male sprague - dawley rats ( charles river , wilmington , ma ) weighing 275350 g were individually housed in the yale animal resource center in temperature - controlled ( 2223c ) and humidity - controlled rooms .
the animals were fed rat chow ( agway prolab 3000 , syracuse , ny ) , given water ad libitum , and acclimatized to a 12-h light / dark cycle .
seven to 10 days before each study , all animals were anesthetized with an injection ( 1 ml / kg i.p . ) of a mixture of xylazine ( 20 mg / ml anased ; lloyd laboratories , shenandoah , ia ) and ketamine ( 100 mg / ml ketaset ; aveco , fort dodge , ia ) , in a ratio of 1:2 ( vol / vol ) , before undergoing vascular surgery for the implantation of vascular catheters in a carotid artery and jugular vein .
microinjection guide cannulae were then bilaterally inserted into the vmh , targeting the ventromedial nucleus ( coordinates from bregma : anteroposterior 2.6 mm , mediolateral 0.8 mm , and dorsoventral 8.0 ) . on the morning of the study , after an overnight fast and 60 min after opening the catheters , 22-gauge microinjection needles , designed to extend 1 mm beyond the tip of the guide cannula ( plastics one , roanoke , va ) , were inserted through the guide cannula bilaterally into each vmh . the rat was then microinjected over 2 min ( 0.5 l / min ) using a cma-102 infusion pump ( cma microdialysis , north chelmsford , ma ) .
the animals were microinjected immediately before the initiation of a hyperinsulinemic - hypoglycemic clamp with 1 l of one the following pharmacologic agents : artificial extracellular fluid as a control ( n = 12 ) ; 1 mol / l concentration of the b2ar antagonist ici-118,551 ( n = 12 ) ; or 1 mol / l concentration of the b2ar agonist formoterol ( n = 10 ) .
the doses of b2ar agonists and antagonists selected were based on preliminary dose - ranging studies using a 1 nmol / l to 200 mol / l concentration of the agonist and antagonist . in another set of studies , we microinjected 1 mol / l cgp 20712 dihydrochloride , a potent , selective b1ar ( n = 7 ) , or 1 mol / l sr59230a hydrochloride , a potent , selective b3ar antagonist ( n = 6 ) . a primed - continuous intravascular infusion of 20 mu  kg  min regular insulin was started , and a variable infusion of 20% dextrose was adjusted in response to 10-min measurements of plasma glucose to reach a target value of 50 mg / dl within 30 min .
thereafter , plasma glucose was monitored at 10-min intervals using a glucose analyzer ( analox instruments , lunenburg , ma ) to adjust the dextrose infusion rate to maintain a stable 50 mg / dl glucose level until the end of the study at 90 min .
blood was drawn at baseline and at 30 , 60 , and 90 min for measurement of plasma glucagon , epinephrine , norepinephrine , and insulin . at the end of experiments ,
the rats were killed with an overdose of sodium pentobarbital ( sleepaway ; fort dodge animal health , fort dodge , ia ) .
catecholamine analysis was performed by high - performance liquid chromatography using electrochemical detection ( esa , acton , ma ) . plasma insulin and glucagon
baseline comparisons were performed using anova . a mixed - model analysis with an unstructured covariance matrix
analyses were performed using sas 9.2 software ( sas institute , inc . , cary , nc ) , with a threshold p < 0.05 considered as statistically significant .
seven to 10 days before each study , all animals were anesthetized with an injection ( 1 ml / kg i.p . ) of a mixture of xylazine ( 20 mg / ml anased ; lloyd laboratories , shenandoah , ia ) and ketamine ( 100 mg / ml ketaset ; aveco , fort dodge , ia ) , in a ratio of 1:2 ( vol / vol ) , before undergoing vascular surgery for the implantation of vascular catheters in a carotid artery and jugular vein .
microinjection guide cannulae were then bilaterally inserted into the vmh , targeting the ventromedial nucleus ( coordinates from bregma : anteroposterior 2.6 mm , mediolateral 0.8 mm , and dorsoventral 8.0 ) .
on the morning of the study , after an overnight fast and 60 min after opening the catheters , 22-gauge microinjection needles , designed to extend 1 mm beyond the tip of the guide cannula ( plastics one , roanoke , va ) , were inserted through the guide cannula bilaterally into each vmh . the rat was then microinjected over 2 min ( 0.5 l / min ) using a cma-102 infusion pump ( cma microdialysis , north chelmsford , ma ) .
the animals were microinjected immediately before the initiation of a hyperinsulinemic - hypoglycemic clamp with 1 l of one the following pharmacologic agents : artificial extracellular fluid as a control ( n = 12 ) ; 1 mol / l concentration of the b2ar antagonist ici-118,551 ( n = 12 ) ; or 1 mol / l concentration of the b2ar agonist formoterol ( n = 10 ) .
the doses of b2ar agonists and antagonists selected were based on preliminary dose - ranging studies using a 1 nmol / l to 200 mol / l concentration of the agonist and antagonist . in another set of studies , we microinjected 1 mol / l cgp 20712 dihydrochloride , a potent , selective b1ar ( n = 7 ) , or 1 mol / l sr59230a hydrochloride , a potent , selective b3ar antagonist ( n = 6 ) .
a primed - continuous intravascular infusion of 20 mu  kg  min regular insulin was started , and a variable infusion of 20% dextrose was adjusted in response to 10-min measurements of plasma glucose to reach a target value of 50 mg / dl within 30 min .
thereafter , plasma glucose was monitored at 10-min intervals using a glucose analyzer ( analox instruments , lunenburg , ma ) to adjust the dextrose infusion rate to maintain a stable 50 mg / dl glucose level until the end of the study at 90 min .
blood was drawn at baseline and at 30 , 60 , and 90 min for measurement of plasma glucagon , epinephrine , norepinephrine , and insulin .
at the end of experiments , the rats were killed with an overdose of sodium pentobarbital ( sleepaway ; fort dodge animal health , fort dodge , ia ) .
catecholamine analysis was performed by high - performance liquid chromatography using electrochemical detection ( esa , acton , ma ) .
plasma insulin and glucagon were measured by radioimmunoassay ( linco , st . charles , mo ) .
baseline comparisons were performed using anova . a mixed - model analysis with an unstructured covariance matrix
analyses were performed using sas 9.2 software ( sas institute , inc . , cary , nc ) , with a threshold p < 0.05 considered as statistically significant .
as summarized in table 1 , the groups of rats did not differ with respect to body weight or concentrations of plasma glucose , insulin , glucagon , epinephrine , and norepinephrine at baseline . during the hyperinsulinemic - hypoglycemic clamp studies , plasma glucose and insulin in each animal group reached levels that were not significantly different ( table 2 ) .
baseline metabolic status of animals in the morning before the study data are presented as mean  sem .
plasma glucose and insulin concentrations during the final 60 min of the hyperinsulinemic - hypoglycemic clamp study for rats in each study group data are presented as mean  sem . as shown in fig .
1a , vmh delivery of the b2ar agonist reduced the exogenous glucose infusion rates ( gir ) required to maintain target glucose concentration by 32% during the entire hypoglycemic clamp ( p < 0.01 ) , whereas b2ar blockade in the vmh raised exogenous glucose requirements by 27% ( p < 0.05 ) compared with the control study .
b2ar modulation in the vmh and its effect on gir and counterregulatory hormones during the hypoglycemic clamp study .
gir ( a ) and hormonal responses for plasma epinephrine ( b ) , glucagon ( c ) , and norepinephrine ( d ) for rats receiving microinjection of the artificial extracellular fluid vehicle ( control ; n = 12 ) , the b2ar antagonist ici-118,551 ( n = 12 ) , or the b2ar agonist formoterol ( n = 10 ) during the hyperinsulinemic - hypoglycemic glucose clamp .
post hoc linear contrasts to localized effects ; * p < 0.05 , * * p < 0.01 , and * * * p < 0.001 vs. controls . these changes were accompanied by alternations in peak and in overall epinephrine responses during hypoglycemia .
delivery of b2ar agonist to the vmh increased peak epinephrine responses by 37% ( p < 0.01 ) and epinephrine levels during the entire 90-min period of hypoglycemia by 50% ( p < 0.01 ) compared with the control group . in contrast , vmh delivery of the b2ar antagonist suppressed the peak by 53% ( p < 0.001 ) and epinephrine levels during the entire 90-min period of hypoglycemia by 32% ( p < 0.05 ) compared with the control group ( fig .
peak and overall glucagon response was also increased by activation of vmh b2ar by 53 and 63% , respectively ( p < 0.01 for both vs. control ) ( fig .
however , administration of the b2ar antagonist had only a small inhibitory effect on the peak glucagon response , which failed to reach statistical significance ( p = 0.051 vs. control at 30 min ) ( fig .
norepinephrine levels were also slightly higher in the b2ar agonist group , but at 90 min only ( p < 0.05 vs. control ) ( fig .
targeted blockade of vmh b1ar or b3ar failed to suppress counterregulatory hormone responses to hypoglycemia or increase the gir needed to maintain hypoglycemia ( fig .
2a ) but did produce a modest increase in the overall epinephrine response ( p < 0.05 vs. control ) ( fig . 2b ) .
vmh b3ar blockade produced a small 21% decrease in the gir required during hypoglycemia ( p < 0.05 vs. control ) .
however , b3ar blockade had no significant effect on the release of epinephrine , glucagon , and norepinephrine during hypoglycemia compared with controls ( fig .
2 ) . effect of vmh b1ar or b3ar blockade on gir and counterregulatory hormones during the hypoglycemic clamp study .
gir ( a ) as well as epinephrine ( b ) , glucagon ( c ) , and norepinephrine ( d ) responses for rats receiving microinjection of the artificial extracellular fluid vehicle ( control ; n = 12 ) , the b1ar antagonist cgp 20712 ( n = 7 ) , and the b3ar antagonist sr59230a ( n = 6 ) during the hyperinsulinemic - hypoglycemic glucose clamp .
previous studies have suggested a potential role for local vmh norepinephrine neurotransmission in modulating glucose counterregulation . this view is supported by data showing that 1 ) norepinephrine delivery into the vmh increases blood glucose and counterregulatory hormone levels ( 16,17 ) ; 2 ) glucopenia stimulates norepinephrine turnover in the mediobasal hypothalamus , including the vmh ( 18,19 ) ; and 3 ) levels of norepinephrine increase in vmh interstitial fluid during hypoglycemia ( 12,13 ) .
the current study was undertaken to assess whether norepinephrine acts to promote glucose counterregulation via specific vmh bars .
the data demonstrate that local vmh delivery of a specific b2ar agonist increases both epinephrine and glucagon responses to acute hypoglycemia , whereas delivery of a b2ar antagonist suppresses epinephrine release and tends to reduce the initial glucagon response .
in contrast , blockade of vmh b1ar or b3ar failed to have a significant inhibitory effect on counterregulatory responses .
it should be noted that for these studies we used similar concentrations of bar agonists and antagonists ( 1 mol / l ) as were used in previous in vivo ( 12,20 ) and in vitro ( 11 ) studies examining the specific receptors mediating the effects of norepinephrine on vmh neurons .
although these concentrations exceed the nanomolar norepinephrine concentrations present in the vmh interstitial fluid , if one takes into account the dilution of the agent in the vmh and in vivo clearance from the tissue during the study , the active local dose level in the vmh was considerably lower .
nevertheless , it seems unlikely that the changes we observed represent off - target effects .
we have observed that if the dose of b2ar antagonist is diminished 10-fold , there is a similar degree of suppression of epinephrine responses to hypoglycemia and that if much higher doses of the compound are given , the effect on epinephrine release is less pronounced ( b.s . , unpublished data ) . taken together
, our data suggest that norepinephrine release into the vmh during hypoglycemia acts to promote glucose recovery , at least in part via stimulation of b2ar .
these findings are consistent with previous studies showing that norepinephrine activates isolated glutamatergic neurons derived from the rat vmh via b2ar ( 11 ) .
it is noteworthy that earlier studies suggested that norepinephrine s central actions to regulate peripheral glucose levels were mediated via 2-receptors .
smythe et al . ( 18,19,21 ) reported that yohimbine , an 2-receptor antagonist , markedly inhibited stress induced hyperglycemia as well as the hyperglycemic effects of 2-deoxyglucose administration . moreover , beverly et al .
( 20 ) reported that during hypoglycemia the norepinephrine rise within the vmh followed a bimodal pattern .
the initial peaks disappeared when yohimbine was locally delivered , whereas the second rise was suppressed in the presence of timolol , a nonselective bar antagonist .
neither of these studies , however , examined the effect of hypothalamic adrenergic receptor modulation specifically on the responses of counterregulatory hormones to hypoglycemia .
it is noteworthy in this regard that previous studies suggest that the activation of an appropriate counterregulatory response to hypoglycemia is associated with inhibition of gaba tone ( 9 ) and activation of glutamate neurons within the vmh ( 10 ) .
it is intriguing to speculate that norepinephrine might act via different adrenergic receptors to exert complementary effects on vmh glutamate and gaba neurotransmission to promote counterregulatory responses to hypoglycemia .
it is now recognized that hypoglycemia is detected by specialized glucose - sensing neurons located within a variety of brain regions ( 6,7 ) as well as periphery ( 5 ) .
watts and donovan , in a recent review ( 14 ) , proposed that brainstem catecholaminergic neurons receive neural input from peripheral glucose sensors and then transmit this information to hypothalamic glucose - sensing neurons , including those in the vmh .
thus , these catecholaminergic projections might serve to integrate the vmh into a wide network glucose - sensing neurons .
we conclude that synaptic release of catecholamines acting via b2ar within the vmh plays an important role in modulating the magnitude of the epinephrine responses and , to lesser extent , glucagon responses to insulin - induced hypoglycemia .
these observations are consistent with previous studies suggesting that administration of the b2ar agonist terbutaline may restore hypoglycemia awareness and reduce nocturnal hypoglycemia ( 22,23 )
. this effect may be particularly useful in humans with homozygosity for glycine at codon 16 ( glygly ) of the b2ar who appear to show reduced b2ar sensitivity to antecedent hypoglycemia ( 24 ) .
moreover , the current data are consistent with recent data indicating that repeated adrenergic receptor activation might also be involved in the generation of hypoglycemia - associated autonomic failure ( 25 ) and raise the possibility that b2ar receptor downregulation might play a role in this phenomenon . in our study
given that hypoglycemia remains the major limiting factor in the use of intensified insulin therapy in the management of diabetes , our study may have potential therapeutic implications for strategies aimed at reducing the risk of severe hypoglycemia in diabetes .
 objectivenorepinephrine is locally released into the ventromedial hypothalamus ( vmh ) , a key brain glucose - sensing region in the response to hypoglycemia . as a result , 
 this neurotransmitter may play a role in modulating counterregulatory responses . 
 this study examines whether norepinephrine acts to promote glucose counterregulation via specific vmh -adrenergic receptors ( bar).research design and methodsawake male sprague - dawley rats received , via implanted guide cannulae , bilateral vmh microinjections of 1 ) artificial extracellular fluid , 2 ) b2ar agonist , or 3 ) b2ar antagonist . 
 subsequently , a hyperinsulinemic - hypoglycemic clamp study was performed . 
 the same protocol was also used to assess the effect of vmh delivery of a selective b1ar or b3ar antagonist.resultsdespite similar insulin and glucose concentrations during the clamp , activation of b2ar in the vmh significantly lowered by 32% ( p < 0.01 ) , whereas vmh b2ar blockade raised by 27% exogenous glucose requirements during hypoglycemia ( p < 0.05 ) compared with the control study . 
 these changes were associated with alternations in counterregulatory hormone release . 
 epinephrine responses throughout hypoglycemia were significantly increased by 50% when the b2ar agonist was delivered to the vmh ( p < 0.01 ) and suppressed by 32% with the b2ar antagonist ( p < 0.05 ) . 
 the glucagon response was also increased by b2ar activation by 63% ( p < 0.01 ) . 
 neither blockade of vmh b1ar nor b3ar suppressed counterregulatory responses to hypoglycemia . 
 indeed , the b1ar antagonist increased rather than decreased epinephrine release ( p < 0.05).conclusionslocal catecholamine release into the vmh enhances counterregulatory responses to hypoglycemia via stimulation of b2ar . 
 these observations suggest that b2ar agonists might have therapeutic benefit in diabetic patients with defective glucose counterregulation . 
RESEARCH DESIGN AND METHODS
Surgery.
Microinjection.
Hyperinsulinemic-hypoglycemic clamp.
Probe placement.
Analytic procedures.
Data analysis.
RESULTS
DISCUSSION