Regulation of Glucagon Secretion at Low Glucose Concentrations: Evidence for Adenosine Triphosphate-Sensitive Potassium Channel Involvement
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《内分泌学杂志》
Departments of Medicine (L.A.-B., A.S.R.) and Molecular and Cellular Biology (A.M., M.H., J.B., L.A.-B.), Baylor College of Medicine, Houston, Texas 77030
The Institute of Child Health (K.H.), Biochemistry Endocrinology and Metabolism Unit, University College London, London WC1N 1EH United Kingdom
Abstract
Glucagon is a potent counterregulatory hormone that opposes the action of insulin in controlling glycemia. The cellular mechanisms by which pancreatic -cell glucagon secretion occurs in response to hypoglycemia are poorly known. SUR1/KIR6.2-type ATP-sensitive K+ (KATP) channels have been implicated in the glucagon counterregulatory response at central and peripheral levels, but their role is not well understood. In this study, we examined hypoglycemia-induced glucagon secretion in vitro in isolated islets and in vivo using Sur1KO mice lacking neuroendocrine-type KATP channels and paired wild-type (WT) controls. Sur1KO mice fed ad libitum have normal glucagon levels and mobilize hepatic glycogen in response to exogenous glucagon but exhibit a blunted glucagon response to insulin-induced hypoglycemia. Glucagon release from Sur1KO and WT islets is increased at 2.8 mmol/liter glucose and suppressed by increasing glucose concentrations. WT islets increase glucagon secretion approximately 20-fold when challenged with 0.1 mmol/liter glucose vs. approximately 2.7-fold for Sur1KO islets. Glucagon release requires Ca2+ and is inhibited by nifedipine. Consistent with a regulatory interaction between KATP channels and intra-islet zinc-insulin, WT islets exhibit an inverse correlation between -cell secretion and glucagon release. Glibenclamide stimulated insulin secretion and reduced glucagon release in WT islets but was without effect on secretion from Sur1KO islets. The results indicate that loss of -cell KATP channels uncouples glucagon release from inhibition by -cells and reveals a role for KATP channels in the regulation of glucagon release by low glucose.
Introduction
THE RELEASE OF glucagon, a small peptide hormone secreted by pancreatic -cells, is stimulated by hypoglycemia and inhibited by hyperglycemia, insulin, and somatostatin. In combination with insulin, glucagon determines the rate of gluconeogenesis and glycogenolysis in the liver and thus plays a key role in the counterregulatory response to hypoglycemia (1). The inhibition of glucagon release after a meal is often blunted and contributes to postprandial hyperglycemia by accelerating glycogenolysis in type 2 diabetes (2). The stimulation of glucagon release by insulin-induced hypoglycemia during the counterregulatory response is impaired in type 1 diabetes and in advanced stages of type 2 diabetes (3, 4). This reduced secretion predisposes individuals to repeated hypoglycemic episodes that may lead to coma or neurological injury (5). Large clinical studies including the Diabetes Control and Complications Trial (6) and the U.K. prospective study (7) advocate aggressive management using an intensive insulin regiment to achieve euglycemia and reduce complications, but this strategy has been associated with an increased frequency of hypoglycemic episodes (6) during which impaired glucagon secretion constitutes a significant barrier to the prevention of, and recovery from, hypoglycemia (4, 8).
The control of glucagon secretion is multifactorial, reportedly regulated by -aminobutyric acid (9, 10), low glucose (11), and sympathetic innervation (12) and by intra-islet insulin (13, 14, 15) or cosecreted zinc (16, 17). Although all are potential regulators, the mechanism(s) by which falling blood glucose controls glucagon secretion is not well understood. One school of thought holds that low glucose sensing in the brain, particularly neurons in the hypothalamus (18, 19), activates autonomic pathways that stimulate glucagon release (20), implying intact innervation of pancreatic islets is required (21). Other evidence suggests that local intra-islet control mechanisms are involved and that glucose and insulin levels, either directly or indirectly via -cell secretion, affect -cell glucagon release independently of central or autonomic control (15, 22, 23).
Recent studies provide evidence for intra-islet control, demonstrating that glucagon release is stimulated strongly by a combination of falling plasma glucose and insulin levels (22, 23, 24). SUR1/KIR6.2-type ATP-sensitive potassium (KATP) channels are known to play a role in regulation of insulin release, but their role in glucagon secretion is less clear. The increase in ATP/ADP ratio associated with increased glucose metabolism inhibits -cell channels, resulting in depolarization, activation of voltage-gated Ca2+ channels, and a transient rise in cytosolic Ca2+, [Ca2+]i, associated with exocytosis (25). A similar mechanism has been suggested to operate in -cells stimulated by pyruvate, a fuel that can readily enter -cells via monocarboxylate transporters not present in -cells (16) and thus selectively increase the metabolic rate of -cells. In this case, inhibitory intra-islet insulin and/or zinc remains low while the increase in -cell fuel metabolism raises ATP/ADP, closing KATP channels and thus increasing [Ca2+]i, which stimulates glucagon release (16).
We have used KATP channel-null Sur1KO mice to study the role of KATP channels in glucagon secretion. SUR1 and KIR6.2-null mice exhibit dysregulation of insulin secretion (26, 27, 28). There is accumulating evidence that ATP-sensitive K+ channels are involved in brain glucose sensing (18, 29, 30, 31), and a recent study shows an impaired glucagon secretory response in Sur1KO mice (32). Here we evaluate the ability of mouse islets to secrete glucagon in response to a hypoglycemic challenge and use Sur1KO islets to establish a role for KATP channels in the glucagon secretory response to hypoglycemia.
Materials and Methods
Experimental animals
Experiments were performed on mice using protocols approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sur1KO mice were generated by homologous recombination as described previously (26). In vivo and in vitro experiments were done on Sur1KO and control C57BL/6 wild-type (WT) 12- to 16-wk-old male mice maintained on a 12-h light, 12-h dark cycle and fed with standard rodent chow. A 6-h fast was initiated between 0800 and 0900 h; experiments were done between 1400 and 1500 h. Experiments with fed mice were done between 1400 and 1500 h. Data points were obtained from mice euthanized at the time points as indicated in the figure legends.
Hormones and compounds
Short-acting human insulin (0.75 IU/kg) (Humulin R; Eli Lilly, Indianapolis, IN) was used throughout. Human glucagon (0.5 mg/kg) (Calbiochem, La Jolla, CA) was injected ip after a 6-h fast. In perfusion experiments, KATP channels were blocked with glibenclamide (1 μM) (Sigma Chemical Co., St. Louis, MO); L-type voltage-dependent calcium channels were inhibited with nifedipine (1 μmol/liter) (Sigma). Insulin and glucagon were dissolved in 0.9% NaCl before application. Glibenclamide and nifedipine stock solutions were prepared in dimethylsulfoxide (1 mmol/liter) and dissolved in perfusion medium at a final concentration of 1 μmol/liter. The final dimethyl sulfoxide concentration was 0.1%.
Islet isolation and culture
Pancreatic islets were isolated by intraductal injection of 1.0 mg/ml collagenase P (Roche Molecular Biochemicals, Indianapolis, IN), hand picked, and transferred to DMEM supplemented with 10% fetal bovine serum and 5 mmol/liter glucose, 2 mmol/liter glutamine, 2 mmol/liter sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Freshly isolated islets were cultured overnight at 37 C in humidified air containing 5% CO2, size matched, divided into groups, and used for static incubation (10 islets) or perfusion experiments (200 islets).
Static determination of insulin and glucagon secretion
Measurements were carried out in freshly prepared Krebs-Ringer bicarbonate HEPES buffer (KRB-HEPES) containing (in mmol/liter) 130 NaCl, 5 KCl, 1.25 KH2PO4, 1.25 MgSO4, 2.68 CaCl2, 5.26 NaHCO3, 10 HEPES (equilibrated with 5% CO2, pH 7.4) supplemented with 0.1% BSA (fraction V), 2 alanine (Ala), 2 arginine-HCl (Arg), and 2 glutamine (Gln) (33, 34) and 100 U/ml penicillin, 100 μg/ml streptomycin, and the indicated concentrations of glucose and reagents. Amino acids were included to simulate conditions in vivo; antibiotics were included to prevent bacterial growth. Islets were washed twice with KRB and preincubated at 37 C with gentle shaking. After 30 min, the medium was replaced with KRB containing 1.0 mmol/liter glucose and incubated for an additional 2 h at which time 0.5-ml aliquots of the supernatant were taken for determination of insulin and glucagon. The islets were homogenized in 70% acid-ethanol and the extracts used to determine total insulin and glucagon contents. Aprotinin (5.0 μg/ml) (Sigma Chemical Co., St. Louis, MO.) was added, and the samples were stored at –20 C for RIA. Determinations were done in duplicate for the number of different islet preparations indicated in the figure legends.
Perfusion assays for insulin and glucagon secretion
Islets were transferred to a column of Bio-Gel P-10 (Bio-Rad) and perfused (0.8 ml/min) with KRB-HEPES containing 2.8 mmol/liter glucose for 30 min to reach a basal state. The glucose concentration was raised to 16.7 mmol/liter for 30 min to stimulate insulin secretion and then lowered to 0.1 mmol/liter glucose for 30 min to stimulate glucagon release. Samples were collected at the indicated time points, aprotinin was added, and the samples were handled as described above.
Glucose measurements
Glucose values were determined from tail vein blood samples using a Free-Style glucometer (Therasense, Alameda, CA).
Insulin and glucagon measurements
Glucagon concentrations in plasma and in samples from static and perfusion experiments were determined by RIA according to the manufacturer’s protocol (Linco, St. Charles, MO.). Plasma insulin was measured in 5.0-μl samples using an ultrasensitive mouse-insulin ELISA kit (ALPCO Diagnostics, Windham, NH). Secreted insulin in samples from static and perfusion experiments was determined using a rat/mouse RIA according to the manufacturer’s protocol (Linco).
Glycogen measurements
Liver glycogen content was determined using the anthrone reaction (35) normalized to protein content determined using the BCA (bicinchoninic acid) assay (Pierce Biotechnology, Inc., Rockford, IL). Values are expressed as micrograms glycogen per microgram protein.
Data analysis
Statistical analyses were performed on paired data using a one-tailed Student’s t test and on grouped data by ANOVA using Bonferroni’s posttest. All data are presented as mean ± SE; P < 0.05 was considered statistically significant. Graphics as well as statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
Results
Sur1KO mice exhibit an impaired counterregulatory response to insulin-induced hypoglycemia
To compare their counterregulatory responses, insulin was administered to fed WT and Sur1KO animals to induce hypoglycemia. The initial blood glucose values were the same in both animals, but administration of insulin (0.75 U/kg) produced a more profound hypoglycemia in the knockout animals (Fig. 1A). Blood glucose values returned to normal in the control animals within approximately 90 min, whereas the Sur1KO mice exhibited a slower rate of recovery (Fig. 1A). Although their initial plasma values were equivalent, 15 min of hypoglycemia prompted a 2-fold increase in glucagon level in Sur1KO mice vs. an approximately 5-fold increase in control animals (Fig. 1B). Similar observations have been made in patients with persistent hyperinsulinism of infancy (36) and another Sur1KO mouse model (32). The initial hepatic glycogen contents were the same in both animals, and insulin produced a comparable transient increase in glycogen content during the first 15 min, presumably as a consequence of increased insulin-dependent glucose uptake (37) or because of a greater hepatic glycogen cycling as a result of inhibition of glycogenolysis (38). This transient increase was followed by a marked decline in glycogen content in both animals, although the rate of glycogen use was reduced in the Sur1KO animals (Fig. 1C). The results extend a study using KIR6.2-null mice (29) and are consistent with the idea that the slower recovery of Sur1KO mice from insulin-induced hypoglycemia is in part a consequence of an impaired counterregulatory response with reduced glucagon secretion and consequent failure to mobilize liver glycogen stores efficiently.
Glycemic response to exogenous glucagon
To determine whether differential hormone sensitivity could account for the impaired response, glucagon (0.5 mg/kg) was administered to 6-h-fasted animals. WT mice exhibited a transient, less than 2-fold, increase in blood glucose that returned to the control value within 60 min, whereas the Sur1KO animals displayed a greater, sustained hyperglycemia (Fig. 2A). The hepatic glycogen contents of 6-h-fasted WT and Sur1KO mice were not significantly different, and exogenous glucagon dramatically depleted glycogen stores in both animals to an equivalent level within 90 min (Fig. 2B). The plasma insulin levels were significantly lower in Sur1KO vs. WT mice (Fig. 2C). The results imply the hepatic response to exogenous glucagon is not impaired in the knockout animals and that the prolonged hyperglycemia observed in the Sur1KO mice is a consequence of their previously reported lack of first-phase insulin release when glucose is elevated (26, 28).
Glucagon and insulin release from isolated islets
A previous study reported that glucagon release from KIR6.2-null islets is higher but not statistically different from WT, with approximately a 2-fold increase in static measurements in 16.7 vs. 1 mmol/liter glucose (29). This report focused on the central nervous system (CNS) component, concluding it is impaired. To assess the secretory capacity of Sur1KO -cells further, isolated islets were tested in both static and perifusion assays. When tested under hypoglycemic conditions (2 h in 1.0 mmol/liter glucose plus 2.0 mmol/liter Ala, Arg, and Gln) (see Refs.33 and 34), Sur1KO islets released significantly more insulin vs. control islets (Fig. 3A); glucagon release was consistently lower from the knockout islets but did not achieve statistical significance (Fig. 3C). The total insulin (WT, 1271 ± 121 ng/10 islets; Sur1KO, 1199 ± 153 ng/10 islets) and total glucagon contents (WT, 43.81 ± 5.15 ng/10 islets; Sur1KO, 54.18 ± 1.22 ng/10 islets) were not significantly different after the incubation period in low glucose (P = 0.12; n = 3).
Perifusion assays show that the Sur1KO -cells respond to changes in glucose level, but their response is blunted. Figure 3B illustrates the normal biphasic insulin response of WT islets to a stepwise change in glucose concentration. By contrast, the knockout islets exhibit a small transient decrease and increase in insulin release at the start and end of the high glucose step, respectively, presumably as a consequence of changes in cytosolic Ca2+ as described previously (39), but insulin release continues even in 0.1 mmol/liter glucose. Figure 3D shows that switching WT islets from low to high glucose (2.8–16.7 mmol/liter) decreased glucagon secretion from 238.4 ± 54.1 to 24.1 ± 0.15 pg/ml per 200 islets within 5 min, an approximately 10-fold decrease (P < 0.05; n = 3). In contrast, glucagon secretion from Sur1KO islets was reduced from 161.3 ± 9.38 to 81.4 ± 10.93 pg/ml per 200 islets over the same period (2-fold decrease) (P < 0.05; n = 3). After exposure to high glucose, a low-glucose challenge produced a marked approximately 20-fold increase of glucagon release in WT islets (26.1 ± 4.8 to 540.7 ± 63.9 pg/ml per 200 islets) concomitant with a decrease in insulin release (4.5 ± 0.8 to 1.0 ± 0.1 ng/ml per 200 islets) within 10 min (Fig. 3, B and D, and Table 1). The equivalent switch with Sur1KO islets produced an increase in glucagon secretion (73.8 ± 4.7 to 196.1 ± 19.73 pg/ml per 200 islets) and an overall decrease in insulin secretion (5.1 ± 0.2 to 3.8 ± 0.4 ng/ml per 200 islets). Note, however, that although the increased glucagon release from WT islets correlates with a monotonic fall in insulin secretion over the first 10 min, the period when the rise in glucagon release is maximal, the Sur1KO islets actually increase their rate of insulin secretion, reaching a peak value of 7.88 ± 1.03 ng/ml per 200 islets. The results show that the glucagon response to low glucose is attenuated and that there is an uncoupling of the communication between - and -cells in the Sur1KO islets.
Increased intra-islet insulin (23, 40) and/or zinc (41, 42) have been implicated in suppression of glucagon release, and our WT data are consistent with a strong inverse correlation between insulin and glucagon secretion that is perturbed or uncoupled in Sur1KO islets. To assess the effect(s) of -cell secretion on glucagon release further, WT and Sur1KO islets were challenged with low glucose plus glibenclamide (1 μmol/liter) to block KATP channels. Glibenclamide strongly stimulates insulin secretion from WT islets in 0.1 mmol/liter glucose and significantly blunts glucagon release the WT glucagon response to a 0.1 mmol/liter glucose challenge (compare responses of WT in Fig. 4A with WT in Fig. 3D, and see Table 1). Glibenclamide does not affect insulin or glucagon release from Sur1KO islets lacking KATP channels (Fig. 4B and Table 1). Note that the levels of glucagon secretion from WT islets treated with glibenclamide mimic the impaired release observed for Sur1KO islets (compare Fig. 4A with Figs. 3D and 4B). The results are consistent with the partial suppression of glucagon release by -cell secretory products acting via KATP channels (43).
Exocytosis of both insulin and glucagon requires Ca2+. To ensure that the hormone release observed in response to profound hypoglycemic challenge was specific, we evaluated the ability of nifedipine, an L-type Ca2+ channel blocker, to suppress secretion induced by low glucose. Figure 5A shows that 1 μmol/liter nifedipine inhibits the marked increase in glucagon release observed when WT islets are challenged with 0.1 mol/liter glucose (see Fig. 3D) and similarly inhibits the smaller glucagon release from Sur1KO. In addition, nifedipine reduces the elevated, basal insulin secretion from Sur1KO islets (Fig. 5B). These observations confirm our earlier reports that nifedipine will suppress persistent insulin release from Sur1KO islets (26, 39). Table 1 summarizes the insulin and glucagon secretion values at 30 min after switching the glucose concentration from 16.7 to 0.1 mmol/liter glucose in the presence of glibenclamide or nifedipine in WT and Sur1KO islets. The Sur1KO islets have an increased output of insulin and a decreased output of glucagon in response to hypoglycemic challenge compared with WT islets. Glibenclamide does not affect hormone secretion from Sur1KO islets after 30 min of incubation, whereas blocking L-type calcium channels with nifedipine effectively inhibits insulin secretion in both WT and Sur1KO islets.
Discussion
Sur1KO mice exhibit a reduced rate of glucagon release in response to insulin-induced hypoglycemia, indicating that SUR1/KIR6.2-type KATP channels play a role in the counterregulatory response. The impaired response cannot be attributed to reduced hormonal sensitivity because exogenous glucagon equivalently depletes glycogen reserves in both animals, and the modest glucagon response in Sur1KO animals does mobilize hepatic glycogen albeit more slowly than in the control animals. Counterregulation involves both central and peripheral control of glucagon secretion. Although we have not quantified the relative contributions, SUR1/KIR6.2 channels are expected to have a role in both central and peripheral control, and these data show that isolated Sur1KO islets do exhibit a deficient secretory response to hypoglycemia consistent with impaired peripheral control. The results extend the analysis reported for KIR6.2-null islets (29); we observe an increase in glucagon release in static experiments under low-glucose conditions and show a pronounced blunting of the glucagon secretory response when islets are subject to a hypoglycemic challenge after exposure to elevated glucose, a protocol described by others (23, 24). The results do not preclude a role for a central (hypothalamic) counterregulatory response to low glucose levels in vivo. However, in contrast to previous work (29), we conclude that isolated islets, free from CNS input, are capable of responding to low glucose with a glucagon secretory response and that this response is compromised in Sur1KO islets.
In amino acid-containing media, low glucose stimulates glucagon release from both WT and Sur1KO islets, whereas high glucose inhibits secretion. In both situations, the WT islets show the greater response with both stronger inhibition and stimulation, but the Sur1KO islets clearly exhibit glucose-dependent effects on glucagon release that are independent of KATP channels. The stronger inhibition in control islets is consistent with the idea that intra-islet insulin or cosecreted zinc acts to suppress glucagon release (13, 43), reviewed by Samols and Stagner (15, 44), specifically that insulin (40, 43) or zinc (41, 42, 43) can activate KATP channels and thus hyperpolarize -cells, which would reduce the activity of voltage-gated Ca2+ channels and lower [Ca2+]i necessary for exocytosis. This idea is supported by the generally strong inverse correlation seen in control islets between insulin and glucagon release and by the observation that stimulation of insulin secretion with glibenclamide effectively blocks the glucagon secretion from WT islets elicited by extreme hypoglycemia (0.1 mmol/liter or 1.8 mg/dl) without significantly affecting release from knockout islets under the same stimulus (Fig. 4). Surprisingly, although the loss of -cell KATP channels appears to uncouple glucagon release from the inhibitory effects of -cell secretion, it does not produce hyperglucagonemia. Given that intra-islet zinc/insulin is expected to be consistently elevated in the Sur1KO islets, an inhibitory mechanism might be invoked to explain their attenuated glucagon release, in which case a KATP-independent inhibitory pathway must be involved. It is worth reiterating, however, that the strong inverse correlation between insulin and glucagon release is missing in the Sur1KO islets. This can be seen clearly, for example, in Fig. 3, B and D, where high glucose reduces Sur1KO glucagon release without altering insulin secretion, and the shift to very low glucose stimulates secretion of insulin during the period when glucagon release is increasing most rapidly. The results support the idea that -cells have a two-tier control system in which -cell glucagon secretion is tightly coupled to release of zinc-insulin by -cells via KATP channels but have an underlying KATP-independent regulatory mechanism that is regulated by fuel metabolism. The nature of the underlying mechanism is not understood but may be similar to the control(s) regulating insulin release in KATP-null -cells (39, 45).
A previous report (46) indicated that glucagon secretion in mouse islets was mediated by N-type Ca2+ channels and only modestly affected by L-type Ca2+ channel antagonists (nifedipine at 20 μmol/liter). Therefore, we attempted to inhibit insulin secretion from Sur1KO islets with nifedipine in an effort to mimic the fall in insulin seen in WT islets and test the idea that falling insulin and falling glucose would enhance glucagon secretion in the absence of KATP channels. However, consistent with a role for L-type Ca2+ channels and a requirement for elevated [Ca2+]i, nifedipine inhibited insulin and glucagon release from both WT and Sur1KO islets. The suppression of glucagon release from Sur1KO islets is more pronounced than the controls possibly as a consequence of tonic inactivation of N- and T-type calcium channels as suggested previously (47). On the other hand, glucagon secretion in response to epinephrine is reported to involve the activation of store-operated currents (48), emphasizing the importance of intracellular calcium changes.
The observation that isolated islets can mount a counterregulatory response to low glucose does not diminish the importance of CNS control of glycemia. The role(s) for hypothalamic KATP channels in counterregulation and control of hepatic gluconeogenesis are well established (30, 31). Clinical data on patients with persistent hyperinsulinemic hypoglycemia of infancy, some with mutations in the SUR1/ABCC8 gene, show that their glucagon counterregulatory response is impaired, although they exhibit appropriate increases in serum catecholamines during hypoglycemia (36). The data are consistent with mechanisms local to the -cell blunting the serum glucagon counterregulatory response and/or that centers outside the ventromedial hypothalamus are involved in the catecholamine response to hypoglycemia.
In summary, pancreatic islets can sense and respond directly to changes in ambient glucose and mount a counterregulatory response in vitro, secreting glucagon in response to hypoglycemia, independent of CNS regulation. Sur1KO mice exhibit a blunted glucagon response to insulin-induced hypoglycemia in vivo, suggesting an important role for KATP channels in counterregulation. Our isolated islet data are consistent with a KATP-dependent -/-cell dialog in which glucagon release is suppressed by intra-islet insulin and/or Zn2+. The blunted counterregulatory response observed in KATP-null mice may thus have multiple causes: uncoupling of the -/-cell dialog, impaired peripheral control secondary to loss of KATP channels (18, 29), or a smaller readily releasable pool of glucagon granules in Sur1KO -cells (49). We suggest that the deficient glucagon counterregulatory response seen in persistent hyperinsulinemic hypoglycemia of infancy (36) could originate either from the loss of peripheral control similar to that observed in Sur1KO animals or, in cases where functional KATP channels remain, could result from suppression of glucagon release by elevated intra-islet insulin- and/or Zn2+-mediated -cell hyperpolarization (17). Additional clinical and laboratory studies are required to understand the detailed interactions between pancreatic - and -cells and the role of their dialog in glucose homeostasis.
Acknowledgments
We thank Rebecca A. Schneider for helpful discussion.
Footnotes
This work was supported by Juvenile Diabetes Research Foundation International (3-2003-508 to A.M. and 1-2005-950 to J.B.), the Sistema Nacional de Investigadores/Mexico (30370 to A.M.), and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (DK57671 to L.A-B., DK52771 to J.B., and DK02285 to A.S.R.).
First Published Online August 25, 2005
1 A.M. and M.H. contributed equally to this work.
Abbreviations: CNS, Central nervous system; KATP, ATP-sensitive K+ channel; WT, wild type.
Accepted for publication August 18, 2005.
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The Institute of Child Health (K.H.), Biochemistry Endocrinology and Metabolism Unit, University College London, London WC1N 1EH United Kingdom
Abstract
Glucagon is a potent counterregulatory hormone that opposes the action of insulin in controlling glycemia. The cellular mechanisms by which pancreatic -cell glucagon secretion occurs in response to hypoglycemia are poorly known. SUR1/KIR6.2-type ATP-sensitive K+ (KATP) channels have been implicated in the glucagon counterregulatory response at central and peripheral levels, but their role is not well understood. In this study, we examined hypoglycemia-induced glucagon secretion in vitro in isolated islets and in vivo using Sur1KO mice lacking neuroendocrine-type KATP channels and paired wild-type (WT) controls. Sur1KO mice fed ad libitum have normal glucagon levels and mobilize hepatic glycogen in response to exogenous glucagon but exhibit a blunted glucagon response to insulin-induced hypoglycemia. Glucagon release from Sur1KO and WT islets is increased at 2.8 mmol/liter glucose and suppressed by increasing glucose concentrations. WT islets increase glucagon secretion approximately 20-fold when challenged with 0.1 mmol/liter glucose vs. approximately 2.7-fold for Sur1KO islets. Glucagon release requires Ca2+ and is inhibited by nifedipine. Consistent with a regulatory interaction between KATP channels and intra-islet zinc-insulin, WT islets exhibit an inverse correlation between -cell secretion and glucagon release. Glibenclamide stimulated insulin secretion and reduced glucagon release in WT islets but was without effect on secretion from Sur1KO islets. The results indicate that loss of -cell KATP channels uncouples glucagon release from inhibition by -cells and reveals a role for KATP channels in the regulation of glucagon release by low glucose.
Introduction
THE RELEASE OF glucagon, a small peptide hormone secreted by pancreatic -cells, is stimulated by hypoglycemia and inhibited by hyperglycemia, insulin, and somatostatin. In combination with insulin, glucagon determines the rate of gluconeogenesis and glycogenolysis in the liver and thus plays a key role in the counterregulatory response to hypoglycemia (1). The inhibition of glucagon release after a meal is often blunted and contributes to postprandial hyperglycemia by accelerating glycogenolysis in type 2 diabetes (2). The stimulation of glucagon release by insulin-induced hypoglycemia during the counterregulatory response is impaired in type 1 diabetes and in advanced stages of type 2 diabetes (3, 4). This reduced secretion predisposes individuals to repeated hypoglycemic episodes that may lead to coma or neurological injury (5). Large clinical studies including the Diabetes Control and Complications Trial (6) and the U.K. prospective study (7) advocate aggressive management using an intensive insulin regiment to achieve euglycemia and reduce complications, but this strategy has been associated with an increased frequency of hypoglycemic episodes (6) during which impaired glucagon secretion constitutes a significant barrier to the prevention of, and recovery from, hypoglycemia (4, 8).
The control of glucagon secretion is multifactorial, reportedly regulated by -aminobutyric acid (9, 10), low glucose (11), and sympathetic innervation (12) and by intra-islet insulin (13, 14, 15) or cosecreted zinc (16, 17). Although all are potential regulators, the mechanism(s) by which falling blood glucose controls glucagon secretion is not well understood. One school of thought holds that low glucose sensing in the brain, particularly neurons in the hypothalamus (18, 19), activates autonomic pathways that stimulate glucagon release (20), implying intact innervation of pancreatic islets is required (21). Other evidence suggests that local intra-islet control mechanisms are involved and that glucose and insulin levels, either directly or indirectly via -cell secretion, affect -cell glucagon release independently of central or autonomic control (15, 22, 23).
Recent studies provide evidence for intra-islet control, demonstrating that glucagon release is stimulated strongly by a combination of falling plasma glucose and insulin levels (22, 23, 24). SUR1/KIR6.2-type ATP-sensitive potassium (KATP) channels are known to play a role in regulation of insulin release, but their role in glucagon secretion is less clear. The increase in ATP/ADP ratio associated with increased glucose metabolism inhibits -cell channels, resulting in depolarization, activation of voltage-gated Ca2+ channels, and a transient rise in cytosolic Ca2+, [Ca2+]i, associated with exocytosis (25). A similar mechanism has been suggested to operate in -cells stimulated by pyruvate, a fuel that can readily enter -cells via monocarboxylate transporters not present in -cells (16) and thus selectively increase the metabolic rate of -cells. In this case, inhibitory intra-islet insulin and/or zinc remains low while the increase in -cell fuel metabolism raises ATP/ADP, closing KATP channels and thus increasing [Ca2+]i, which stimulates glucagon release (16).
We have used KATP channel-null Sur1KO mice to study the role of KATP channels in glucagon secretion. SUR1 and KIR6.2-null mice exhibit dysregulation of insulin secretion (26, 27, 28). There is accumulating evidence that ATP-sensitive K+ channels are involved in brain glucose sensing (18, 29, 30, 31), and a recent study shows an impaired glucagon secretory response in Sur1KO mice (32). Here we evaluate the ability of mouse islets to secrete glucagon in response to a hypoglycemic challenge and use Sur1KO islets to establish a role for KATP channels in the glucagon secretory response to hypoglycemia.
Materials and Methods
Experimental animals
Experiments were performed on mice using protocols approved by the Institutional Animal Care and Use Committee of Baylor College of Medicine and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sur1KO mice were generated by homologous recombination as described previously (26). In vivo and in vitro experiments were done on Sur1KO and control C57BL/6 wild-type (WT) 12- to 16-wk-old male mice maintained on a 12-h light, 12-h dark cycle and fed with standard rodent chow. A 6-h fast was initiated between 0800 and 0900 h; experiments were done between 1400 and 1500 h. Experiments with fed mice were done between 1400 and 1500 h. Data points were obtained from mice euthanized at the time points as indicated in the figure legends.
Hormones and compounds
Short-acting human insulin (0.75 IU/kg) (Humulin R; Eli Lilly, Indianapolis, IN) was used throughout. Human glucagon (0.5 mg/kg) (Calbiochem, La Jolla, CA) was injected ip after a 6-h fast. In perfusion experiments, KATP channels were blocked with glibenclamide (1 μM) (Sigma Chemical Co., St. Louis, MO); L-type voltage-dependent calcium channels were inhibited with nifedipine (1 μmol/liter) (Sigma). Insulin and glucagon were dissolved in 0.9% NaCl before application. Glibenclamide and nifedipine stock solutions were prepared in dimethylsulfoxide (1 mmol/liter) and dissolved in perfusion medium at a final concentration of 1 μmol/liter. The final dimethyl sulfoxide concentration was 0.1%.
Islet isolation and culture
Pancreatic islets were isolated by intraductal injection of 1.0 mg/ml collagenase P (Roche Molecular Biochemicals, Indianapolis, IN), hand picked, and transferred to DMEM supplemented with 10% fetal bovine serum and 5 mmol/liter glucose, 2 mmol/liter glutamine, 2 mmol/liter sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin. Freshly isolated islets were cultured overnight at 37 C in humidified air containing 5% CO2, size matched, divided into groups, and used for static incubation (10 islets) or perfusion experiments (200 islets).
Static determination of insulin and glucagon secretion
Measurements were carried out in freshly prepared Krebs-Ringer bicarbonate HEPES buffer (KRB-HEPES) containing (in mmol/liter) 130 NaCl, 5 KCl, 1.25 KH2PO4, 1.25 MgSO4, 2.68 CaCl2, 5.26 NaHCO3, 10 HEPES (equilibrated with 5% CO2, pH 7.4) supplemented with 0.1% BSA (fraction V), 2 alanine (Ala), 2 arginine-HCl (Arg), and 2 glutamine (Gln) (33, 34) and 100 U/ml penicillin, 100 μg/ml streptomycin, and the indicated concentrations of glucose and reagents. Amino acids were included to simulate conditions in vivo; antibiotics were included to prevent bacterial growth. Islets were washed twice with KRB and preincubated at 37 C with gentle shaking. After 30 min, the medium was replaced with KRB containing 1.0 mmol/liter glucose and incubated for an additional 2 h at which time 0.5-ml aliquots of the supernatant were taken for determination of insulin and glucagon. The islets were homogenized in 70% acid-ethanol and the extracts used to determine total insulin and glucagon contents. Aprotinin (5.0 μg/ml) (Sigma Chemical Co., St. Louis, MO.) was added, and the samples were stored at –20 C for RIA. Determinations were done in duplicate for the number of different islet preparations indicated in the figure legends.
Perfusion assays for insulin and glucagon secretion
Islets were transferred to a column of Bio-Gel P-10 (Bio-Rad) and perfused (0.8 ml/min) with KRB-HEPES containing 2.8 mmol/liter glucose for 30 min to reach a basal state. The glucose concentration was raised to 16.7 mmol/liter for 30 min to stimulate insulin secretion and then lowered to 0.1 mmol/liter glucose for 30 min to stimulate glucagon release. Samples were collected at the indicated time points, aprotinin was added, and the samples were handled as described above.
Glucose measurements
Glucose values were determined from tail vein blood samples using a Free-Style glucometer (Therasense, Alameda, CA).
Insulin and glucagon measurements
Glucagon concentrations in plasma and in samples from static and perfusion experiments were determined by RIA according to the manufacturer’s protocol (Linco, St. Charles, MO.). Plasma insulin was measured in 5.0-μl samples using an ultrasensitive mouse-insulin ELISA kit (ALPCO Diagnostics, Windham, NH). Secreted insulin in samples from static and perfusion experiments was determined using a rat/mouse RIA according to the manufacturer’s protocol (Linco).
Glycogen measurements
Liver glycogen content was determined using the anthrone reaction (35) normalized to protein content determined using the BCA (bicinchoninic acid) assay (Pierce Biotechnology, Inc., Rockford, IL). Values are expressed as micrograms glycogen per microgram protein.
Data analysis
Statistical analyses were performed on paired data using a one-tailed Student’s t test and on grouped data by ANOVA using Bonferroni’s posttest. All data are presented as mean ± SE; P < 0.05 was considered statistically significant. Graphics as well as statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA).
Results
Sur1KO mice exhibit an impaired counterregulatory response to insulin-induced hypoglycemia
To compare their counterregulatory responses, insulin was administered to fed WT and Sur1KO animals to induce hypoglycemia. The initial blood glucose values were the same in both animals, but administration of insulin (0.75 U/kg) produced a more profound hypoglycemia in the knockout animals (Fig. 1A). Blood glucose values returned to normal in the control animals within approximately 90 min, whereas the Sur1KO mice exhibited a slower rate of recovery (Fig. 1A). Although their initial plasma values were equivalent, 15 min of hypoglycemia prompted a 2-fold increase in glucagon level in Sur1KO mice vs. an approximately 5-fold increase in control animals (Fig. 1B). Similar observations have been made in patients with persistent hyperinsulinism of infancy (36) and another Sur1KO mouse model (32). The initial hepatic glycogen contents were the same in both animals, and insulin produced a comparable transient increase in glycogen content during the first 15 min, presumably as a consequence of increased insulin-dependent glucose uptake (37) or because of a greater hepatic glycogen cycling as a result of inhibition of glycogenolysis (38). This transient increase was followed by a marked decline in glycogen content in both animals, although the rate of glycogen use was reduced in the Sur1KO animals (Fig. 1C). The results extend a study using KIR6.2-null mice (29) and are consistent with the idea that the slower recovery of Sur1KO mice from insulin-induced hypoglycemia is in part a consequence of an impaired counterregulatory response with reduced glucagon secretion and consequent failure to mobilize liver glycogen stores efficiently.
Glycemic response to exogenous glucagon
To determine whether differential hormone sensitivity could account for the impaired response, glucagon (0.5 mg/kg) was administered to 6-h-fasted animals. WT mice exhibited a transient, less than 2-fold, increase in blood glucose that returned to the control value within 60 min, whereas the Sur1KO animals displayed a greater, sustained hyperglycemia (Fig. 2A). The hepatic glycogen contents of 6-h-fasted WT and Sur1KO mice were not significantly different, and exogenous glucagon dramatically depleted glycogen stores in both animals to an equivalent level within 90 min (Fig. 2B). The plasma insulin levels were significantly lower in Sur1KO vs. WT mice (Fig. 2C). The results imply the hepatic response to exogenous glucagon is not impaired in the knockout animals and that the prolonged hyperglycemia observed in the Sur1KO mice is a consequence of their previously reported lack of first-phase insulin release when glucose is elevated (26, 28).
Glucagon and insulin release from isolated islets
A previous study reported that glucagon release from KIR6.2-null islets is higher but not statistically different from WT, with approximately a 2-fold increase in static measurements in 16.7 vs. 1 mmol/liter glucose (29). This report focused on the central nervous system (CNS) component, concluding it is impaired. To assess the secretory capacity of Sur1KO -cells further, isolated islets were tested in both static and perifusion assays. When tested under hypoglycemic conditions (2 h in 1.0 mmol/liter glucose plus 2.0 mmol/liter Ala, Arg, and Gln) (see Refs.33 and 34), Sur1KO islets released significantly more insulin vs. control islets (Fig. 3A); glucagon release was consistently lower from the knockout islets but did not achieve statistical significance (Fig. 3C). The total insulin (WT, 1271 ± 121 ng/10 islets; Sur1KO, 1199 ± 153 ng/10 islets) and total glucagon contents (WT, 43.81 ± 5.15 ng/10 islets; Sur1KO, 54.18 ± 1.22 ng/10 islets) were not significantly different after the incubation period in low glucose (P = 0.12; n = 3).
Perifusion assays show that the Sur1KO -cells respond to changes in glucose level, but their response is blunted. Figure 3B illustrates the normal biphasic insulin response of WT islets to a stepwise change in glucose concentration. By contrast, the knockout islets exhibit a small transient decrease and increase in insulin release at the start and end of the high glucose step, respectively, presumably as a consequence of changes in cytosolic Ca2+ as described previously (39), but insulin release continues even in 0.1 mmol/liter glucose. Figure 3D shows that switching WT islets from low to high glucose (2.8–16.7 mmol/liter) decreased glucagon secretion from 238.4 ± 54.1 to 24.1 ± 0.15 pg/ml per 200 islets within 5 min, an approximately 10-fold decrease (P < 0.05; n = 3). In contrast, glucagon secretion from Sur1KO islets was reduced from 161.3 ± 9.38 to 81.4 ± 10.93 pg/ml per 200 islets over the same period (2-fold decrease) (P < 0.05; n = 3). After exposure to high glucose, a low-glucose challenge produced a marked approximately 20-fold increase of glucagon release in WT islets (26.1 ± 4.8 to 540.7 ± 63.9 pg/ml per 200 islets) concomitant with a decrease in insulin release (4.5 ± 0.8 to 1.0 ± 0.1 ng/ml per 200 islets) within 10 min (Fig. 3, B and D, and Table 1). The equivalent switch with Sur1KO islets produced an increase in glucagon secretion (73.8 ± 4.7 to 196.1 ± 19.73 pg/ml per 200 islets) and an overall decrease in insulin secretion (5.1 ± 0.2 to 3.8 ± 0.4 ng/ml per 200 islets). Note, however, that although the increased glucagon release from WT islets correlates with a monotonic fall in insulin secretion over the first 10 min, the period when the rise in glucagon release is maximal, the Sur1KO islets actually increase their rate of insulin secretion, reaching a peak value of 7.88 ± 1.03 ng/ml per 200 islets. The results show that the glucagon response to low glucose is attenuated and that there is an uncoupling of the communication between - and -cells in the Sur1KO islets.
Increased intra-islet insulin (23, 40) and/or zinc (41, 42) have been implicated in suppression of glucagon release, and our WT data are consistent with a strong inverse correlation between insulin and glucagon secretion that is perturbed or uncoupled in Sur1KO islets. To assess the effect(s) of -cell secretion on glucagon release further, WT and Sur1KO islets were challenged with low glucose plus glibenclamide (1 μmol/liter) to block KATP channels. Glibenclamide strongly stimulates insulin secretion from WT islets in 0.1 mmol/liter glucose and significantly blunts glucagon release the WT glucagon response to a 0.1 mmol/liter glucose challenge (compare responses of WT in Fig. 4A with WT in Fig. 3D, and see Table 1). Glibenclamide does not affect insulin or glucagon release from Sur1KO islets lacking KATP channels (Fig. 4B and Table 1). Note that the levels of glucagon secretion from WT islets treated with glibenclamide mimic the impaired release observed for Sur1KO islets (compare Fig. 4A with Figs. 3D and 4B). The results are consistent with the partial suppression of glucagon release by -cell secretory products acting via KATP channels (43).
Exocytosis of both insulin and glucagon requires Ca2+. To ensure that the hormone release observed in response to profound hypoglycemic challenge was specific, we evaluated the ability of nifedipine, an L-type Ca2+ channel blocker, to suppress secretion induced by low glucose. Figure 5A shows that 1 μmol/liter nifedipine inhibits the marked increase in glucagon release observed when WT islets are challenged with 0.1 mol/liter glucose (see Fig. 3D) and similarly inhibits the smaller glucagon release from Sur1KO. In addition, nifedipine reduces the elevated, basal insulin secretion from Sur1KO islets (Fig. 5B). These observations confirm our earlier reports that nifedipine will suppress persistent insulin release from Sur1KO islets (26, 39). Table 1 summarizes the insulin and glucagon secretion values at 30 min after switching the glucose concentration from 16.7 to 0.1 mmol/liter glucose in the presence of glibenclamide or nifedipine in WT and Sur1KO islets. The Sur1KO islets have an increased output of insulin and a decreased output of glucagon in response to hypoglycemic challenge compared with WT islets. Glibenclamide does not affect hormone secretion from Sur1KO islets after 30 min of incubation, whereas blocking L-type calcium channels with nifedipine effectively inhibits insulin secretion in both WT and Sur1KO islets.
Discussion
Sur1KO mice exhibit a reduced rate of glucagon release in response to insulin-induced hypoglycemia, indicating that SUR1/KIR6.2-type KATP channels play a role in the counterregulatory response. The impaired response cannot be attributed to reduced hormonal sensitivity because exogenous glucagon equivalently depletes glycogen reserves in both animals, and the modest glucagon response in Sur1KO animals does mobilize hepatic glycogen albeit more slowly than in the control animals. Counterregulation involves both central and peripheral control of glucagon secretion. Although we have not quantified the relative contributions, SUR1/KIR6.2 channels are expected to have a role in both central and peripheral control, and these data show that isolated Sur1KO islets do exhibit a deficient secretory response to hypoglycemia consistent with impaired peripheral control. The results extend the analysis reported for KIR6.2-null islets (29); we observe an increase in glucagon release in static experiments under low-glucose conditions and show a pronounced blunting of the glucagon secretory response when islets are subject to a hypoglycemic challenge after exposure to elevated glucose, a protocol described by others (23, 24). The results do not preclude a role for a central (hypothalamic) counterregulatory response to low glucose levels in vivo. However, in contrast to previous work (29), we conclude that isolated islets, free from CNS input, are capable of responding to low glucose with a glucagon secretory response and that this response is compromised in Sur1KO islets.
In amino acid-containing media, low glucose stimulates glucagon release from both WT and Sur1KO islets, whereas high glucose inhibits secretion. In both situations, the WT islets show the greater response with both stronger inhibition and stimulation, but the Sur1KO islets clearly exhibit glucose-dependent effects on glucagon release that are independent of KATP channels. The stronger inhibition in control islets is consistent with the idea that intra-islet insulin or cosecreted zinc acts to suppress glucagon release (13, 43), reviewed by Samols and Stagner (15, 44), specifically that insulin (40, 43) or zinc (41, 42, 43) can activate KATP channels and thus hyperpolarize -cells, which would reduce the activity of voltage-gated Ca2+ channels and lower [Ca2+]i necessary for exocytosis. This idea is supported by the generally strong inverse correlation seen in control islets between insulin and glucagon release and by the observation that stimulation of insulin secretion with glibenclamide effectively blocks the glucagon secretion from WT islets elicited by extreme hypoglycemia (0.1 mmol/liter or 1.8 mg/dl) without significantly affecting release from knockout islets under the same stimulus (Fig. 4). Surprisingly, although the loss of -cell KATP channels appears to uncouple glucagon release from the inhibitory effects of -cell secretion, it does not produce hyperglucagonemia. Given that intra-islet zinc/insulin is expected to be consistently elevated in the Sur1KO islets, an inhibitory mechanism might be invoked to explain their attenuated glucagon release, in which case a KATP-independent inhibitory pathway must be involved. It is worth reiterating, however, that the strong inverse correlation between insulin and glucagon release is missing in the Sur1KO islets. This can be seen clearly, for example, in Fig. 3, B and D, where high glucose reduces Sur1KO glucagon release without altering insulin secretion, and the shift to very low glucose stimulates secretion of insulin during the period when glucagon release is increasing most rapidly. The results support the idea that -cells have a two-tier control system in which -cell glucagon secretion is tightly coupled to release of zinc-insulin by -cells via KATP channels but have an underlying KATP-independent regulatory mechanism that is regulated by fuel metabolism. The nature of the underlying mechanism is not understood but may be similar to the control(s) regulating insulin release in KATP-null -cells (39, 45).
A previous report (46) indicated that glucagon secretion in mouse islets was mediated by N-type Ca2+ channels and only modestly affected by L-type Ca2+ channel antagonists (nifedipine at 20 μmol/liter). Therefore, we attempted to inhibit insulin secretion from Sur1KO islets with nifedipine in an effort to mimic the fall in insulin seen in WT islets and test the idea that falling insulin and falling glucose would enhance glucagon secretion in the absence of KATP channels. However, consistent with a role for L-type Ca2+ channels and a requirement for elevated [Ca2+]i, nifedipine inhibited insulin and glucagon release from both WT and Sur1KO islets. The suppression of glucagon release from Sur1KO islets is more pronounced than the controls possibly as a consequence of tonic inactivation of N- and T-type calcium channels as suggested previously (47). On the other hand, glucagon secretion in response to epinephrine is reported to involve the activation of store-operated currents (48), emphasizing the importance of intracellular calcium changes.
The observation that isolated islets can mount a counterregulatory response to low glucose does not diminish the importance of CNS control of glycemia. The role(s) for hypothalamic KATP channels in counterregulation and control of hepatic gluconeogenesis are well established (30, 31). Clinical data on patients with persistent hyperinsulinemic hypoglycemia of infancy, some with mutations in the SUR1/ABCC8 gene, show that their glucagon counterregulatory response is impaired, although they exhibit appropriate increases in serum catecholamines during hypoglycemia (36). The data are consistent with mechanisms local to the -cell blunting the serum glucagon counterregulatory response and/or that centers outside the ventromedial hypothalamus are involved in the catecholamine response to hypoglycemia.
In summary, pancreatic islets can sense and respond directly to changes in ambient glucose and mount a counterregulatory response in vitro, secreting glucagon in response to hypoglycemia, independent of CNS regulation. Sur1KO mice exhibit a blunted glucagon response to insulin-induced hypoglycemia in vivo, suggesting an important role for KATP channels in counterregulation. Our isolated islet data are consistent with a KATP-dependent -/-cell dialog in which glucagon release is suppressed by intra-islet insulin and/or Zn2+. The blunted counterregulatory response observed in KATP-null mice may thus have multiple causes: uncoupling of the -/-cell dialog, impaired peripheral control secondary to loss of KATP channels (18, 29), or a smaller readily releasable pool of glucagon granules in Sur1KO -cells (49). We suggest that the deficient glucagon counterregulatory response seen in persistent hyperinsulinemic hypoglycemia of infancy (36) could originate either from the loss of peripheral control similar to that observed in Sur1KO animals or, in cases where functional KATP channels remain, could result from suppression of glucagon release by elevated intra-islet insulin- and/or Zn2+-mediated -cell hyperpolarization (17). Additional clinical and laboratory studies are required to understand the detailed interactions between pancreatic - and -cells and the role of their dialog in glucose homeostasis.
Acknowledgments
We thank Rebecca A. Schneider for helpful discussion.
Footnotes
This work was supported by Juvenile Diabetes Research Foundation International (3-2003-508 to A.M. and 1-2005-950 to J.B.), the Sistema Nacional de Investigadores/Mexico (30370 to A.M.), and the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (DK57671 to L.A-B., DK52771 to J.B., and DK02285 to A.S.R.).
First Published Online August 25, 2005
1 A.M. and M.H. contributed equally to this work.
Abbreviations: CNS, Central nervous system; KATP, ATP-sensitive K+ channel; WT, wild type.
Accepted for publication August 18, 2005.
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