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Am J Physiol Endocrinol Metab 289: E570-E577, 2005. First published June 7, 2005; doi:10.1152/ajpendo.00102.2005
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Impaired glucagon secretory responses in mice lacking the type 1 sulfonylurea receptor

Chiyo Shiota, Jonathan V. Rocheleau, Masakazu Shiota, David W. Piston, and Mark A. Magnuson

Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee

Submitted 9 March 2005 ; accepted in final form 30 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pancreatic {alpha}-cells, like {beta}-cells, express ATP-sensitive K+ (KATP) channels. To determine the physiological role of KATP channels in {alpha}-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic {alpha}-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca2+ concentration ([Ca2+]i) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null {alpha}-cells showed continuous oscillations in [Ca2+]i regardless of the glucose concentration. These findings indicate that KATP channels in {alpha}-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack KATP channels maintain euglycemia.

pancreatic {alpha}-cell; adenosine 5'-triphosphate-sensitive potassium channel; sulfonylurea receptor 1; pancreas perfusion; glucagon

ATP-SENSITIVE K+ (KATP) CHANNELS are present in a variety of tissues and cell types where they act to couple intracellular metabolic changes to the electrical activity of the plasma membrane (1, 46). In the pancreatic {beta}-cell, KATP channels consist of a heterooctameric complex of both type 1 sulfonylurea receptors (Sur1) and inwardly rectifying K+ channel (Kir6.2) subunits (3). These channels play a key role in glucose-stimulated insulin secretion by coupling a rise in glucose metabolism to {beta}-cell depolarization (32). They are also the molecular target for a widely used class of drugs, the sulfonylureas (2, 19) and the glinides (16, 30).

Pancreatic {alpha}-cells comprise ~10–20% of the cells within the islets of Langerhans. These cells contribute to blood glucose homeostasis through the secretion of glucagon, a major catabolic and hyperglycemic hormone. In patients with type 1 diabetes, impaired glucagon secretion increases the susceptibility to episodes of severe insulin-induced hypoglycemia, which is a limiting factor for intensive insulin therapy. Like pancreatic {beta}-cells, {alpha}-cells are electrically excitable. However, action potentials in {alpha}-cells occur in the absence of glucose (47), and are inhibited as the glucose concentration rises (5, 7). Although depolarization-induced Ca2+ influx through voltage-gated channels is essential for exocytosis in both {alpha}-cells and {beta}-cells (5), the mechanisms that contribute to glucagon secretion in {alpha}-cells are not as well understood. KATP channels are present in the {alpha}-cells of many species (8, 40, 41). In addition, {alpha}-cells express glucokinase, which is essential for glucose-induced insulin secretion by {beta}-cells (17), thereby suggesting that both molecules play a role in the regulation of glucagon secretion by glucose, although the physiological functions of these cells oppose each other.

We have previously described the generation of KATP channel-deficient mice by introducing a null mutation in the Sur1 gene (36). In this study, we have examined the role of KATP channels in glucagon secretion. We report that pancreata from Sur1 null (Sur1–/–) mice lack glucagon secretory responses to a low concentration of glucose and to synergistic stimulation by arginine. These findings indicate that KATP channels in {alpha}-cells play a key role in the regulation of glucagon secretion. Moreover, because Sur1–/– mouse pancreata retain their ability to secrete glucagon in response to epinephrine, we suggest that KATP channel-independent glucagon secretion may contribute to the maintenance of euglycemia in these animals, which is otherwise paradoxical given the central role of KATP channel in both pancreatic {alpha}- and {beta}-cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Mice containing the Sur1 null allele, originally present in a mixed 129/SvJ and C57BL/6J strain background (36), were backcrossed a minimum of eight times into a C57BL/6J background for this study. Age- and sex-matched C57BL/6J mice, or wild-type littermates obtained from heterozygote matings, were used as wild-type (Sur1+/+) controls. All experimental protocols were approved by the Vanderbilt University Institutional Animal Care and Use Committee.

Preparation of single islet cells. Islets of Langerhans were isolated from 3-mo-old mice by a collagenase technique (22) and dispersed into single cells in PBS with 0.05% trypsin and 0.53 mM EDTA (GIBCO/Invitrogen, Carlsbad, CA).

Single-cell RT-PCR. Dispersed islet cells were individually picked under an inverted phase-contrast microscope using a pipette adjusted to 2 µl. Each cell was transferred to a tube containing 8 µl RT buffer (50 mM Tris·HCl, 75 mM KCl, and 3 mM MgCl2, pH 8.3) with 1 U/µl RNasin (Promega, Madison, WI) and 10 mM dithiothreitol (DTT), and frozen immediately on dry ice. First-strand cDNA synthesis was performed by adding 10 µl RT buffer with 10 µM random hexamer primers (Applied Biosystems, Foster City, CA), 1 mM of each of dNTPs, 1 U/µl RNasin, 10 mM DTT, and 10 U/µl SuperScript II RT (Invitrogen) to the frozen cell samples. RT reaction was carried for 60 min at 42°C, followed by incubation at 75°C for 15 min to inactivate RT. To determine specific endocrine cell types, nested PCR was performed for insulin, glucagon, somatostatin, and pancreatic polypeptide. The first round of PCR was multiplexed (21) and contained 4 µl RT reaction and four pairs of outside primer (0.2 µM each in final concentration) in a total volume of 20 µl. The second round of PCR was performed for each hormone individually using 0.2 µl of the first-round PCR mixture and inside primers. The cycling conditions were 35 cycles of 95°C for 15 s and 60°C for 1 min after denaturing at 95°C for 10 min for both rounds. The RT reactions that were positive only in insulin or glucagon were further subjected to nested PCR for Sur1. Parallel reactions using buffer alone and a whole islet from each cell preparation were served as negative and positive controls, respectively. A list of the primers used is shown in Table 1.


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  		Table 1. Oligonucleotides used in the single-cell multiplex RT-PCR analyses

 
Pancreas perfusion. Mice (3–5 mo old) were fasted for 16 h before experimentation. Perfusion of isolated pancreas in situ was performed as previously described (36). Data for males and females were not significantly different in the perfusion studies and were combined.

Measurement of changes in intracellular free Ca2+ concentration. Single islet cells were suspended in RPMI 1640 medium (GIBCO/Invitrogen) supplemented with 10% FBS, 11 mM glucose, 100 IU/ml penicillin, and 100 µg/ml streptomycin and allowed to attach to poly-L-lysine-coated gridded glass-bottom culture dishes (MatTek, Ashland, MA) for 2 days at 37°C in an atmosphere of 5% CO2. Cells were loaded with 2 µM fura 2-AM (Molecular Probes, Eugene, OR) and 0.02% Pluronic F-127 (wt/vol; Molecular Probes) in a HEPES-buffered Krebs-Ringer solution (KRH) (in mM: 118 NaCl, 5.4 KCl, 2.4 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, and 20 HEPES, pH 7.4) supplemented with 0.1% BSA and 5.5 mM glucose for 30 min at room temperature. The dish was placed on a flexiglass-enclosed stage of a Nikon TE300 inverted epifluorescence microscope with a x40 oil immersion objective lens and superfused with KRH containing 0.1% BSA and the indicated concentration of glucose. The enclosed stage was kept at 37°C. The cells were successively excited at 340 and 380 nm, and the fluorescent image emitted at 510 nm was collected at 2-s intervals by a CCD camera. Quantitative changes in intracellular free Ca2+ concentration ([Ca2+]i) were inferred from the ratio of the fluorescence intensity at two wavelengths. After collecting images, cells were subjected to immnocytochemisty for insulin and glucagon to identify cell type.

Immunostaining. Tissues or cells were fixed in 4% paraformaldehyde. The {alpha}- and {beta}-cells were detected with rabbit anti-glucagon antibody (Linco Research, St. Louis, MO) and guinea pig anti-insulin antibody (Linco Research), respectively, with a Cy3-conjugated donkey anti-rabbit IgG and a Cy2-conjugated donkey anti-guinea pig IgG as a secondary antibody (Jackson Immunoresearch, West Grove, PA).

Measurements of glucose, HbA1c, and hormones. Blood glucose concentrations were determined by a glucose oxidase method using a blood glucose analyzer (Hemocue, Mission Viejo, CA). The fraction of HbA1c was determined by a DCA 2000 analyzer (Bayer, Tarrytown, NY). Glucagon concentrations were measured by RIA (Linco Research). Insulin concentrations were determined by RIA using anti-insulin-coated tubes (ICN, Orangeburg, NY) and radiolabeled insulin (Diagnostic Products, Los Angeles, CA).

Statistical analysis. Data are expressed as means ± SE. Differences within and between groups were determined using paired or unpaired Student's t-test, respectively. P < 0.05 denoted statistical significance.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sur1 in mouse {alpha}-cells. Although electrophysiological studies have indicated the presence of functional KATP channels in mouse {alpha}-cells, Sur1 gene expression in {alpha}-cells has only been reported using clonal cell lines (29, 31). Thus, to confirm the expression of Sur1 gene in mouse {alpha}-cells, freshly isolated single islet cells were analyzed by RT-PCR. Cell types were identified by PCR for four islet hormones, and expression of Sur1 gene was then assessed in the insulin- and glucagon-positive cells. As shown in Fig. 1, a 110-bp Sur1 cDNA fragment was amplified from both {alpha}- and {beta}-cells of Sur1+/+ mice, and Sur1 cDNA was not detected in {alpha}-cells of Sur1–/– mice.



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  		Fig. 1. Type 1 sulfonylurea receptor (Sur1) gene expression in mouse pancreatic {alpha}-cells. Sur1 mRNA was detected by single-cell RT-PCR in both insulin-positive cells ({beta}) and glucagon-positive cells ({alpha}) from Sur1+/+ mice. No RT-PCR amplification for Sur1 mRNA was seen in glucagon-positive cells from Sur1–/– mice. B, buffer only; I, whole islet; PP, pancreatic polypeptide.

 
Unchanged plasma glucagon levels in Sur1–/– mice during fasting. Average blood glucose levels in Sur1–/– mice were assessed by measuring HbA1c. There was no difference in HbA1c level between Sur1+/+ and Sur1–/– mice at 2 mo of age in either sex (Fig. 2A). Plasma glucagon levels were measured in 3- and 20-h (overnight) fasted male animals along with blood glucose levels. Prolonged fasting caused a significant decrease in blood glucose concentration in both Sur1+/+ and Sur1–/– mice (Fig. 2B). In a 3-h fasted condition, Sur1–/– mice have a higher level of plasma glucagon than Sur1+/+ mice. Plasma glucagon levels increased significantly in Sur1+/+ mice after overnight fast, whereas they did not change in Sur1–/– mice (Fig. 2B).



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  		Fig. 2. Lack of glucagon response to hypoglycemia in Sur1–/– mice. A: HbA1c levels in 2-mo-old Sur1+/+ and Sur1–/– mice (n = 10–11). B: blood glucose and plasma glucagon levels in 3- and 20-h-fasted male Sur1+/+ and Sur1–/– mice (n =7–8). *P < 0.05 and **P < 0.01.

 
Islet structure and glucagon content in Sur1–/– pancreata. Immunohistochemical staining showed a marked disturbance of islet structure in adult Sur1–/– mice, as reported in other KATP-deficient mouse lines (26, 27, 35). Glucagon-positive cells were found scattered throughout Sur1–/– islets instead of forming a mantle around a core of insulin-positive cells, as seen in Sur1+/+ islets (Fig. 3A). The abnormal islet architecture was observed as early as 1 mo of age, but became more pronounced with age. This morphological abnormality, however, did not cause any significant changes of pancreatic glucagon content in 3-mo-old animals (Fig. 3B).



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  		Fig. 3. Abnormal islet structure but normal glucagon contents in Sur1–/– mouse pancreata. A: structure of 3-mo-old Sur1+/+ and Sur1–/– mouse islets. Immunohistochemical staining for insulin (green) and glucagon (red) reveals abnormal structure in Sur1–/– mouse islet. Bar = 50 µm. B: glucagon contents of Sur1+/+ and Sur1–/– mouse pancreas at 3 mo of age (n = 4).

 
Impaired glucagon response to low glucose in Sur1–/– pancreata. To determine how the lack of KATP channels in {alpha}-cells affects glucagon secretion, we perfused isolated pancreata, and measured several stimulant-induced hormonal responses. We first examined the effect of glucose on the glucagon secretory response. The Sur1+/+ pancreas secreted glucagon in a clear biphasic manner when glucose concentration was decreased from 5.5 to 1.7 mM (Fig. 4). Glucagon secretion was diminished (<5 pg/min) when glucose concentration was returned to 5.5 mM and further raised to 16.7 mM. In contrast, there was no significant change in glucagon secretion from Sur1–/– pancreata throughout the experimental period. As previously reported (35, 36), the insulin secretory response to glucose is impaired in Sur1–/– pancreata. Consistent with this, only a small amount of insulin secretion was observed via KATP channel-independent mechanisms (Fig. 4).



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  		Fig. 4. Glucagon and insulin secretion from perfused Sur1+/+ and Sur1–/– pancreata in response to glucose (n = 4–5). Concentration of glucose was changed stepwise, as indicated. Significant differences (P < 0.05) in glucagon secretion between Sur1+/+ and Sur1–/– pancreata are observed at time 1–6, 8–11, and 13–31 min. Significant differences (P < 0.05 ) in insulin secretion between Sur1+/+ and Sur1–/– pancreata are observed at time 47–52 and 54–66 min.

 
Attenuated glucagon secretory response to arginine in Sur1–/– pancreata. We next examined the response to 20 mM arginine, which is known to potentiate both glucagon and insulin secretion from normal islets (12). In the presence of 20 mM arginine, glucagon secretion in response to 1.7 mM glucose was significantly increased in both Sur1+/+ and Sur1–/– pancreata (Fig. 5). However, although the average rate of glucagon secretion was 542 ± 140 pg/min in Sur1+/+ pancreata, it was only 91 ± 22 pg/min in Sur1–/– pancreata (P < 0.05). In Sur1+/+ pancreata, the effect of arginine was inversely dependent on glucose concentration. On the other hand, glucagon secretion from Sur1–/– pancreata in response to arginine was maintained at the same level regardless of glucose concentration. There was no significant difference in the rate of arginine-stimulated glucagon secretion between Sur1+/+ and Sur1–/– pancreata in the presence of 16.7 mM glucose. Insulin secretion was increased by arginine in both Sur1+/+ and Sur1–/– pancreata (Fig. 5). In the presence of 1.7 mM glucose, the average rate of arginine-stimulated insulin secretion in Sur1–/– pancreata was higher than Sur1+/+ pancreata (0.13 ± 0.07 ng/min in Sur1+/+ and 0.98 ± 0.21 ng/min in Sur1–/–; P < 0.05). In the presence of 16.7 mM glucose, the average rate of arginine-stimulated insulin secretion in Sur1–/– pancreata was lower than Sur1+/+ pancreata (4.08 ± 0.38 ng/min in Sur1+/+ and 2.32 ± 0.35 ng/min in Sur1–/–; P < 0.05).



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  		Fig. 5. Glucagon and insulin secretion from perfused Sur1+/+ and Sur1–/– pancreata in response to arginine. Arginine (20 mM) was perfused with different concentrations of glucose as indicated (n = 4). Significant differences (P < 0.05) in glucagon secretion between Sur1+/+ and Sur1–/– pancreata are observed at time 11, 13–18, 20, 22, 23, 29–37, and 39 min. Significant differences (P < 0.05) in insulin secretion between Sur1+/+ and Sur1–/– pancreata are observed at time 14–26, 28, 29, 42–44, 49, and 54 min.

 
Epinephrine-stimulated glucagon secretion from Sur1–/– pancreata. Epinephrine is known to stimulate glucagon secretion but inhibit insulin secretion from normal islets. In the presence of 5.5 mM glucose, we found that epinephrine stimulated glucagon secretion similarly in both Sur1+/+ and Sur1–/– pancreata during the first 3 min (Fig. 6A). After this first-phase secretion, the second-phase secretion was reduced to <50% of the first phase in Sur1+/+ pancreata, whereas sustained secretion was observed in the second phase in Sur1–/– pancreata. In the presence of 1.7 mM glucose, epinephrine evoked glucagon secretion in both Sur1+/+ and Sur1–/– pancreata (Fig. 6B). However, the amount of secretion from Sur1+/+ pancreata was larger than that from Sur1–/– pancreata during the first 4 min and became similar thereafter. Comparison of the results shown in Fig. 6, A and B, reveals that glucagon secretion in response to epinephrine was not affected by glucose in Sur1–/– pancreata. In contrast, the rate of epinephrine-stimulated glucagon secretion from Sur1+/+ pancreata was higher in the presence of 1.7 mM glucose than the presence of 5.5 mM glucose. As expected, insulin secretion was not stimulated during perfusion of epinephrine.



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  		Fig. 6. Glucagon and insulin secretion from perfused Sur1+/+ and Sur1–/– pancreata in response to epinephrine. Epinephrine (1 µM) was perfused in the presence of 5.5 mM glucose (A, n = 4) or 1.7 mM glucose (B, n = 4). Significant differences (P < 0.05) in glucagon secretion between Sur1+/+ and Sur1–/– pancreata are observed at time 1–7, 12–14, and 45–50 min in A and 5–11, 13, 14, and 16 min in B.

 
Lack of [Ca2+]i responses to changes of glucose concentration in Sur1–/– {alpha}-cells. Because the Sur1–/– mice lack KATP channels in {beta}-cells and {alpha}-cells, the absence of glucagon response to glucose in Sur1–/– pancreata might be caused by impaired influence from the {beta}-cells rather than a primary defect in {alpha}-cells. To determine if this is the case, we examined the responsiveness of isolated single {alpha}-cells to glucose by monitoring [Ca2+]i. After fura 2 was loaded, the cells were perifused with a sequence of KRH buffer that contained 3, 0, and 16.7 mM glucose. Sur1+/+ {beta}-cells typically responded to 16.7 mM glucose with elevation of [Ca2+]i (Fig. 7A), whereas Sur1+/+ {alpha}-cells exhibited oscillations in [Ca2+]i in the absence of glucose and became silent when glucose concentration was increased to 16.7 mM (Fig. 7B). As reported previously (36), Sur1–/– {beta}-cells exhibited continuous oscillations in [Ca2+]i in the absence of glucose (Fig. 7C). Similarly, Sur1–/– {alpha}-cells showed continuous oscillations in [Ca2+]i throughout the recording period. The oscillations were smaller in some Sur1–/– {alpha}-cells (Fig. 7D) compared with other cells (Fig. 7E). None of the Sur1–/– {alpha}-cells responded to 16.7 mM glucose with a reduction of [Ca2+]i. Recent studies by Gromada et al. (15) suggest that [Ca2+]i oscillations in Sur1–/– {alpha}-cells are caused by Ca2+ influx through voltage-gated Ca2+ channels and that the voltage-gated Ca2+ channels are activated by Na+ channel-dependent action potentials.



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  		Fig. 7. Effect of glucose on intracellular free Ca2+ concentration ([Ca2+]i) in isolated {beta}-cells and {alpha}-cells from Sur1+/+ and Sur1–/– mice. Traces are representative of at least 15 cells from 3 independent experiments in each group. A: Sur1+/+ {beta}-cell. B: Sur1+/+ {alpha}-cell. C: Sur1–/– {beta}-cell. D and E: Sur1–/– {alpha}-cell.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The role of KATP channels in glucose-stimulated insulin secretion by the {beta}-cell is well established. However, the mechanisms that regulate the secretion of glucagon by the {alpha}-cell are not as well understood. Because several lines of evidence (14, 29, 31, 40, 41), including the analysis here of single islet cells by RT-PCR, have established that KATP channels are also present in mouse {alpha}-cells, we have examined the role of this channel in glucagon secretion using Sur1 null mice as a model of a KATP channel deficiency.

The {alpha}-cell KATP channel has a role in glucagon response to hypoglycemia.

Although Sur1+/+ pancreata responded to a low concentration of glucose with a 20-fold increase in glucagon secretion, we found that Sur1–/– pancreata did not respond. Previously, Miki et al. (25) examined the glucagon response to low glucose of islets isolated from Kir6.2 knockout mice and did not find any difference in glucagon secretion between wild-type and Kir6.2 knockout mouse islets. However, these findings are difficult to interpret because of the fact that islets from control animals in their studies showed only a twofold increase in glucagon secretion, which suggests the process of isolating islets, or their culture, may have attenuated the responsiveness of {alpha}-cells to a low concentration of glucose. By using a perfusion method that avoids the need to isolate islets, we were able to better assess the impact that the deficiency of KATP channels has on {alpha}-cell function.

Although pancreatic {beta}-cells are electrically coupled with each other through gap junctions and exhibit synchronized [Ca2+]i oscillations (34), there is little evidence to date for direct electrical coupling between {alpha}- and {beta}-cells (13, 28). However, glucagon secretion is known to be regulated by paracrine stimulation from {beta}-cells. Insulin (24, 39, 44) and cosecreted zinc (20) have been shown to negatively modulate glucagon secretion. {gamma}-Aminobutyric acid, which is released from microvesicles in {beta}-cells by Ca2+-dependent exocytosis, also acts to inhibit glucagon secretion (33, 45). Recently, Banarer et al. (4) have suggested that a sudden drop in intraislet insulin concentration may be an essential signal for glucagon secretion in response to hypoglycemia. Studies using streptozotocin (STZ)-administrated rats as a model of defective counterregulation (18, 48) showed that exposure of islets to a high concentration of glucose and insulin in an antecedent period restored the glucagon response to glucose deprivation, thereby supporting this "switch-off" hypothesis. In our studies, all animals were fasted overnight before experimentation, and we did not observe any differences between Sur1+/+ and Sur1–/– pancreata in insulin secretion before starting experiments. Thus lack of glucagon response to a low concentration of glucose, which was seen only in Sur1–/– pancreata, is not caused by a defect in insulin secretion.

Islet morphology in Sur1–/– mice is markedly disturbed, with {alpha}-cells becoming localized to the core of islets. Similar morphological abnormalities have been reported in other diabetic models, including STZ-administrated animals (23). Restoration of glucagon response in STZ-administrated rat islets by providing a switch-off signal, as mentioned above, indicates that the abnormal location of {alpha}-cells does not affect their functions. Furthermore, Sur1–/– {alpha}-cells did not show normal [Ca2+]i response to changes of glucose concentration in an isolated single-cell preparation. Taken together, the impaired glucagon response we observed in Sur1–/– pancreata is likely intrinsic to Sur1–/– {alpha}-cells rather than a secondary effect of KATP deficiency in {beta}-cells.

Potentiation of glucagon secretion by arginine is dependent on the {alpha}-cell KATP channel.

The electrogenic transport of the cationic amino acid arginine produces an inward current that depolarizes the membrane, thereby increasing [Ca2+]i (38). Although arginine stimulates glucagon secretion similarly in both Sur1+/+ and Sur1–/– pancreata in the presence of an inhibitory concentration of glucose (16.7 mM), an exaggerated glucagon secretion was caused by arginine at a stimulatory concentration of glucose (1.7 mM) only in Sur1+/+ pancreata. During perfusion of arginine, Sur1–/– pancreata secreted more insulin than Sur1+/+ pancreata when the glucose concentration was low (1.7 mM). On the other hand, Sur1+/+ pancreata secreted more insulin than Sur1–/– pancreata when glucose concentration was high (16.7 mM). Because insulin inhibits glucagon secretion (24, 39, 44), these differences in insulin secretion between Sur1+/+ and Sur1–/– pancreata during perfusion of arginine may underlie the differences in glucagon secretion. However, the marked difference in glucagon secretion between Sur1+/+ and Sur1–/– pancreata cannot be explained solely by the differences in insulin secretion. In the presence of 5.5 mM glucose, glucagon response to arginine was significantly higher in Sur1+/+ pancreata than Sur1–/– pancreata even though there was no difference in insulin secretion. Thus our studies suggest that the synergistic effect of arginine on glucagon secretion in response to hypoglycemia is dependent on KATP channels in {alpha}-cells.

Glucagon secretory response to epinephrine is independent of the {alpha}-cell KATP channel.

Epinephrine is another important counterregulatory hormone to hypoglycemia. This hormone elevates blood glucose levels by stimulating hepatic glucose production, inhibiting insulin secretion and stimulating glucagon secretion. We did not observe any major differences in glucagon response to epinephrine in the presence of 5.5 mM glucose between Sur1+/+ and Sur1–/– pancreata. Lowering the glucose concentration to 1.7 mM did not change epinephrine-stimulated glucagon secretion from Sur1–/– pancreata, whereas it increased that from Sur1+/+ pancreata. Comparison of glucagon secretion from Sur1+/+ pancreata under different conditions (1.7 mM glucose, 1.7 mM glucose with epinephrine, and 5.5 mM glucose with epinephrine) suggests that the response to low glucose and epinephrine is additive rather than synergistic. These data also suggest that epinephrine stimulates glucagon secretion mainly through a KATP channel-independent mechanism. In {beta}-cells, epinephrine hyperpolarizes {beta}-cells in the absence of KATP channels via activation of low-conductance BaCl2-sensitive K+ channels that are regulated by pertussis toxin-sensitive G proteins (37). Although it has been reported that the stimulatory effect of epinephrine on glucagon secretion involves both {alpha}1- and {beta}-adrenergic components in mouse {alpha}-cells (43), the precise mechanism is not yet known.

Brain KATP channels and glucagon secretion.

The central nervous system also plays an important role in sensing glucopenia and triggering counterregulatory hormone release during hypoglycemia (6). The ventromedial hypothalamic nucleus (VMH) is thought to be the specific region in the brain for this role (9–11). This region stimulates glucagon secretion by sympathetic and parasympathetic innervations in islets and by activation of the hypothalamic-pituitary-adrenal axis (42). VMH neurons possess KATP channels made of Sur1 and Kir6.2 subunits. Miki et al. (25) have shown that Kir6.2-deficient mice lack glucagon response to either systemic hypoglycemia induced by insulin injection or neuroglycopenia induced by intracerebroventricular administration of 2-deoxy-D-glucose, although they maintain an intact responsiveness to hypoglycemia with elevation of plasma epinephrine levels. Based on these findings in Kir6.2-deficient mice, it is likely that Sur1–/– mice have a similar defect in the central regulation of glucagon secretion. Thus lack of glucagon response to overnight fasting observed in the Sur1–/– mice may be due to, at least in part, KATP channel deficiency in the brain in these mice.

In conclusion, we have demonstrated that Sur1–/– pancreata lack glucagon response to hypoglycemia and fail to respond appropriately to arginine. The results indicate the essential role of {alpha}-cell KATP channel in glucose sensing and regulation of glucagon secretion. We have also shown that epinephrine stimulates glucagon secretion through a KATP channel-independent pathway. When considered together with the studies of Gromada et al. (15), who have also observed an impairment of glucagon secretion in Sur1–/– mouse islets, it has become clear that KATP channels may play as important a role pancreatic {alpha}-cells, as has long been known to be the case for pancreatic {beta}-cells.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) Grants DK-42502 to M. A. Magnuson, DK-60667 to M. Shiota, and DK-53434 to D. W. Piston. The Vanderbilt Cell Imaging Core Resource and the Vanderbilt Mouse Metabolic Physiology Center are supported by NIDDK Grants DK-20593 and DK-59637, respectively.


   ACKNOWLEDGMENTS
 
We thank Wendell E. Nicholson and W. Steven Head for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Magnuson, Vanderbilt Univ. School of Medicine, Dept. of Molecular Physiology and Biophysics, 747 Light Hall, Nashville, TN 37232-0615 (e-mail: mark.magnuson{at}vanderbilt.edu)

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
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aguilar-Bryan L and Bryan J. Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 20: 101–135, 1999.[Abstract/Free Full Text]
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