabetes (14). In addition, the glucagon secretory response is impaired when circulating glucose drops (11), which may result in life-threatening hypoglycemia in patients treated with insulin. This unresponsiveness to hypoglycemia makes precise glycemic management difficult (8), although strict control is known to be essential for preventing diabetic complications (38). Therefore, it is important to understand the mechanisms whereby glucagon secretion is regulated by nutrients.

Three types of regulatory mechanisms have been proposed by which nutrients, such as glucose, suppress glucagon secretion. The first is a direct action of glucose on {alpha}-cells (16, 23). Glucose metabolism in {alpha}-cells is considered to generate signals that inhibit glucagon secretion, whereas glucose metabolism increases insulin secretion in {beta}-cells. Therefore, intracellular signaling arising from glucose metabolism might differ between the two cell types, although {alpha}-cells also express molecules essential for stimulus-secretion coupling in {beta}-cells, including ATP-sensitive K+ (KATP) channels (3, 5). The second mechanism involves modulation by neighboring endocrine cells, such as {beta}- (2, 10, 13, 17, 33, 39) and {delta}-cells (7, 34). Several molecules, including insulin (2, 13, 33), Zn2+ (10, 17), {gamma}-aminobutyric acid (GABA; see Ref. 39), and somatostatin (7, 34), have been postulated to be mediators of these inhibitory effects. Autonomic regulation is the third mechanism (6, 37) and might be clinically important for responses to hypoglycemia, although in humans the glucagon response to hypoglycemia from a transplanted (denerved) pancreas is intact, arguing against this possibility (9).

Studies of stimulus-secretion coupling in {beta}-cells, the predominant cell type of islets, have made great progress in recent decades (20). In contrast, {alpha}-cell research has been hampered because of difficulties in getting sufficient numbers of this cell type. Nonetheless, an earlier study has found important characteristics of {alpha}-cells (32), and several recent studies have discovered interesting features of this cell type. Characterization of electrical activity and calcium dynamics revealed a unique ion channel composition in {alpha}-cells (12). In addition, pyruvate induces glucagon secretion from {alpha}-cells (17) but does not stimulate insulin secretion from {beta}-cells. This is probably because {alpha}-cells have a transporting system for pyruvate but {beta}-cells do not. This observation suggests that metabolized nutrients can induce exocytosis in {alpha}-cells as is the case in {beta}-cells. However, when the pancreas is perfused or islets are stimulated with metabolized nutrients such as glucose, insulin secretion is stimulated, whereas glucagon secretion is suppressed.

In this study, to gain insight into the regulatory mechanism governing glucagon secretion in islets, we have established a method to activate cellular metabolism in {beta}-cells and non-{beta}-cells separately. For this purpose, we have expressed Na+-dependent dicarboxylate transporter (NaDC)-1 or glycerol kinase (GlyK) in {beta}-cells and/or non-{beta}-cells. Using this method, we showed rat {alpha}-cells to secret glucagon when metabolically activated in the absence of {beta}-cell activation. In addition, basal glucagon secretion was shown for the first time to be suppressed by {beta}-cell activation. These data contribute to our understanding of the regulation of islet hormone secretion, providing insights that are anticipated to be of value in managing hypoglycemia and hyperglycemia in subjects with diabetes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of recombinant adenoviruses bearing rat GlyK cDNA (AdRIPHAGlyK and AdCAGlxHAGlyKlx) and NaDC-1 cDNA (AdRIPNaDC and AdCAGlxNaDClx). Rat GlyK cDNA (31) was amplified using rat liver total RNA. An entire coding region was sequenced and subcloned downstream of the hemagglutinin (HA)-epitope sequence. Rat NaDC-1 cDNA was as described previously (36). A SphI-SpeI fragment of HA-tagged GlyK (HAGlyK) cDNA and a SalI-SmaI fragment of NaDC-1 cDNA were ligated between the 410-bp fragment of the rat insulin 1 promoter and the rabbit {beta}-globin poly(A) signal region. The resulting expression units were used for generation of AdRIPHAGlyK and AdRIPNaDC by the methods described previously (27). Rat GlyK and NaDC-1 cDNA were also subcloned between two loxP sequences and ligated under the CAG (a transcriptional unit composed of the cytomegalovirus enhancer, the actin promoter, and the globin intron) promoter unit (28). Recombinant viruses harboring these expression units were then generated (AdCAGlxHAGlyKlx and AdCAGlxNaDClx). AdCAGlacZ (27) expressing {beta}-galactosidase was used as a control adenovirus. AdRIPNCre was renamed from AdInsPNCre generated as described previously (17). Adenovirus titers were measured by the method described previously (27).

Isolation of rat islets and infection with recombinant adenoviruses. Rat islets were prepared by retrograde collagenase infusion through the common bile duct and hand picked under the microscope. Isolated islets were infected with the recombinant adenoviruses at 1.2 x 106 plaque-forming units (PFU)/islet in 1.0 ml medium for 60 min. In the case of combined infection of AdCAGlxNaDClx plus AdRIPNCre or AdCAGlxHAGlyKlx plus AdRIPNCre, the amount of AdRIPNCre was four times greater than the others, with a total amount of 1.2 x 106 PFU/islet.

Immunoblot analysis. INS-1E cells (25) were infected with either AdRIPHAGlyK or AdRIPNaDC at multiplicity of infection (MOI) of 100, cultured for 2 days, and directly dissolved in the SDS sample buffer. Proteins were subjected to SDS-PAGE and were transferred to nitrocellulose membranes. Membranes were probed with rabbit anti-rat NaDC-1 antibody raised against the carboxy-terminal peptide (1:500; see Ref. 36) or with anti-HA tag antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature and then incubated for 1 h with anti-rabbit IgG (1:1,000) conjugated with horseradish peroxidase, respectively. Detection was accomplished with chemiluminescence (ECL; Amersham Biosciences, Piscataway, NJ).

Immunocytochemical analyses. INS-1E cells infected with either AdRIPHAGlyK or AdRIPNaDC at an MOI of 100 were incubated with anti-rat NaDC-1 antibody (1:500) or with anti-HA tag antibody (1:200) for 1 h at room temperature and then incubated for 1 h with FITC-conjugated anti-rabbit IgG (1:500; Jackson ImmunoResearch, West Grove, PA). Islets infected with AdRIPHAGlyK, AdCAGlxHAGlyKlx alone, or AdCAGlxHAGlyKlx plus AdRIPNCre were dispersed on coverslips. Cells were then fixed with 4% paraformaldehyde and incubated with anti-HA tag antibody (1:200) followed by incubation with FITC-conjugated anti-rabbit IgG. Insulin and glucagon were also stained using mouse monoclonal antibodies against these hormones (1:1,000; Sigma-Aldrich, Tokyo, Japan) and Texas red-conjugated anti-mouse IgG (1:500; Jackson ImmunoResearch).

Hormone secretion. INS-1E cells (0.2 x 106 cells/well of 24-well plates) or islets (10 islets/tube) infected with recombinant adenoviruses were incubated over a period of 60 min in 1 ml of Krebs-Ringer-bicarbonate-HEPES buffer [140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 2 mM NaHCO3, 10 mM HEPES (pH 7.4), and 0.1% BSA] containing 2.5 mM glucose plus indicated stimulators. Insulin and glucagon were detected by RIA kits (Linco, St. Louis, MO).

Statistical analyses. Data are presented as means ± SE. Differences between groups were assessed by Student's t-test for unpaired data.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Expression of NaDC-1 resulted in cell activation in response to dicarboxylates. We first sought to establish a means of activating metabolism in specific cell types of pancreatic islets to study 1) the roles of {alpha}-cell nutrient metabolism in glucagon secretion and 2) whether activation of neighboring {beta}-cells in response to nutrient metabolism modulates {alpha}-cell secretion. It was previously shown that {beta}-cells expressing monocarboxylate transporter (MCT-1) metabolize pyruvate and secrete insulin in response to the monocarboxylate (18). Similarly, insulin secretion is reportedly stimulated in {beta}-cells expressing GlyK in response to glycerol (1, 29). These data suggested that cells normally unresponsive to some nutrients can be activated by expressing protein(s) needed for their metabolism. We tested whether tricarboxylic acid (TCA) cycle intermediates alter insulin and glucagon secretion in isolated rat islets and found {alpha}-ketoglutarate, succinate, fumarate, and malate to have no effects on hormone secretion in wild-type islets (Fig. 1). A membrane-permeable analog of succinate, methylsuccinate, is known to stimulate insulin secretion (24), suggesting that inability of TCA cycle intermediates to activate {beta}-cells is attributable to low or no expression of membrane transporters for these compounds. Therefore, to activate cells, a recombinant adenovirus harboring cDNA encoding rat NaDC-1 under the rat insulin promoter (AdRIPNaDC) was constructed, with the aim of activating the cells with TCA cycle intermediates.



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  		Fig. 1. Tricarboxylic acid (TCA) cycle intermediates had no effects on either insulin or glucagon secretion. Isolated islets were cultured overnight and challenged with glucose (20 mM), pyruvate (10 mM), and various TCA cycle intermediates (10 mM). Insulin (A) and glucagon (B) secreted during a 60-min incubation were measured. G2.5, 2.5 mM glucose; G20, 20 mM glucose; Cit, citrate; {alpha}KG, {alpha}-ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Pyr, pyruvate. Data are means ± SE; n = 3~7. *P < 0.05 and **P < 0.01.

 
We first employed rat insulinoma INS-1E cells to study whether the recombinant adenovirus induces functional expression of NaDC-1, leading to increased cellular metabolism and thereby promoting insulin secretion. Western blotting using an antibody against NaDC-1 showed strong expression of NaDC-1, with the expected protein size, in insulinoma INS-1E cells infected with AdRIPNaDC (Fig. 2A). Immunocytochemical analysis revealed strong staining at the cell surface, although weak staining was observed inside the cell, suggesting improper targeting of some expressed membrane proteins because of forced expression (Fig. 2B). As shown in Fig. 2C, adenovirus-mediated expression of NaDC-1 made INS-1E cells responsive to various TCA cycle intermediates. Citrate, one of the tricarboxylates, existing partly in a divalent form at pH 7.4 (36), could be transported into INS-1E cells expressing NaDC-1, and thereby induced insulin secretion. A nonmetabolizable dicarboxylate, glutarate, failed to induce insulin secretion from INS-1E cells expressing NaDC-1, indicating insulin secretion evoked by dicarboxylates to be the result of activation of metabolism. In addition, insulin secretion evoked by malate was abolished by 2 mM NaN3, a metabolic inhibitor (data not shown), further supporting this notion.



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  		Fig. 2. Adenovirus-mediated Na+-dependent dicarboxylate transporter (NaDC)-1 or glycerol kinase (GlyK) expression in INS-1E cells. A: INS-1E cells infected with either AdCAGlacZ (lane 1) or AdRIPNaDC (lane 2) were subjected to SDS-PAGE and probed with an anti-NaDC-1 antibody. B: INS-1E cells infected with either AdCAGlacZ (left) or AdRIPNaDC (right) were stained with anti-NaDC-1 antibody. Bars, 4 µm. C: INS-1E cells infected with either AdCAGlacZ (open bars) or AdRIPNaDC (filled bars) were challenged with 20 mM glucose or various TCA cycle intermediates (10 mM). Insulin secreted during a 60-min incubation was measured. Data are means ± SE; n = 5. **P < 0.01. Glut, glutarate. D: INS-1E cells infected with either AdCAGlacZ (lane 1) or AdRIPHAGlyK (lane 2) were subjected to SDS-PAGE and probed with an anti-hemagglutinin (HA) antibody. E: INS-1E cells infected with either AdCAGlacZ (left) or AdRIPHAGlyK (right) were stained with an anti-HA antibody. Bars, 4 µm. F: INS-1E cells infected with either AdCAGlacZ (open bars) or AdRIPHAGlyK (filled bars) were challenged with 20 mM glucose, 10 mM glycerol (Gly), or 10 mM dihydroxyacetone (DHA). Insulin secreted during a 60-min incubation was measured. Data are means ± SE; n = 4. **P < 0.01.

 
Expression of GlyK resulted in cell activation in response to glycerol. We also expressed rat GlyK to activate the cellular glycolytic pathway. Glycerol and dihydroxyacetone can enter the glycolytic pathway after GlyK-mediated conversion to glycerol 3-phosphate and dihydroxyacetone phosphate, respectively. Adenovirus-mediated introduction of HAGlyK cDNA resulted in expression of this protein in the INS-1E cell cytosol (Fig. 2, D and E). Expressed HAGlyK was functional, since INS-1E cells expressing HAGlyK secreted insulin in response to glycerol or dihydroxyacetone (Fig. 2F), as was reported in INS-1E cells expressing Escherichia coli GlyK (1, 29).

Taken together, these data indicate NaDC-1 and GlyK expressions to be effective in activating cellular metabolism in response to certain nutrients.

Cell type-specific expressions of genes in isolated islets. To study the stimulus-secretion coupling in {alpha}-cells and possible cross-talk with other pancreatic endocrine cells, we next sought to express the genes of interest in {alpha}- and {beta}-cells separately. As was reported previously (17), the rat insulin 1 promoter has high transcription activity and specificity for {beta}-cell-restricted expression of foreign genes. Therefore, {beta}-cell-specific expression of NaDC-1 or HAGlyK was achieved using recombinant adenovirus vectors with the rat insulin 1 promoter (Fig. 3A). When islets were infected with AdRIPHAGlyK, >60% of insulin-positive cells were stained with HA (Fig. 3D), but none of the glucagon-positive cells expressed HAGlyK (Fig. 3E).



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  		Fig. 3. Selective gene expression method for {beta}- and non-{beta}-cells in islets. A–C: schematic representation of adenoviruses for expression in {beta}-cells (A), whole islet cells (B), and non-{beta}-cells (C). NCre, nuclear targeted Cre recombinase; CAG, a transcriptional unit composed of the cytomegalovirus enhancer, the actin promoter, and the globin intron (28). D and E: islets infected with AdRIPHAGlyK were dispersed and stained with an anti-HA (green) antibody (D and E) together with anti-insulin (red; D) or anti-glucagon (red; E) antibody. F and G: islets infected with AdCAGlxHAGlyKlx alone were dispersed and stained with an anti-HA (green) antibody (F and G) together with anti-insulin (red; F) or anti-glucagon (red; G) antibody. H and I: islets infected with AdCAGlxHAGlyKlx plus AdRIPNCre were dispersed and stained with an anti-HA (green) antibody (H and I) together with anti-insulin (red; H) or anti-glucagon (red; I) antibody. Bars, 10 µm. Colocalization resulted in yellow.

 
In contrast to the insulin 1 promoter for {beta}-cells, the glucagon promoter (1.6 kbp) did not have high transcriptional activities specific for {alpha}-cells when placed in the adenoviral genome (data not shown). To increase expression in {alpha}-cells, a dual-adenovirus approach was previously developed (17); one adenovirus produces Cre recombinase under the glucagon promoter, and the other virus expresses the desired genes under the potent CAG promoter unit (28) once the intervening sequence is excised by Cre recombinase (17). Although the strategy increased the expression levels of reporter genes, such as luciferase, in {alpha}-cells, it did not significantly increase the activities of cellular enzymes or transporters, such as glucokinase and MCT-1 (data not shown). We then raised the recombinant virus titer. However, a high titer of virus with the glucagon promoter also induced expression in cells other than {alpha}-cells (data not shown). We therefore did not employ the glucagon promoter in this study but rather devised a method employing the insulin 1 promoter and the Cre-loxP system.

As shown in Fig. 3C, a cDNA floxed with loxP sequences was placed downstream from the CAG promoter unit (28) that enables transcription in any cell type. This expression unit was then introduced into islet cells, together with the insulin promoter-Cre adenovirus (AdRIPNCre; see Ref. 17). The cDNA was expected to be removed from the unit by the Cre recombinase in the {beta}-cell, allowing expression of the genes of interest in non-{beta}-cells, a cell population where {alpha}-cells are predominant. Indeed, when rat islets were infected with AdCAGlxHAGlyKlx and AdRIPNCre, ~70% of glucagon-positive cells was stained with HA (Fig. 3, H and I). More than 80% of HA-positive cells were observed to be stained with glucagon, and <10% were insulin positive, although HA staining was occasionally observed in somatostatin-positive cells and fibroblast-like cells (data not shown). When islets were infected with AdCAGlxHAGlyKlx alone (Fig. 3B), ~60% of {beta}-cells (Fig. 3F) and 65% of {alpha}-cells (Fig. 3G) expressed HAGlyK.

{alpha}-Cell activation triggered glucagon secretion when {beta}-cells remained nonactivated. To study the role of nutrient metabolism in glucagon secretion from {alpha}-cells, isolated rat islets were infected with AdCAGlxNaDClx plus AdRIPNCre and challenged with succinate or malate. As shown in Fig. 4, A and B, glucagon secretion was increased by 80%, without changes in insulin secretion. These effects were abolished by 2 mM NaN3, indicating the observed glucagon secretion to be due to activation of cellular metabolism of the dicarboxylates. When islets were infected with AdCAGlxHAGlyKlx plus AdRIPNCre and then challenged with 10 mM glycerol, insulin secretion did not change (Fig. 4C) and glucagon secretion tended to increase, but the differences did not reach statistical significance (Fig. 4D).



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  		Fig. 4. Selective {alpha}-cell activation induced glucagon secretion. A and B: isolated islets (10 islets/tube) infected with AdCAGlacZ plus AdRIPNCre (open bars) or AdCAGlxNaDClx plus AdRIPNCre (filled bars) were challenged with 20 mM glucose, 10 mM malate (Mal10), or 10 mM succinate (Suc10) with or without 2 mM NaN3. Insulin (A) and glucagon (B) secreted during a 60-min incubation were measured; n = 3~5. *P < 0.05. C and D: isolated islets (10 islets/tube) infected with AdCAGlacZ plus AdRIPNCre (open bars) or AdCAGlxHAGlyKlx plus AdRIPNCre (filled bars) were challenged with 20 mM glucose, or 10 mM glycerol (Gly10). Insulin (C) and glucagon (D) secreted during a 60-min incubation were measured; n = 4.

 
In contrast, when {alpha}-cells were activated together with {beta}-cells by infecting islets with AdCAGlxNaDClx alone, i.e., without AdRIPNCre (Fig. 5, A and B), insulin secretion was increased by more than threefold in response to 10 mM malate or succinate, whereas glucagon release was unchanged. Similarly, AdCAGlxHAGlyKlx infection increased insulin (by 2.5-fold) but not glucagon secretion with a 10 mM glycerol challenge (Fig. 5, C and D). These data indicate that {alpha}-cell activation dose not lead to glucagon secretion when {beta}-cells are activated simultaneously.



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  		Fig. 5. Simultaneous {alpha}- and {beta}-cell activation induced insulin but not glucagon secretion. A and B: isolated islets (10 islets/tube) infected with AdCAGlacZ (open bars) or AdCAGlxNaDClx (filled bars) were challenged with 20 mM glucose, 10 mM malate, or 10 mM succinate. Insulin (A) and glucagon (B) secreted during a 60-min incubation were measured; n = 4~6. *P < 0.05 and **P < 0.01. C and D: isolated islets (10 islets/tube) infected with AdCAGlacZ (open bars) or AdCAGlxHAGlyKlx (filled bars) were challenged with 20 mM glucose, or 10 mM glycerol. Insulin (C) and glucagon (D) secreted during a 60-min incubation were measured; n = 4. *P < 0.05.

 
Recent studies have postulated several molecules, including insulin (2, 13, 33), Zn2+ (10, 17), and GABA (39), as mediators of {beta}-cell inhibitory effects on glucagon secretion. During succinate stimulation, insulin secreted from {beta}-cells expressing NaDC-1 amounted to ~25 ng/ml (Fig. 5A). We therefore examined whether this amount of insulin inhibits glucagon secretion from islets expressing NaDC-1 in {alpha}- but not {beta}-cells. As shown in Fig. 6, succinate-stimulated glucagon secretion from islets infected with AdCAGlxNaDClx plus AdRIPNCre was significantly suppressed by 25 ng/ml insulin, indicating that insulin mediates inhibitory effects of {beta}-cells on glucagon secretion. However, the suppression seemed incomplete, suggesting another molecule(s) is also important in suppression of glucagon secretion by {beta}-cell activation.



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  		Fig. 6. Insulin suppressed succinate-stimulated glucagon secretion. Isolated islets (10 islets/tube) infected with AdCAGlacZ plus AdRIPNCre (open bars) or AdCAGlxNaDClx plus AdRIPNCre (filled bars) were challenged with 20 mM glucose, 10 mM succinate alone, or 10 mM succinate with 25 ng/ml insulin. Glucagon secreted during a 60-min incubation was measured; n = 4. *P < 0.05.

 
{beta}-Cell activation increased insulin secretion and decreased basal glucagon secretion. We next studied whether basal glucagon secretion was altered by {beta}-cell activation. For this purpose, NaDC-1 was expressed only in {beta}-cells by infecting islets with AdRIPNaDC. When these islets were challenged with 10 mM malate or succinate, insulin secretion more than doubled (Fig. 7A). Interestingly, glucagon secretion from the same islets was reduced significantly, by 30% (Fig. 7B). Similar modulation of hormone secretion was observed when islets were infected with AdRIPGlyK; insulin secretion was increased significantly, by 1.7-fold, in response to 10 mM glycerol (Fig. 7C), whereas glucagon secretion tended to decrease, although not to a statistically significant degree (Fig. 7D).



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  		Fig. 7. Selective {beta}-cell activation inhibited glucagon secretion. A and B: isolated islets (10 islets/tube) infected with AdCAGlacZ (open bars) or AdRIPNaDC (filled bars) were challenged with 20 mM glucose, 10 mM malate, or 10 mM succinate. Insulin (A) and glucagon (B) secreted during a 60-min incubation were measured. *P < 0.05; n = 5. C and D: isolated islets (10 islets/tube) infected with AdCAGlacZ (open bars) or AdRIPHAGlyK (filled bars) were challenged with 20 mM glucose or 10 mM glycerol. Insulin (C) and glucagon (D) secreted during a 60-min incubation were measured. *P < 0.05; n = 4.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Abnormal nutrient-mediated modulation of glucagon secretion is often associated with diabetes mellitus. However, the mechanisms whereby nutrients modulate glucagon secretion remain poorly understood. Paracrine modulation by {beta}- or {delta}-cells is among the postulated mechanisms. Herein we provide further evidence that glucagon secretion from {alpha}-cells is stimulated by nutrient metabolism in the absence of {beta}-cell activation but it is suppressed when {beta}-cells are activated.

We first demonstrated, employing NaDC-1 expression, that TCA cycle intermediates induce insulin secretion from {beta}-cells engineered to transport these substrates. It was previously reported that pyruvate and lactate stimulate insulin secretion from {beta}-cells expressing MCT-1 and lactate dehydrogenase (LDH) but not from normal {beta}-cells (18). This was interpreted as low levels of MCT-1 and LDH expression protecting {beta}-cells from the stimulatory effects of pyruvate and lactate, which could otherwise cause undesired insulin secretion in catabolic states, such as during exercise. Similarly, the present data suggest that low levels of NaDC-1 expression protect {beta}-cells from the stimulatory effects of dicarboxylates and confer glucose selectivity on insulin secretion.

A major part of the present study was based on the method we devised to activate cellular metabolism in {beta}-cells and non-{beta}-cells, separately, in primary rat islets by specifically expressing NaDC-1 or GlyK in {beta}-cells and non-{beta}-cells. Employing this innovative approach, we showed TCA cycle intermediates, succinate and malate, to induce glucagon secretion when NaDC-1 is expressed in {alpha}-cells. Stimulation of glucagon secretion was previously demonstrated in intact islets challenged with another mitochondrial substrate, pyruvate, which exerts essentially no stimulatory effects on {beta}-cells (17). In subjects with type 1 diabetes, glucose reportedly failed to suppress, or even slightly stimulated, glucagon secretion (14). Abnormal glucagon secretion in response to glucose was also reported in islets from insulin-deficient Chinese hamsters (19). The present data, obtained employing NaDC-1 expression, thus support the concept of {alpha}-cells having an inherent capacity to increase glucagon secretion in response to nutrients under certain circumstances, i.e., in the absence of {beta}-cell effects. GlyK expression in {alpha}-cells tended to increase glucagon secretion in response to glycerol, but not to a statistically significant degree (Fig. 3D). This might be because the coupling of glycolysis and mitochondrial metabolism is less efficient in {alpha}-cells than in {beta}-cells, as previously suggested (35). In contrast, dicarboxylates directly stimulate mitochondrial metabolism in cells expressing NaDC-1, thereby possibly producing a significant increase in glucagon secretion.

The concept of {alpha}-cells having an inherent capacity to increase glucagon secretion in response to nutrients has been reinforced recently by the demonstration of glucagon secretion stimulated by glucose from purified rat {alpha}-cells (10). An earlier study (32), however, reported that glucose inhibited glucagon secretion induced by an amino acid mixture in purified {alpha}-cells. Thus direct action on {alpha}-cells could be multiple, both inhibitory and stimulatory in nature. Glucose reportedly promotes the filling of the endoplasmic reticulum Ca2+ stores in {alpha}-cells (23) as in {beta}-cells (20). In the presence of an amino acid mixture, glucose inhibitory effects could attenuate the rise in cytosolic Ca2+ induced by amino acids, whereas glucose stimulatory effects could be masked by amino acid-stimulated Ca2+ elevation.

Glucagon secretion stimulated by pyruvate was previously shown to be suppressed by activation of {beta}-cells expressing MCT-1 (17). Similar inhibition of activated glucagon secretion by {beta}-cell secretory activities was recently reported in {beta}-cell-specific Foxa2 knockout mice (22). Islets from these mice secreted insulin in response to an amino acid mixture, and, interestingly, the glucagon secretion that is normally seen in the wild-type islets in response to amino acids was abolished in the mutant islets. This result is consistent with the notion that suppression of activated glucagon secretion is attributable to {beta}-cell secretory activities. In the present study, for the first time, we have shown basal glucagon secretion to also be suppressed by {beta}-cell activation. In addition, in NaDC-1-expressing cells, glucose stimulated insulin secretion more potently (an ~4.5-fold increase) than dicarboxylates (an ~2-fold increase; Fig. 7A), whereas glucose and dicarboxylates suppressed glucagon secretion to a similar extent (~30%). We speculated that this is because, when islets were challenged with glucose, {alpha}-cells were also activated for glucagon secretion, which counteracted the suppressing effect exerted by {beta}-cell secretory activities. Recent studies demonstrated that insulin (2, 13, 33), Zn2+ (10, 17), and GABA (39) are candidates for {beta}-cell-derived inhibitory substances of glucagon secretion in rat islets. Our observation of inhibitory effects of insulin on succinate-stimulated glucagon secretion from islets expressing NaDC-1 in {alpha}- but not {beta}-cells supports this notion about the role of insulin. To study roles of Zn2+ and GABA, it is crucial to determine amounts of these molecules secreted from {beta}-cells during nutrient stimulation. Further studies are needed to elucidate the molecular basis of {beta}-cell inhibitory effects.

Glucagon secretion was reported to depend differentially on Ca2+ influx through N- and L-type Ca2+ channels (12, 16). N-type Ca2+ channels operate predominantly under basal conditions and L-type Ca2+ channels in the stimulated state. {beta}-Cell activation suppressed glucagon secretion regardless of whether {alpha}-cells were in the basal (Fig. 7B) or the stimulated state (Fig. 4B; see Refs. 17 and 22), suggesting the suppressed glucagon secretion to possibly be due to direct inhibition of two Ca2+ channels or to indirect inhibition of Ca2+ channels resulting from prevention of membrane depolarization. The latter could be achieved by opening of GABAA receptor Cl– channels in the {alpha}-cell (39). In addition, prevention of membrane depolarization is also brought about by activation of KATP channels, which is reportedly induced by the {beta}-cell secretory products, Zn2+ (4, 10) and insulin (10, 21). However, involvement of KATP channels in regulating glucagon secretion is controversial, since different glucagon responses were demonstrated in the following two mutant islets lacking functional KATP channels: preserved glucagon responses from islets deficient in one of the KATP channel subunits, Kir6.2 (26), and no response from islets deficient in another subunit, sulfonylurea receptor 1 (16).

Although inhibition of glucagon secretion by activation of {beta}-cells expressing NaDC-1 supports the paracrine mechanism, it does not exclude a direct inhibitory effect of glucose metabolism on glucagon secretion, especially at relatively low glucose concentrations and in the presence of stimulators of glucagon secretion, such as an amino acid mixture (see above). Two different mechanisms by which glucose directly suppresses glucagon secretion have been proposed. One involves a store-operated current, which controls a depolarizing cascade leading to opening of L-type Ca2+ channels in {alpha}-cells (23). Thus glucose-induced ATP generation stimulates Ca2+ sequestration in endoplasmic reticulum and modulates a store-operated current. Another is based on low KATP channel activity and the special ion channel composition of the {alpha}-cell (5, 15); KATP channel closure by ATP produced during glucose metabolism causes modest depolarization, which inactivates, instead of activating, voltage-gated Na+, T- and N-type Ca2+, and A-type K+ channels participating in action potential generation. Both models are based on data obtained in mouse {alpha}-cells, in which the KATP channel density is much less than that in rat {alpha}-cells. Rat {alpha}-cells were calculated to have nearly 100-fold more KATP channels than mouse {alpha}-cells and double the number in rat {beta}-cells (3, 5). KATP channels couple nutrient metabolism to membrane depolarization. Therefore, in rat {alpha}-cells with a greater number of KATP channels, nutrient metabolism could induce greater changes in membrane potential compared with those in mouse {alpha}-cells, thereby allowing glucagon secretion. Thus the importance of paracrine inhibition might be species dependent. It is essential to establish the level of KATP channel expression in human {alpha}-cells and whether this channel contributes to the regulation of glucagon secretion in humans. In this context, it is noteworthy that KATP channel-blocking agents stimulated glucagon secretion in subjects with insulin-deficient type 1 diabetes (30).

In summary, our findings provide further evidence supporting the concept that {alpha}-cell exocytosis can be modulated by {beta}-cells via a paracrine mechanism. Future studies should focus on detailed molecular analyses of stimulus-secretion coupling in {alpha}-cells under paracrine regulation. This is a promising approach to identifying new drug targets for treating {alpha}-cell abnormalities in diabetic patients.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a grant from Suzuken Memorial Foundation to H. Ishihara and a Grant-in-Aid for Scientific Research (15659213) from the Ministry of Education, Science, Sports, and Culture of Japan and a Grant-in-Aid for Research on Human Genome, Tissue Engineering (H17-genome-003) from the Ministry of Health, Labor and Welfare to Y. Oka.


   ACKNOWLEDGMENTS
 
We are grateful to Y. Nagura for expert assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. Ishihara, Div. of Molecular Metabolism and Diabetes, Tohoku Univ. Graduate School of Medicine, 2–1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan (e-mail: ishihara-tky{at}umin.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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