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Endocrinology 147(6):2781–2788
Copyright © 2006 by The Endocrine Society
doi: 10.1210/en.2005-1247
Antecedent Hypoglycemia, Catecholamine Depletion,
and Subsequent Sympathetic Neural Responses
Judith A. Herlein, Donald A. Morgan, Bradley G. Phillips, William G. Haynes, and William I. Sivitz
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
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 ⴝ 15 per group) on d ⴚ2 and ⴚ1 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
T
HE SYNDROME OF impaired glycemic counterregulation and hypoglycemic unawareness has been termed
hypoglycemia-associated autonomic failure (HAAF), a wellrecognized 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, deFirst 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.
Endocrinology is published monthly by The Endocrine Society (http://
www.endo-society.org), the foremost professional society serving the
endocrine community.
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 ⴝ 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.
(Endocrinology 147: 2781–2788, 2006)
spite 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
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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
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.
Rats were transiently anesthetized at 0800 on d 0 with ip ketamine/
xylazine (91/9.1 mg/kg). Additional ketamine/xylazine sodium was
administered iv every 10 min, to sustain the level of anesthesia, until 30
min after the surgical procedure was completed. Catheters were placed
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.
Herlein et al. • Antecedent Hypoglycemia and Hypotension
in the carotid artery and jugular vein. A nerve branch to the left adrenal
nerve was exposed through a flank incision, and the platinum-iridium
electrode was attached. When an adequate recording was obtained, the
electrode was fixed in place using silicon gel (Kwikcast; World Precision
Instruments, Sarasota, FL), the wound was closed with 4.0 silk, and skin
was glued shut with Vetbond tissue adhesive (3M, St. Paul, MN). The
vascular catheters and electrode wire were tunneled to exit the skin at
the nape of the neck and protected by a spring tether connected to a
swivel mount apparatus (Instech Laboratories, Inc., Plymouth Meeting,
PA) at the top of the cage. The rats were loosely restrained, allowing
movement but not 180 degree rotation, to protect the electrode lead. The
procedure itself required approximately 20 min, and recovery from
anesthesia occurred over approximately 60 min. Adrenal SNA was continuously recorded until the completion of the experiment.
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 ␮g in the rats exposed to antecedent hypoglycemia and 373 ⫾ 24 ␮g 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
Herlein et al. • Antecedent Hypoglycemia and Hypotension
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 ␮l of centrifuged plasma to a glass extraction
vial containing 20 mg of acid-washed alumina (AAO; Bioanalytical
Systems, West Lafayette, IN), 20 ␮l 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 ␮l of 4% acetic acid. After centrifugal
microfiltration using individual 0.2 ␮m regenerated cellulose membranes, each sample was chromatographed using a Phenomonex Synergi Hydro-RP C-18 column (4 ␮m, 150 ⫻ 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 ⫻ 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
Endocrinology, June 2006, 147(6):2781–2788
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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-␮m 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 –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
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.
The EPI and NE responses to NTP-induced hypotension in
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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).
the two groups of rats are shown in Fig. 4. The data show that
the plasma EPI response to NTP-induced hypotension was
decreased 2- to 3-fold in the rats exposed to antecedent hypoglycemia compared with sham. In contrast, the NE response to hypotension was nearly identical in the two groups
of animals. Baseline (before NTP) values for plasma EPI and
NE in the hypoglycemia and sham rats did not differ. PostNTP plasma EPI, but not NE, was significantly less in the rats
exposed to antecedent hypoglycemia compared with sham.
Effects of antecedent hypoglycemia on adrenal
catecholamine content
Antecedent hypoglycemia, compared with sham hypoglycemia, reduced adrenal EPI content (Fig. 5). In contrast,
antecedent hypoglycemia had no effect on NE content. These
results were similar whether expressed as content per microgram of wet weight or as absolute content per gland (Fig.
5). Adrenal wet weight did not differ between rats exposed
to antecedent hypoglycemia or sham treatment (mean ⫾ sem,
65 ⫾ 4 mg for hypoglycemia and 62 ⫾ 8 mg for sham).
Effects of antecedent hypoglycemia on enzyme expression
and phosphorylation
The immunoblot results for the nonphosphorylated and
Ser40-phosphorylated forms of TH are illustrated in Fig. 6.
Densitometric quantification of the data revealed no significant difference between adrenal extracts of rats exposed to
antecedent hypoglycemia compared with sham-treated rats.
Likewise, we observed no difference in PNMT expression
Herlein et al. • Antecedent Hypoglycemia and Hypotension
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).
(Fig. 6). We obtained very clean immunoblots for TH and
phosphorylated TH (pTH). PNMT immunoblotting was less
distinct but did reveal a predominant band that migrated as
expected for the 37.1-kD protein.
Discussion
A major finding reported here is the effect of antecedent
hypoglycemia to blunt the subsequent adrenomedullary response (EPI release) to a nonhypoglycemic stimulus, hypotension. However, we also report that this occurred in the
absence of any impairment in sympathetic nerve traffic to the
adrenal gland. If anything, sympathetic nerve traffic was
slightly greater before and during NTP infusion in the animals exposed to antecedent hypoglycemia (Fig. 3). Hence,
the data suggest that the impaired adrenal EPI release resulted, not from impaired sympathetic neural input, but,
more likely, from perturbed physiology within the adrenal
medullae per se. Presumably, this involved impaired catecholamine production and/or depleted adrenal catecholamine stores after antecedent hypoglycemia.
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 catechol-
Herlein et al. • Antecedent Hypoglycemia and Hypotension
Endocrinology, June 2006, 147(6):2781–2788
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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 twofactor ANOVA for A.
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.
amine 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 re-
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Herlein et al. • Antecedent Hypoglycemia and Hypotension
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.
current 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.
Herlein et al. • Antecedent Hypoglycemia and Hypotension
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 –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
Endocrinology, June 2006, 147(6):2781–2788
2787
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.
Acknowledgments
Received September 30, 2005. Accepted February 28, 2006.
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:
[email protected].
Disclosure statement: J.A.H., D.A.M., B.G.P., W.G.H., and W.I.S. have
nothing to declare.
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