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Antecedent Hypoglycemia, Catecholamine Depletion, and Subsequent Sympathetic Neural Responses

Department of Internal Medicine, Divisions of Endocrinology and Cardiovascular Disease, and Department of Pharmacology, University of Iowa and Iowa City Veterans Affairs Medical Center, Iowa City, Iowa 52246

Address all correspondence and requests for reprints to: Dr. William Sivitz, Department of Internal Medicine, The University of Iowa Hospitals and Clinics, 3E-17 VA, Iowa City, Iowa 52246. E-mail: William-Sivitz{at}uiowa.edu.

	Abstract

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Antecedent hypoglycemia is well known to impair sympathetic responses to subsequent hypoglycemia. However, it is less clear whether this occurs through altered sympathetic neural traffic or through decreased adrenal catecholamine release per se. It is also not clear whether antecedent hypoglycemia impairs sympathetic responsiveness to subsequent nonhypoglycemic sympathetic stimuli. We exposed rats to two episodes of insulin-induced hypoglycemia or sham hypoglycemia (n = 15 per group) on d –2 and –1 before exposure to transient (10 min) hypotension on d 0. Adrenal sympathetic nerve activity (SNA) was directly recorded in the conscious state and plasma catecholamine concentrations were assessed. We also examined the effect of antecedent hypoglycemia on phosphorylated and nonphosphorylated tyrosine hydroxylase (TH) protein expression as well as the expression of phenylethanolamine N-methyltransferase. Adrenal SNA was not significantly altered by antecedent hypoglycemia either at baseline of d 0 (before hypotension) or in response to hypotension. In contrast, plasma epinephrine (EPI) responsiveness was impaired by more than 50% (P = 0.025) in rats exposed to antecedent vs. sham hypoglycemia. Antecedent hypoglycemia had no effect on norepinephrine responsiveness to hypotension. In studies of adrenal tissue from separate rats, antecedent hypoglycemia decreased adrenal EPI content but did not significantly alter the expression of TH, phosphorylated TH, or phenylethanolamine N-methyltransferase. In summary, antecedent hypoglycemia impaired EPI responsiveness to subsequent hypotension despite no reduction in adrenal SNA and in association with reduced adrenal EPI content. Thus, antecedent hypoglycemia impaired responsiveness to a subsequent nonhypoglycemic sympathetic stimulus, an effect mediated at the level of the adrenal medullae.

	Introduction

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THE SYNDROME OF impaired glycemic counterregulation and hypoglycemic unawareness has been termed hypoglycemia-associated autonomic failure (HAAF), a well-recognized problem complicating the management of patients with diabetes (1). The cause of HAAF remains unclear and it is not certain whether the syndrome results primarily from defective adrenal catecholamine release, impaired neural responsiveness, or combined defects in adrenal and neural responses.

The adrenal catecholamine response to hypoglycemia is well known to be impaired as little as 24 h after antecedent hypoglycemia (2). However, less is known concerning the effect of antecedent hypoglycemia on adrenal sympathetic nerve activity (SNA) per se and the consequences of potentially altered SNA toward adrenal catecholamine release. Past studies in our laboratory using direct adrenal nerve recording in conscious rats showed that adrenal SNA was acutely increased by insulin-induced hypoglycemia (3). More importantly, adrenal SNA measured 24 h after antecedent insulin-induced hypoglycemia was not reduced, but actually remained elevated, in comparison to control rats exposed to antecedent sham hypoglycemia. However, despite this persistent adrenal SNA, we found (as expected) that adrenal catecholamine responsiveness to subsequent hypoglycemia determined as plasma epinephrine (EPI) was impaired by prior hypoglycemia (3). Thus, our data suggested that antecedent hypoglycemia and persistent adrenal sympathetic neural traffic impaired EPI responsiveness to subsequent hypoglycemia through direct effects on the adrenal medullae to limit catecholamine synthesis or release. This is consistent with older data in rodents showing that adrenal catecholamine stores were reduced after antecedent hypoglycemia (4, 5).

Hence, we reasoned that, if hypoglycemia reduced the capacity of the adrenal to release EPI in response to a subsequent stimulus, then that stimulus should not be restricted to repeat hypoglycemia per se. Therefore, we hypothesized that antecedent hypoglycemia would reduce the subsequent ability of the adrenal to release EPI in response to the nonhypoglycemic sympathetic stimulus, hypotension. In addition, we hypothesized that antecedent hypoglycemia would not reduce adrenal SNA in response to subsequent hypotension. Furthermore, we hypothesized that antecedent hypoglycemia would reduce adrenal catecholamine content.

Realizing that hypotension is also a potent stimulus to norepinephrine (NE) release from peripheral postganglionic nerve endings, we reasoned that antecedent hypoglycemia might also impair this response as well. Alternatively, the effect of antecedent hypoglycemia might be specific to the adrenal. Peripheral nerve endings release NE rather than EPI (6) whereas, the adrenal medullae, at least in the rat, preferentially releases EPI (7). Therefore, an effect of antecedent hypoglycemia to impair the EPI, but not the NE, response to hypotension would indicate that antecedent hypoglycemia specifically impairs the adrenomedullary response.

In the current studies, we exposed normal rats to two episodes of antecedent hypoglycemia and subsequently to transient hypotension induced by intravenous nitroprusside (NTP). Plasma catecholamines were determined before and after the hypotensive episodes, whereas adrenal nerve activity was continuously assessed by direct neural recording in the conscious state. We also measured the adrenal content of catecholamines. Finally, to attempt to learn more of the intraadrenal physiology subsequent to hypoglycemia, we measured the expression of phosphorylated and nonphosphorylated tyrosine hydroxylase (TH) protein and the expression of phenylethanolamine N-methyltransferase (PNMT).

	Materials and Methods

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Experimental animals
Rats were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were fed and maintained according to standard National Institutes of Health guidelines, and the protocol was approved by the University of Iowa Animal Care Committee. Room temperature was maintained at 25 C. Groups of animals were designated as group I, II, and III. Male rats were used in all experiments.

Group 1 experiments: effects of antecedent hypoglycemia on sympathetic neural and catecholamine responses to subsequent hypotension
Studies were performed as depicted in Fig. 1. All rats were subject to two episodes of insulin-induced hypoglycemia (n = 15, mean weight 451 ± 10 g) or sham hypoglycemia (n = 15, 447 ± 9 g). Hypoglycemia or sham hypoglycemia was induced by sc injection of 2.0 U of regular human insulin at 0830 h on d –2 and 1.0 U at 0830 h on d –1 or an equal volume of saline (sham treatment). Blood glucose was determined 150 min after injection on tail vein blood using a glucose reagent strip and meter considered accurate to glucose readings as low as 40 mg/100 ml. Food was removed from the cages after insulin (or saline) injection and returned after the glucose determination at 150 min. Blood glucose in the rats exposed to hypoglycemia ranged from less than 40–48 mg/100 ml on d –2 and from less than 40–50 mg/100 ml on d –1, compared with readings in the sham hypoglycemic rats of 93 ± 2 on d –2 and 93 ± 1 on d –1.

View larger version (21K): [in this window] [in a new window]   FIG. 1. Schematic diagram depicting group 1 experiments designed to determine the effect of antecedent hypoglycemia upon subsequent adrenal SNA and catecholamine release. Group 2 and 3 experiments were carried out in the same way, except that rats were killed on the morning of d 0 rather than anesthetized for further procedures.

At 1100 h, all rats received an iv bolus injection of NTP (Abbott Labs, North Chicago, IL), which was continued for 10 min. The exact amount (mean ± SEM, 340 ± 21 µg in the rats exposed to antecedent hypoglycemia and 373 ± 24 µg in the rats exposed to sham treatment; difference not significant) was determined by adjusting the rate of infusion to target a near 50% drop in mean arterial pressure, which was continuously monitored. One milliliter of blood was obtained for catecholamines from the carotid arterial line before and 10 min after initiation of NTP, and volume was replaced by infusing 1.0 ml of saline. Glucose concentrations determined on arterial blood using a glucose analyzer (Yellow Springs Instruments, Inc, Yellow Springs, OH) before NTP injection were 132 ± 9 and 140 ± 8 mg/100 ml in the rats that had been exposed to antecedent hypoglycemia or sham hypoglycemia, respectively (difference not significant).

Sympathetic nerve recordings.
Sympathetic activity innervating the adrenal nerve was measured by multifiber recording as we have previously described (8). SNA was recorded at 5-min intervals as average activity over the preceding 1 min. Using a dissecting microscope, a nerve branch to the left adrenal was carefully dissected, and the bipolar electrode was placed. After an optimum recording of SNA was obtained, the electrode was fixed in place using silicon gel. The electrode was connected to a high impedance probe (HIP-511; Grass Instruments, Warwick, RI), amplified by 10⁵, and filtered at low- and high-frequency cutoffs of 100 and 1000 Hz with a nerve traffic analysis system (model 662-C, University of Iowa Bioengineering, Iowa City, IA). The filtered, amplified nerve signal was routed: 1) to an oscilloscope (model 54501A; Hewlett-Packard, Palo Alto, CA) for monitoring; 2) to a MacLab analog-digital converter (CB Sciences, Inc., Milford, MA) for permanent recording of the neurogram on a Macintosh 9500 computer; and 3) to a nerve traffic analyzer (model 706C; University of Iowa Bioengineering) that counts action potentials above a threshold voltage level set just above background (determined postmortem). To document that the nerve recordings represent sympathetic nerve impulses, ganglionic blockade was induced with chlorisondamine, 5 mg/kg iv at the end of each experiment. This reduced nerve activity to low-grade background "noise" that is subtracted from the recorded measurements. Also, there is a characteristic burst activity pattern seen as a result of sympathetic outflow which, although subjective, provides a measure of confirmation. Further, in past studies (8) and in pilot experiments in rats under anesthesia, we transected adrenal nerves distal to the recording site (electrode). In six separate determinations, the neurograms were not altered documenting the efferent rather than afferent origin of the neural signals.

BP and heart rate determinations.
These parameters were continuously monitored along with adrenal SNA in all studies. This was accomplished using a pressure transducer (Gould Stathan P23ID) attached to the carotid arterial line, and the data was acquired by computer through the MacLab analog-digital converter. BP and heart rate were recorded every 5 min as average values over 1-min intervals.

Catecholamine determination.
Plasma EPI and NE concentrations were determined by adding 500 µl of centrifuged plasma to a glass extraction vial containing 20 mg of acid-washed alumina (AAO; Bioanalytical Systems, West Lafayette, IN), 20 µl of a solution containing the internal standard (3,4 dihydroxy-benzylamine in 0.01 N HCl), 1.5 ml of phosphate buffer [0.l M (pH 7.0), plus 0.05 M EDTA] and l ml Tris buffer [1.5 M (pH 8.6), plus 0.05 M NaEDTA]. After immediate gentle shaking for 10 min, the alumina was allowed to settle, and the supernatant was aspirated to waste. After two washes with water, catecholamines were eluted from the aluminia with 200 µl of 4% acetic acid. After centrifugal microfiltration using individual 0.2 µm regenerated cellulose membranes, each sample was chromatographed using a Phenomonex Synergi Hydro-RP C-18 column (4 µm, 150 x 4.6 mm) and mobile phase of 75 mM monobasic sodium phosphate, 0.12 mM NaEDTA, 10 mM citric acid, 15% acetonitrile, 10% methanol, and 1.5 mM sodium dodecyl sulfate as the ion pairing agent. The catecholamines were detected with a Coulochem II Dual Potentiostat Electrochemical Detector (ESA, Inc., Chelmsford, MA). Peaks were integrated using Shimadzu Class VP 7.2.1 Chomatography software. A standard curve for extracted catecholamines (0, 125, 250, 500, 750, 1000, 1500, and 2000 pg of each catecholamine) was prepared using "blank" rat plasma (dialyzed to remove endogenous catecholamines) and linear regression analysis was used to determine sample plasma concentrations. The assay has interassay and intraassay coefficients of variation of 3.4 and 3.1%, respectively, and a lower limit of detection of 20 pg/ml.

Group 2 experiments: effects of antecedent hypoglycemia on adrenal catecholamine content
Studies were performed just as for the group 1 rats (Fig. 1), except that rats were killed on the morning of d 0 rather than anesthetized for further procedures. Both adrenal glands were dissected free, rinsed, and blotted, and weighed before storage at –70 C for determination of catecholamine content. Blood glucose in the rats exposed to hypoglycemia (n = 9, mean weight 427 ± 12 g) was less than 40 mg/100 ml in all animals on d –2 and ranged from less than 40–46 mg/100 ml on d –1. Blood glucose in the rats exposed to sham hypoglycemia (n = 8, mean weight 427 ± 11 g) was 82 ± 2 on d –2 and 76 ± 1 on d –1. Blood glucose on d 0 was 87 ± 2 and 82 ± 2 mg/100 ml in the antecedent hypoglycemia and sham groups, respectively (difference not significant). All glucose determinations were performed using a reagent strip and meter as described for the group 1 rats. At 1100–1200 h on d 0, rats were anesthetized with pentobarbital (150 mg/kg) and killed by cardiac puncture, and adrenal glands were isolated and frozen at –70 C.

Measurement of adrenal EPI and NE content.
For each animal, adrenal glands were thawed and immediately homogenized at 4 C in 5 ml of freshly prepared 0.4 M perchloric acid containing 6 mM reduced glutathione. After centrifugation of the homogenate (1000 x g, 30 min), the entire supernatant was removed to a 50-ml polypropylene centrifuge tube containing 100 mg of acid-washed alumina and the mixture was neutralized by adding 5 ml of Tris buffer (1.5 M, pH 8.5). After immediate gentle shaking for 10 min, the alumina was allowed to settle and the supernatant was aspirated to waste. After three washes with 5 ml water, catecholamines were eluted from the alumina with 2 ml of 0.05 M perchloric acid containing 0.1 mM sodium metabisulfite. The eluate containing the tissue catecholamines was further diluted 1:1000 in 4% acetic acid and chromatographed in a similar fashion as plasma catecholamines (group 1 experiments). Tissue extract peak areas for NE and EPI were compared with the average peak areas determined from the injection of 100 pg of pure standards. Results were corrected for extract dilutions and tissue wet weights and expressed as micrograms per gram.

Group 3 experiments: effects of antecedent hypoglycemia on TH expression and phosphorylation and PNMT expression
An additional set of rats were subject to the same manipulations as in group 2, except that adrenal glands were used to study the expression of the Ser⁴⁰-phosphorylated and nonphosphorylated forms of TH and the expression of PNMT. Blood glucose in the rats exposed to hypoglycemia (n = 6, mean weight 523 ± 15 g) was less than 40 mg/100 ml in all animals on d –2 and on d –1. Blood glucose in the rats exposed to sham hypoglycemia (n = 6, mean weight 523 ± 11 g) was 79 ± 2 on d –2 and 85 ± 1 on d –1. Blood glucose on d 0 was 91 ± 1 and 89 ± 3 mg/100 ml in the antecedent hypoglycemia and sham groups, respectively (difference not significant). All glucose determinations were performed using a reagent strip and meter as described for the group 1 rats. At 1100–1200 h on d 0, rats were killed and left adrenal glands were isolated and frozen as described for group 2 animals.

To determine enzyme expression and phosphorylation, adrenal glands were thawed and homogenized in RIPA (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS), pH 7.4, containing Novagen PhosphoSafe (Novagen, Inc., Madison, WI) using 10 strokes with a Teflon homogenizer. Homogenates were separated on a 12.5% polyacrylamide gel and transferred to 0.45-µm nitrocellulose.

For TH, blots were incubated in TTBS (20 mM Tris base, 0.14M NaCl, and 0.1% Tween 20) in 2.5% BSA for 18 h at 4 C with mouse anti-TH Ab (Sigma T1299), 1:10,000, and then for 2.5 h at room temperature with antimouse IgG-horseradish peroxidase (HRP), 1:25,000, as secondary Ab using 5% fat-free milk as a blocking agent. Blots were washed and developed by enhanced chemiluminescence (ECL) using a standard kit (ECL; Amersham, Pharmacia Biotech, Piscataway, NJ). To determine TH phosphorylation at Ser⁴⁰, immunoblotting was carried out in similar fashion using rabbit anti-phospho-TH Ser⁴⁰ (2791S; Cell Signaling, Danvers, MA) at 1:1000 in TTBS with 2.5% BSA for 18 h at 4 C as first antibody, followed by incubation with goat antirabbit IgG-HRP (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:25,000 for 2.5 h at room temperature using 5% fat-free milk as a blocking agent. Ser⁴⁰ appears to be of major importance as it undergoes phosphorylation through several second messenger pathways, increases cofactor binding, and increases enzyme activity (9, 10, 11). For PNMT expression, immunoblots were exposed to antibody directed against the C-terminal 20 residues of PNMT (sc-16458; Santa Cruz Biotechnology, Inc.) at a dilution of 1:200 for 18 h at 4 C, followed by incubation with donkey antigoat IgG-HRP (Santa Cruz Biotechnology, Inc.) at 1:25,000 for 2.5 h at room temperature using 5% fat-free milk as a blocking agent. Detection required a more sensitive ECL technique using West Femto Maximum Sensitivity Substrate (Pierce, Inc., Rockford, IL).

Statistics
Data were analyzed by t test or ANOVA as indicated in the figure legends.

	Results

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Effects of antecedent hypoglycemia on sympathetic neural and catecholamine responses to subsequent hypotension
As shown in Fig. 2, NTP infusion rapidly reduced mean arterial pressure in rats exposed to either antecedent hypoglycemia or sham hypoglycemia with no differences between the groups. Likewise, NTP appeared to induce a slight transient increase in heart rate in both groups of animals. Corresponding changes in adrenal SNA are shown in Fig. 3. NTP infusion rapidly and transiently increased adrenal SNA. However, there were no differences between animals exposed to antecedent hypoglycemia compared with controls irrespective of whether SNA was expressed either in absolute terms (spikes per second) or as percent of baseline.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Mean arterial pressure and heart rate in rats exposed to antecedent hypoglycemia (antecedent insulin) compared with sham hypoglycemia (antecedent saline). NTP was infused for 10 min beginning at time 0 min. Data represent mean ± SEM (n = 15 rats per group).

View larger version (24K): [in this window] [in a new window]   FIG. 3. Adrenal SNA in absolute terms (spikes per minute) and as percent of baseline (average of four values at times –15, –10, –5, and 0 min) in rats (Fig. 2) exposed to antecedent hypoglycemia (antecedent insulin) compared with sham hypoglycemia (antecedent saline). NTP was infused for 10 min beginning at time 0 min. Data represent mean ± SEM (n = 15 rats per group).

View larger version (42K): [in this window] [in a new window]   FIG. 4. Changes in plasma EPI (Epi) and NE between time 0 (immediately before infusion of NTP) and 10 min (end of NTP infusion) in rats (Figs. 2 and 3) exposed to antecedent (Ant) insulin-induced hypoglycemia compared with sham hypoglycemia (antecedent saline). A and B, Baseline and posttreatment (NTP) EPI (A) and incremental increase in EPI (B). C and D, Baseline and posttreatment NE (C) and incremental increase in NE (D). Data represent mean ± SEM, n = 15 rats per group. *, P < 0.05; or **, P < 0.01 by two-factor ANOVA (factors are time and antecedent treatment) with repeated measures for time (A and C). *, P < 0.05, by two-tailed, unpaired t test (B). The incremental effect in B is of particular importance because there was significant interaction (P = 0.037) between the effects of time (NTP infusion) and treatment (insulin or saline) in the two-factor ANOVA for A.

View larger version (73K): [in this window] [in a new window]   FIG. 5. Adrenal EPI and NE content expressed per unit wet weight (upper panels) and as content per gland (lower panels) in rats exposed to antecedent hypoglycemia (antecedent insulin, n = 9) compared with sham hypoglycemia (antecedent saline, n = 8). Data represent mean ± SEM, *, P < 0.05 compared with saline by unpaired t test. Values for an individual rat represent the average catecholamine content of the left and right adrenals from that animal.

View larger version (48K): [in this window] [in a new window]   FIG. 6. Immunoreactive expression of nonphosphorylated TH, TH phosphorylated at Ser40 (pTH), and PNMT in adrenal tissue of rats exposed to antecedent insulin-induced hypoglycemia or sham. A, Individual immunoblots for TH, pTH, and PNMT; each lane representing a single animal exposed to antecedent insulin (I) or saline (S). B, Densitometric quantification of the data in rats exposed to insulin (INS) compared with saline (SAL). Data represent mean ± SEM, n = 6 for each group. Two-tailed, unpaired t test revealed no significant differences for TH, pTH, or PNMT.

	Discussion

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To our knowledge, this effect of prior hypoglycemia to blunt a nonhypoglycemic adrenomedullary response in the absence of altered adrenal SNA has not been previously reported. Galassetti et al. (12) reported that antecedent hypoglycemia in human subjects abolished glucagon and decreased the EPI, NE, and cortisol responses to exercise while increasing the amount of exogenous glucose needed to maintain euglycemia. Neural activity was not studied in these human experiments.

Our results also suggest that the impairment in catecholamine responsiveness to hypotension induced by antecedent hypoglycemia was specific for adrenal catecholamine release, as opposed to catecholamine release from postganglionic nerve endings. Prior work has shown that peripheral postganglionic sympathetic nerve endings release NE but not EPI (7). Hence, specificity for the adrenal response follows from Fig. 4, which shows that plasma NE responsiveness to NTP was unaffected by prior hypoglycemia, whereas EPI release was decreased 2- to 3-fold. Conceivably, this conclusion could be confounded if, in fact, the adrenals of our rats also released large amounts of NE obscuring any potential peripheral response. However, it has been shown that the rat adrenal, unlike the human, preferentially releases EPI (6).

Antecedent hypoglycemia is well known to impair subsequent EPI responses to insulin-induced hypoglycemia both in rats (3, 13, 14) and humans (2). We now show that antecedent hypoglycemia impairs the EPI response to subsequent hypotension. As in our past studies of subsequent hypoglycemia (3), this impairment of EPI in response to hypotension occurred despite no decrease in adrenal SNA. A difference in our prior study compared with the current study was that basal adrenal SNA (over 15 min before the stimulus of recurrent hypoglycemia) was actually greater in rats exposed to prior hypoglycemia vs. sham. In the current studies, we also observed that adrenal SNA before the recurrent stimulus (in this case hypotension) was greater in the rats exposed to antecedent hypoglycemia (Fig. 3). However, in the current work, this increase was not statistically significant. In any case, both our current and past (3) studies support the concept that impaired adrenal catecholamine responsiveness after antecedent hypoglycemia involves pathophysiology at the level of the adrenal medullae and is not due to impaired sympathetic neural input.

A limitation to our studies is that, although the rats we studied were conscious at the time of blood sampling for EPI and NE (Fig. 1), there was undoubtedly some degree of stress-induced baseline (prehypotension) catecholamine stimulation. This was not avoidable, because the rats had to be prepared for nerve recording. Nonetheless, it was still clearly possible to study EPI and NE responses to hypotension as the posthypotension values were severalfold elevated relative to baseline (Fig. 4).

Adrenal catecholamine content after prior hypoglycemia was not assessed in our past studies (3). Hence, our current data showing that adrenal EPI content is reduced after antecedent hypoglycemia (Fig. 5) provides further evidence for the concept that prior activation of adrenal sympathetic nerve traffic reduces the adrenal capacity for subsequent sympathetic responsiveness. These data on EPI content are in agreement with older studies in the cat (5) and rat (4) that show that adrenal EPI stores are reduced for 24–96 h after acute hypoglycemia.

Hence, the aforementioned suggests that one explanation for impaired adrenal sympathetic responses after antecedent hypoglycemia may be as follows. With an antecedent hypoglycemic episode, adrenal SNA initially increases and is followed by persistent or increased SNA for at least 24 h. This results in depleted catecholamine stores and reduced capacity to respond to sympathetic stimuli.

To further examine adrenal physiology subsequent to hypoglycemia, we measured the expression of phosphorylated and nonphosphorylated TH protein and the expression of PNMT. TH catalyzes the first and rate-limiting step in catecholamine biosynthesis and is regulated in part through feedback inhibition (9). TH activity is enhanced after prior sympathetic input and catecholamine release, an apparent means of compensation for catecholamine depletion (4, 9, 10, 15, 16). Activation of TH follows phosphorylation at several sites, notably at Ser⁴⁰ (9, 10, 11). PMNT catalyzes the conversion of NE to EPI. The enzyme is inducible by glucocorticoids and by neural signals through nicotinic and muscarinic receptors (17, 18) and adrenal PNMT mRNA has been found to be reduced after hypoglycemia in diabetic rats (19).

Based on the aforementioned, we hypothesized that TH and/or its phosphorylation might be up-regulated as compensation for catecholamine depletion consequent to antecedent hypoglycemia and that PNMT protein expression might be reduced. However, our data do not support these hypotheses. Of course, it is important to point out these findings address only a small part of the overall regulation of catecholamine production. Thus, enzyme stability and activation by a variety of factors apart from phosphorylation per se (9, 10) may be important in the adrenal response to depleted catecholamines. Moreover, we only examined TH and PNMT expression at one time point and it is possible that a detailed time course after hypoglycemia may provide different results.

Our studies of adrenal catecholamine content revealed depletion of EPI but not NE. This would fit nicely with the aforementioned report of reduced PNMT mRNA after antecedent hypoglycemia. However, as above, we were not able to document a difference in the protein content of this enzyme. It is possible that our immunoblotting technology was not sensitive enough to detect a reduction in expression, or that enzyme activity, rather than amount expressed, was reduced. It is also possible that PNMT expression or activity had been reduced at a time point before but not at the exact point of our measurement, another scenario that might account for selected depletion of adrenal EPI.

The concept that the adrenal capacity for EPI release per se, independent of neural input, is an important component of hypoglycemic counterregulation is also evident in humans. There is evidence that type 1 diabetic patients with impaired EPI response to hypoglycemia have reduced adrenomedullary capacity to secrete EPI. This is derived from measurements of metanephrine in diabetic subjects based on the principal that metanephrine levels reflect adrenomedullary EPI content when catechol-O-methyl transferase activity is normal (20).

Despite the aforementioned support for an adrenomedullary contribution to the etiology of HAAF, we must acknowledge evidence that this may not be the only component. First, it is difficult to be sure our data in the rat translate to human physiology. Moreover, glucose sensing neurons appear to be present in the rodent hypothalamus and influence glucose counterregulation (21, 22). Also, there is evidence that ATP-sensitive potassium channels are important in sensing glucose (23) in the rat brain, and closure of these channels may impair EPI and glucagon responses to both brain glucopenia and systemic hypoglycemia (24). It has also been shown that blunted activation of the paraventricular nucleus of the hypothalamus through lidocaine anesthesia impairs the EPI response to insulin-induced hypoglycemia in the rat (25). Further, it was recently reported that brain glucoprivation induced by intracerebroventricular 2-deoxyglucose (compared with saline) reduced the subsequent EPI response to insulin (26). Thus, we do not exclude a CNS component to the etiology of hypoglycemia-induced autonomic dysfunction. However, our current and past (3) studies do suggest that reduced adrenal sympathetic nerve traffic to the adrenal just before and during subsequent autonomic stimuli do not contribute to reduced sympathetic responsiveness after antecedent hypoglycemia as induced by our methods in the rat.

In summary, our data show that antecedent hypoglycemia impaired EPI responsiveness to a subsequent, nonhypoglycemic stimulus, transient NTP-induced hypotension. This occurred despite no reduction in adrenal SNA and was specific for EPI as opposed to NE. We conclude that the hypoglycemia-induced defect in autonomic responsiveness was not due to decreased adrenal nerve activity and more likely resulted from adrenal EPI depletion. Our data also show that the effect of antecedent hypoglycemia to impair subsequent hypotension-induced catecholamine release was specific for the adrenal medullae rather than peripheral postganglionic sympathetic nerve endings.

	Footnotes

 
Disclosure statement: J.A.H., D.A.M., B.G.P., W.G.H., and W.I.S. have nothing to declare.

First Published Online March 9, 2006

Abbreviations: ECL, Enhanced chemiluminescence; EPI, epinephrine; HAAF, hypoglycemia-associated autonomic failure; HRP, horseradish peroxidase; NE, norepinephrine; NTP, nitroprusside; PNMT, phenylethanolamine N-methyltransferase; pTH, phosphorylated TH; SNA, sympathetic nerve activity; TH, tyrosine hydroxylase.

Received September 30, 2005.

Accepted for publication February 28, 2006.

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