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Published online January 24, 2007
Diabetes 56:1120-1126, 2007
DOI: 10.2337/db06-1102
© 2007 by the American Diabetes Association
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ATP-Sensitive K+ Channels Regulate the Release of GABA in the Ventromedial Hypothalamus During Hypoglycemia

Owen Chan1, Marcus Lawson2, Wanling Zhu1, Joseph Lee Beverly2, and Robert S. Sherwin1

1 Department of Internal Medicine, Section of Endocrinology, Yale University School of Medicine, New Haven, Connecticut
2 Department of Animal Sciences, University of Illinois at Urbana, Champaign, Urbana, Illinois

Address correspondence and reprint requests to Dr. Robert S. Sherwin, Yale University School of Medicine, Department of Internal Medicine, Section of Endocrinology, 300 Cedar St., TAC S141, New Haven, CT. E-mail: robert.sherwin{at}yale.edu

Abbreviations: BIC, bicuculline methiodide; KATP channel, ATP-sensitive K+ channel; VMH, ventromedial hypothalamus


   ABSTRACT
TOP
 ABSTRACT
RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OBJECTIVE—To determine whether alterations in counterregulatory responses to hypoglycemia through the modulation of ATP-sensitive K+ channels (KATP channels) in the ventromedial hypothalamus (VMH) are mediated by changes in GABAergic inhibitory tone in the VMH, we examined whether opening and closing KATP channels in the VMH alter local GABA levels and whether the effects of modulating KATP channel activity within the VMH can be reversed by local modulation of GABA receptors.

RESEARCH DESIGN AND METHODS—Rats were cannulated and bilateral guide cannulas inserted to the level of the VMH. Eight days later, the rats received a VMH microinjection of either 1) vehicle, 2) the KATP channel opener diazoxide, 3) the KATP channel closer glybenclamide, 4) diazoxide plus the GABAA receptor agonist muscimol, or 5) glybenclamide plus the GABAA receptor antagonist bicuculline methiodide (BIC) before performance of a hypoglycemic clamp. Throughout, VMH GABA levels were measured using microdialysis.

RESULTS—As expected, diazoxide suppressed glucose infusion rates and increased glucagon and epinephrine responses, whereas glybenclamide raised glucose infusion rates in conjunction with reduced glucagon and epinephrine responses. These effects of KATP modulators were reversed by GABAA receptor agonism and antagonism, respectively. Microdialysis revealed that VMH GABA levels decreased 22% with the onset of hypoglycemia in controls. Diazoxide caused a twofold greater decrease in GABA levels, and glybenclamide increased VMH GABA levels by 57%.

CONCLUSIONS—Our data suggests that KATP channels within the VMH may modulate the magnitude of counterregulatory responses by altering release of GABA within that region.

The established benefits of maintaining near-normal blood glucose levels in patients with diabetes are limited by the risk of severe hypoglycemia (1,2). The problem is compounded by the fact that with each reoccurring episode of hypoglycemia, the body's ability to restore blood glucose levels to normal is compromised (3). Therefore, it is essential that we achieve a better understanding of how the body senses and activates defense mechanisms against hypoglycemia so that we can develop therapeutic strategies to more effectively prevent or minimize hypoglycemia in diabetic patients.

Specific regions within the brain (4–12) and periphery (13–15) have been shown to respond to changes in glucose concentrations. One brain region in particular, the ventromedial hypothalamus (VMH), contains glucose-responsive neurons that detect changes in ambient glucose levels and then alter their firing rate accordingly (5,6,16–19). Two predominant subtypes of glucose-sensitive neurons exist within the brain: the glucose-excited neurons that increase and the glucose-inhibited neurons that decrease their firing rate as glucose levels rise (19,20). Kang et al. (18) have demonstrated that these neurons contain much of the same glucose-sensing machinery (e.g., glucokinase, ATP-sensitive K+ channels [KATP channels], etc.) as pancreatic ß-cells.

The KATP channels provide a close link between neuronal metabolism and the regulation of membrane potential in many tissues (21). KATP channels are comprised of two subunits: a sulfonylurea receptor around the periphery and an inwardly rectifying K+ channel forming the K+ conducting pore (22). In the pancreas, the KATP channel has been shown to play a key role in insulin release in response to changing glucose levels (23,24). In the ß-cell, the KATP channel indirectly senses glucose fluctuations at least in part through changes in the intracellular ATP-to-ADP ratio (23,24), which cause the channel to open or close.

KATP channels are also expressed throughout the brain, including in hypothalamic regions thought to be involved in glucose sensing (16,25–29). Electrophysiological studies of rat (30–32) and mouse brain-slice preparations (33) have demonstrated that sulfonylureas can stimulate the firing of glucose-excited neurons and can alter the response of glucose-excited neurons to changes in ambient glucose levels. Moreover, Kir6.2-knockout mice show impaired glucose counterregulation (33). In keeping with these data, we have recently shown in vivo that pharmacological manipulation of KATP channel activity in the VMH can suppress (glybenclamide) or enhance (diazoxide) hormonal counterregulatory hormone responses to systemic insulin-induced hypoglycemia (34,35).

It is noteworthy that our laboratory has also recently reported that pharmacological manipulation of GABAergic inhibitory tone in the VMH can have a profound effect on the magnitude of glucose counterregulatory responses to hypoglycemia (36). More specifically, microinjection of the GABAA receptor agonist muscimol into the VMH suppressed, whereas pharmacological blockade of GABAergic inhibition with bicuculline methiodide amplified, glucagon and epinephrine responses to hypoglycemia.

These findings raised the possibility that the impact of opening and closing of KATP channels within the VMH on glucose counterregulation might be mediated by local modulation of GABA release during hypoglycemia. The current findings link these three latter studies and provide intriguing support for this hypothesis.


   RESEARCH DESIGN AND METHODS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Male Sprague-Dawley rats (Charles River, Richmond, VA) weighing 300–350 g were individually housed in the Yale Animal Resources Center in temperature- (22–23°C) and humidity-controlled rooms. The animals were fed rat chow (Agway Prolab 3,000; Syracuse, NY) and water ad libitum, were acclimatized to handling, and were kept on a 12-h light/dark cycle (lights on between 0700 and 1900 h) for a period of 1 week before experimental manipulation. Principles of laboratory animal care were followed, and experimental protocols were approved by the Institutional Animal Care and Use Committee at Yale University.

Surgery.
Approximately 8–10 days before the experiment, the animals underwent aseptic surgery to have both vascular catheters and steel microdialysis guide cannulas implanted.

Vascular surgery.
Vascular catheters were prepared using a piece of polyethylene tubing (PE-50) attached to a 3-cm piece of silicone tubing. The silicone end of the tubing was inserted into the left carotid artery for blood sampling and into the right jugular vein for infusion, as described previously (36). Catheters were then tunneled subcutaneously and exteriorized at the back of the neck.

Stereotaxic surgery.
The animals were placed into a stereotaxic frame (David Kopf Instruments, Tujunga, CA), and stainless steel guide cannulas (Eicom, Kyoto, Japan) for microdialysis and microinjection (from bregma: 2.6 mm posterior, 3.8 mm lateral, and 8.9 mm ventral, at an angle of 16°) were bilaterally inserted down to the level of the VMH and secured in place with dental acrylic (35).

Microdialysis.
Twenty-four hours before the microdialysis experiment, a combination microdialysis-microinjection probe (Eicom) that extends 1 mm beyond the tip of the guide cannula was inserted into the brain and left in place for 5 min before being removed and replaced with a stylet until the start of the experiment on the subsequent day. This insertion is timed to give optimal measurement conditions the following day (37) and to minimize both tissue disturbance and glial scarring at the probe site (38). The next day the animals were connected to infusion pumps, and then bilateral microdialysis-microinjection probes were inserted down to the level of the VMH. Artificial extracellular fluid was then perfused through the microdialysis probe at a constant rate of 1.5 µl/min for 2.5–3 h to allow for GABA levels to stabilize before the start of microdialysate sample collection (37). Collection of microdialysate samples at 10-min intervals began 45 min before the start of the insulin infusion (0') to establish stable baseline GABA concentrations. The values collected during this period were then averaged together to determine a single "baseline" value. Ten-minute microdialysate collections continued for the duration of the study.

Microinjection.
Just before the start of the insulin infusion, blood was withdrawn from the arterial line to assess baseline hormone levels. The animals were then microinjected over the course of 1 min with 0.1 µl of 1) artificial extracellular fluid with 0.5% DMSO vehicle (controls, n = 7), 2) the KATP channel opener diazoxide (1 nmol per side; n = 8), 3) diazoxide plus the GABAA receptor agonist muscimol (1 nmol each per side; n = 7), 4) the KATP channel inhibitor glybenclamide (1 nmol per side; n = 7), or 5) glybenclamide plus the GABAA receptor antagonist bicuculline methiodide (BIC) (1 nmol glybenclamide and 12.5 pmol BIC per side; n = 6) using a CMA/102 pump (CMA Microdialysis, North Chelmsford, MA). The dose of BIC used here is a subconvulsive dose determined in a previously described pilot study (36).

Hypoglycemic clamp.
Following microinjection, a constant infusion of regular human insulin (50 mU · kg–1 · min–1; Eli Lilly, Indianapolis, IN) and a variable 20% glucose infusion were started and plasma glucose levels lowered and maintained at 45 mg/dl for 90 min. Blood samples were collected at 30-min intervals throughout the study for subsequent measurement of plasma glucagon, catecholamine, and corticosterone responses and at 60-min intervals for plasma insulin concentrations. Following each sample collection, the erythrocytes were resuspended in an equivalent volume of artificial plasma (39) and reinfused back into the animal to prevent volume depletion and anemia. At the end of the study, following collection of the final blood and microdialysate samples, the animals were killed with an overdose of sodium pentobarbital and the brains removed and frozen in dry ice. Subsequently, the accuracy of probe placements was determined histologically by visual inspection of coronal brain sections. Only data obtained from those animals where the microdialysis probes were positioned beside the VMH were used.

Hormone and microdialysate analysis.
Plasma catecholamine concentrations were analyzed by high-performance liquid chromatography using electrochemical detection, while plasma hormone concentrations were determined using commercially available radioimmunoassay kits (40).

VMH GABA concentrations from microdialysate samples were determined using high-performance liquid chromatography (41,42). Changes in VMH GABA levels were calculated as follows. GABA concentrations collected during the basal period from –45 to 0 min, just before the start of the insulin infusion, were averaged together to create a single "baseline value." Subsequently, the changes in GABA concentration were determined by calculating percentage differences in GABA concentrations during the clamping period from that single baseline value.

Statistical analysis.
Treatment effects was analyzed using one- or two-way ANOVA for independent or repeated measures as appropriate, followed by post hoc analysis using the Statistica suite of analytical software for personal computers by StatSoft. P < 0.05 was set as the criterion for statistical significance.


   RESULTS
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Baseline.
Baseline plasma glucose and hormone concentrations, as well as extracellular GABA concentrations, were similar between all five treatment groups (Table 1).


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  		TABLE 1 Baseline plasma hormone and extracellular GABA concentrations of animals microinjected with artificial extracellular fluid (control), diazoxide, diazoxide and the GABAA receptor agonist muscimol, glybenclamide, or glybenclamide and the GABAA receptor antagonist BIC

 
Hypoglycemia.
Plasma glucose and insulin concentrations between the five treatment groups were maintained at similar levels throughout the study (Figs. 1 and 2A). Nevertheless, average glucose infusion rates over the final 90 min of the hypoglycemic clamp varied significantly among the different treatment groups (Fig. 2B). Prior VMH microinjection of the KATP channel opener diazoxide decreased glucose infusion rates by ~38% (P < 0.01). This effect was reversed by the addition of the GABAA receptor agonist muscimol; glucose infusion requirements rose ~30% above control levels (P < 0.04). In contrast, closure of KATP channels in the VMH with glybenclamide increased (~28%, P < 0.04) and coadministration of the GABAA receptor antagonist BIC with glybenclamide reduced (~39%, P < 0.01) exogenous glucose requirements compared with control levels.


Figure 1
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  		FIG. 1. Plasma glucose concentrations of controls (n = 7), diazoxide (DZ; n = 8), diazoxide + GABAA receptor agonist muscimol (DZ + MUS; n = 7), glybenclamide (GLYB; n = 7), and glybenclamide + the GABAA receptor antagonist BIC (GLYB + BIC; n = 6) during the hyperinsulinemic-hypoglycemic glucose clamp.

 

Figure 2
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  		FIG. 2. Average plasma insulin concentrations (A) and glucose infusion rates (B) during the final 90 min of the hypoglycemic clamp in controls (n = 7), diazoxide (DZ; n = 8), diazoxide + GABAA receptor agonist muscimol (DZ + MUS; n = 7), glybenclamide (GLYB; n = 7), and glybenclamide + the GABAA receptor antagonist BIC (GLYB + BIC; n = 6). Results are presented as mean ± SEM. *P < 0.01 vs. controls. {dagger}P < 0.04 vs. controls.

 
Changes in glucose infusion rates were corroborated by changes in peak plasma glucagon and epinephrine levels. During hypoglycemia, diazoxide increased plasma glucagon and epinephrine responses by ~51–56% (P < 0.05), whereas addition of muscimol suppressed both glucagon and epinephrine responses by ~50–57% (P < 0.05) (Fig. 3A and B). Conversely, microinjection of glybenclamide alone suppressed glucagon and epinephrine responses to hypoglycemia by ~45–56% (P < 0.05), and coadministration of the GABA receptor antagonist with glybenclamide amplified these counterregulatory responses ~54–140% (P < 0.05) above control values (Fig. 3A and B).


Figure 3
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  		FIG. 3. Plasma glucagon (A) and epinephrine (B) responses during the hypoglycemic clamp in controls (n = 7), diazoxide (DZ; n = 8), diazoxide + GABAA receptor agonist muscimol (DZ + MUS; n = 7), glybenclamide (GLYB; n = 7), and glybenclamide + the GABAA receptor antagonist BIC (GLYB + BIC; n = 6). Results are presented as mean ± SEM. *P < 0.05 vs. controls. {dagger}P < 0.001 vs. controls. §P < 0.01 vs. controls.

 
During these studies, no changes in the response of plasma norepinephrine (control 569 ± 79, diazoxide 729 ± 84, diazoxide + muscimol 467 ± 75, glybenclamide 706 ± 148, and glybenclamide + BIC 817 ± 158 pg/ml) or corticosterone (control 564 ± 27, diazoxide 487 ± 26, diazoxide + muscimol 540 ± 30, glybenclamide 506 ± 27, and glybenclamide + BIC 555 ± 55 ng/ml) to hypoglycemia were detected.

Interestingly, changes in plasma hormone concentrations during hypoglycemia were inversely associated with changes in VMH extracellular GABA concentrations (Fig. 4). As plasma glucose levels fell to a nadir of 45 mg/dl, VMH GABA concentrations decreased 22% from baseline levels in control animals (P < 0.05). Opening VMH KATP channels with diazoxide further decreased GABA levels by another 2.2-fold (P < 0.05). On the other hand, closure of these channels with glybenclamide raised VMH GABA levels to ~62% above baseline values. Despite the fact that the GABAA receptor agonist and antagonist had such dramatic effects on counterregulatory hormone responses to hypoglycemia, these compounds did not further influence VMH GABA levels beyond that observed with the KATP channel modulators alone when they were co-administered.


Figure 4
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  		FIG. 4. Percentage change from baseline levels of VMH GABA concentrations in controls (n = 7), diazoxide (DZ; n = 8), diazoxide + GABAA receptor agonist muscimol (DZ + MUS; n = 7), glybenclamide (GLYB; n = 7), and glybenclamide + the GABAA receptor antagonist BIC (GLYB + BIC; n = 6). Results are presented as mean ± SEM. *P < 0.05 vs. controls. {dagger}P < 0.05 vs. baseline.

 

   DISCUSSION
TOP
 ABSTRACT
 RESEARCH DESIGN AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
KATP channels are present in brain cells and play a role in neurosecretion at nerve terminals (43). Early studies by Margaill et al. (44) showed that the sulfonylurea glyburide increased GABA release in hippocampal slices cultured under aglycemic conditions. These observations led the authors to suggest that KATP channels may play a role in regulating GABAergic activity during hypoglycemia. A subsequent study by During et al. (45) showed that perfusion of either 10 mmol/l of glucose or glipizide, a sulfonylurea, into the substantia nigra increased and that insulin-induced hypoglycemia decreased GABA release, suggesting that glucose may act as a signaling molecule in this brain region. The current study extends these findings to the VMH, a region of the brain demonstrated to play an important role in regulating the hormonal response to hypoglycemia. Here, we show that KATP channels in this crucial brain glucose–sensing region can modulate the magnitude of counterregulatory responses to hypoglycemia in association with changes in GABA release. The use of a combination microdialysis-microinjection technique allowed us to locally deliver substances into the VMH to pharmacologically modulate KATP channel gating while simultaneously microdialyzing that region for changes in GABA concentrations. We showed during hyperinsulinemic-hypoglycemic conditions that closing KATP channels in the VMH increases local GABA levels, while opening these channels lowers GABA levels. Of particular interest is that these changes in GABA concentrations were associated with a respective suppression and amplification of glucagon and epinephrine release in response to insulin-induced hypoglycemia. Whether alterations in VMH GABA levels can influence counterregulatory hormone levels in the absence of hypoglycemia remains to be determined.

In keeping with earlier studies from our laboratory, local VMH delivery of diazoxide increased and glybenclamide decreased counterregulatory responses to hypoglycemia (34,35). Of particular interest, when the GABAA receptor agonist muscimol was co-administered with diazoxide, it completely reversed the amplified glucagon and epinephrine responses to hypoglycemia seen with diazoxide alone. On the other hand, the reduced hormonal responses seen with VMH delivery of glybenclamide were reversed and actually increased above control values by the addition of the GABAA receptor antagonist. These findings suggest that the capacity of KATP channels within the VMH to modulate the amplitude of counterregulatory responses during hypoglycemia may be, at least in part, mediated by altering GABAergic tone in the VMH.

To determine whether KATP channel gating can affect GABA release in the VMH, we used microdialysis to quantify changes in extracellular GABA concentrations in the VMH following microinjection of KATP channel modulators. During hypoglycemia, VMH GABA concentrations decreased 22% below baseline levels. The decrease in VMH GABA concentrations corresponded to activation of both glucagon and epinephrine responses and thus may be required for full activation of peripheral counterregulatory responses as glucose levels fall. This possibility is consistent with our previously published findings demonstrating that microinjection of a GABAA receptor agonist into the VMH suppressed, whereas microinjection of a GABAA receptor antagonist amplified, counterregulatory responses to hypoglycemia (36).

Further support for this hypothesis is derived from the current studies using modulators of KATP channel activity. The activation of KATP channels with diazoxide further decreased VMH GABA levels compared with controls, and this corresponded to an amplification of counterregulatory hormone responses and a lowering of glucose infusion rates during the clamp. Conversely, inactivation of KATP channels with glybenclamide paradoxically increased VMH GABA levels and suppressed counterregulatory responses. Interestingly, reversal of the diazoxide effects on counterregulation by coadministration of muscimol was not associated with a change in VMH GABA levels. Similarly, BIC also did not alter VMH GABA levels beyond what was seen with glybenclamide alone, yet it did amplify counterregulatory responses above control levels. These findings are consistent with the idea that GABAA receptors may be located downstream of KATP channels. Thus, falling brain glucose levels may activate KATP channels within glucose responsive neurons of the VMH. This in turn would hyperpolarize the neuron, decrease GABA output, and allow for full activation of peripheral counterregulatory responses.

This interpretation may at first glance appear to differ from that reported by Beverly and colleagues (42,46,47). In those studies, it was shown that during an acute episode of hypoglycemia, VMH GABA levels increased in a biphasic manner, and the rise may be mediated by increases in norepinephrine levels. It is noteworthy that we used different coordinates than Beverly and colleagues for our microdialysis, and thus we may be examining the effects of different subpopulations of neurons within the VMH. One might speculate that that there may be multiple GABAergic synapses within the VMH, both noradrenergic inputs onto GABAergic neurons as well as GABAergic regulation of these noradrenergic inputs.

In keeping with earlier studies, none of our treatments affected corticosterone responses to hypoglycemia, suggesting that activation of the hypothalamo-pituitary-adrenal axis is not mediated through changes in the activation of GABAergic neurons within the VMH and may require signals emanating from neurons residing outside of the VMH and possibly from the dorsomedial nucleus and/or the paraventricular nucleus itself (48). In accordance with this hypothesis, we noted that manipulation of GABAergic tone within the hypothalamic paraventricular nucleus specifically altered adrenocortical responses to hypoglycemia and not those of glucagon or epinephrine (O.C., unpublished observations). This would be consistent with the idea of a redundant failsafe mechanism.

While in vivo microinjection certainly provides a greater specificity by targeting specific brain regions, it is not possible to completely exclude effects outside a region of interest. The slow rate of delivery, small volume of injection (0.1 µl), and rapid fall in drug concentration from the injection site suggest that a primary action in neighboring glucose-sensing regions (e.g., dorsomedial hypothalamus and paraventricular nucleus) is unlikely. The fact that we did not observe changes in corticosterone responses in the current study suggests that diffusion to these brain regions is unlikely to have occurred. Moreover, the authors do caution that the current study does rely on pharmacological agents that can alter channel activity in neurons that do not normally either sense glucose as a signal and/or in neurons such as glucose-inhibited ones that do not normally utilize the KATP channel to control their firing rates.

In conclusion, we have demonstrated that during hyperinsulinemic-hypoglycemia, the activation state of KATP channels in the VMH acts to modulate GABAergic output in the VMH, and this in turn appears to regulate the magnitude of glucagon and epinephrine responses during hypoglycemia. Such data findings may have important implications for diabetic patients with defective glucose counterregulation. Studies in the streptozotocin-induced diabetic rat, a model of type 1 diabetes, have shown that VMH GABAergic systems are impaired in these animals (41,49). Hence, a better understanding of the mechanisms underlying defective GABAergic function within the VMH may offer novel therapeutic targets for restoring counterregulatory responses in diabetes.


   ACKNOWLEDGMENTS
 
This work was generously supported by research grants from the National Institutes of Health (DK20495 and P30DK45735) and the Juvenile Diabetes Research Foundation Center for the Study of Hypoglycemia. O.C. is a recipient of the Canadian Institutes of Health Research Post-Doctoral Research Fellowship.

The authors are grateful to Ralph Jacob, Aida Groszmann, and Andrea Belous for their excellent technical support and assistance.


   FOOTNOTES
 
Published ahead of print at http://diabetes.diabetesjournals.org on 24 January 2007. DOI: 10.2337/db06-1102.

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

Received for publication August 8, 2006 and accepted in revised form January 12, 2007


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