Download Hypothalamic thyrotropin-releasing hormone mRNA responses to

Am J Physiol Endocrinol Metab 283: E85–E93, 2002.
First published March 19, 2002; 10.1152/ajpendo.00558.2001.
Hypothalamic thyrotropin-releasing hormone mRNA
responses to hypothyroxinemia induced by sleep deprivation
CAROL A. EVERSON1 AND THADDEUS S. NOWAK, JR.2
Department of Neurology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226;
and 2Department of Neurology, University of Tennessee Health Sciences Center,
Memphis, Tennessee 38163
1
Received 19 December 2001; accepted in final form 8 March 2002
THERE IS NEAR UNIVERSAL CONCURRENCE that profound and
chronic sleep deprivation is a risk factor for illness (1,
2) and that it impairs recovery (5, 14). Although
specific physiological changes that may contribute to
pathology in the clinical population have yet to be
extensively investigated, characteristic findings have
emerged from studies in laboratory animals. A typical
3-wk survival period in sleep-deprived rats is marked
by hypercatabolism, manifested by increased food intake and loss of body weight without diabetes or malabsorption of calories (8, 22, 27). Body temperature
initially increases slightly, by ⬍0.5°C (8). Despite this
resemblance to hyperthyroidism, plasma thyroxine
(T4) and triiodothyronine (T3) instead decline progressively to very low concentrations (8, 23). Plasma norepinephrine increases (45), whereas plasma corticosteroids remain unchanged (23). Resistance to infectious
disease is decreased, revealed by early and continual
infection of the mesenteric lymph nodes by bacteria
translocated from the intestine, and periodic transient
infections of major organs (26). The endocrine changes
and the septic state eventuate in an acute condition of
advanced morbidity and hypothermia that precede
death. Terminal signs and positive blood cultures are
consistent with lethal septicemia (21).
The declines in both plasma T4 and T3 in sleepdeprived rats are considered grossly abnormal, not
only because of their progressiveness and severity but
also because of the apparent lack of pituitary activation. The free forms of T4 and T3 decline in parallel
with the total concentrations, indicating that changes
in binding characteristics are not responsible for low T4
(23). Plasma reverse T3 concentrations are not increased into a range that would indicate T4 inactivation (23). T4 shows a greater decline than does T3, to
75% below basal concentrations by the onset of advanced morbidity, compared with a 55% decline in T3
(8). The increased T3-to-T4 ratio likely involves local T3
production by brown adipose tissue (BAT), a major
T4-to-T3 conversion pathway in the rat, which can
contribute 40% or more of circulating T3 (54). Indeed,
the T4-to-T3 conversion enzyme, type II 5⬘-deiodinase,
in BAT is increased 100-fold in sleep-deprived rats (6),
a level consistent with experimental hypothyroidism
(13, 54).
Low plasma T4 is a potent stimulator of thyroidstimulating hormone (TSH) secretion from the pituitary (61). A previous study in sleep-deprived rats
indicated that the plasma TSH concentration remains
Address for reprint requests and other correspondence: C. A.
Everson, MCW Neurology Research 151, VA Medical Center, Bldg.
70, 5000 West National Ave., Milwaukee, WI 53295 (E-mail:
[email protected]).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
central hypothyroidism; preprothyrotropin-releasing hormone messenger ribonucleic acid; paraventricular nucleus;
thyroid hormones
http://www.ajpendo.org
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society
E85
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
Everson, Carol A. and Thaddeus S. Nowak, Jr. Hypothalamic thyrotropin-releasing hormone mRNA responses to
hypothyroxinemia induced by sleep deprivation. Am J
Physiol Endocrinol Metab 283: E85–E93, 2002. First published March 19, 2002; 10.1152/ajpendo.00558.2001.—Sleep
deprivation in rats results in progressive declines in circulating concentrations of both total and free thyroxine (T4) and
triiodothyronine (T3) without an expected increase in plasma
thyroid-stimulating hormone (TSH). Administration of thyrotropin-releasing hormone (TRH) results in appropriate
increases in plasma TSH, free T4, and free T3 across experimental days, suggesting deficient endogenous TRH production and/or release. This study examined transcriptional responses related to TRH regulation following sleep
deprivation. In situ hybridization was used to detect and
quantitate expression of mRNAs encoding prepro-TRH and
5⬘-deiodinase type II (5⬘-DII) in brain sections of six rats
sleep deprived for 16–21 days, when there was marked
hypothyroxinemia, and in sections from animals yoked to the
experimental protocol as well as from sham controls. TRH
transcript levels in the paraventricular nucleus (PVN) were
essentially unchanged at 15–16 days but increased to about
threefold control levels in three of four rats sleep deprived for
20–21 days, a change comparable to that typically found in
prolonged experimental hypothyroidism. There was no evidence for suppression of 5⬘-DII mRNA levels, which would be
a sign of T3 feedback downregulation of neurons in the PVN.
A failure to increase serum TSH in response to hypothyroxinemia and to increased prepro-TRH mRNA expression indicates that alterations in posttranscriptional stages of TRH
synthesis, processing, or release likely mediate the central
hypothyroidism induced by sleep deprivation.
E86
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
METHODS
Animals. All procedures were carried out in accordance
with the National Institutes of Health guidelines on the care
and use of animals and with an animal study protocol approved by the University of Tennessee Animal Care and Use
Committee. Eighteen adult male Sprague-Dawley rats (Harlan, Indianapolis, IN) weighing 464 ⫾ 33 (⫾SD) g and aged
24 ⫾ 2.5 wk were surgically prepared, of which six pairs
served as sleep deprived and experimentally yoked animals,
respectively, with six surgical controls. Anesthesia and analgesia were induced by ketamine 䡠 HCl (100 mg/kg ip),
xylazine 䡠 HCl (2.4 mg/kg im), and atropine sulfate (0.1 mg/kg
im). Supplementary doses of ketamine 䡠 HCl (10 mg/kg ip)
were provided as needed. A solution of 1–2% lidocaine 䡠 HCl
(2.4 mg/kg) was also administered subcutaneously at an
abdominal incision site through which a low-frequency telemetric transmitter (Barrows, San Jose, CA) was implanted to
detect hypothermia, one indication of advanced morbidity (9,
21). Cortical and muscle electrodes were implanted for monAJP-Endocrinol Metab • VOL
itoring sleep and wakefulness. Animals were allowed to recover from surgery for ⱖ7 days.
Animal environmental conditions. Rats were kept under
conditions of constant light to diminish the amplitude of the
circadian rhythm. This was to avoid the influence of phase of
circadian rhythm on dependent variables under study and
the influence of sleep deprivation on the phase, which would
differ between subject groups. Ambient air temperature was
maintained at 28°C, within the thermoneutral zone established for rats (56), with thermostatically controlled heat
lamps. Food and water were available ad libitum. Rats were
fed a balanced, purified diet that was isocaloric to a normal
diet and augmented with protein, as used in previous studies
(23, 27).
Procedure for producing sleep-deprived, yoked, and sham
control animals. The apparatus and procedures for sleep
deprivation are detailed elsewhere (9, 27). In brief, amplitude
changes in electroencephalogram (EEG) variables were electronically processed to detect sleep onset in the rat to be sleep
deprived, which triggered slow rotation of a platform on
which the rats were housed, forcing both the sleep-deprived
and the yoked rats to move for several seconds. The yoked rat
experienced forced locomotion at the same time as the sleepdeprived rat, but it could sleep whenever the platform was
stationary because the sleep-deprived partner was awake.
This experimental paradigm results in consistent sleep loss
across subject groups (21, 22, 24, 27). During a 7-day baseline
period, with 6 s of rotation every hour, rats are awake 47% of
the time. Thereafter, sleep-deprived rats are kept awake 90%
of total time, and much of the remaining sleep is highly
fragmented and/or consists of low-amplitude EEG transitional sleep. Yoked rats are awake 58% of total time, and
their sleep is fragmented because of frequent arousals from
sleep due to the paired conditions and are thus considered
partially sleep deprived. Sham controls are permitted to
sleep normally throughout the baseline and experimental
periods.
Sleep-deprived rats exhibit a consistent progression of
clinical signs, but individual animals do so at different rates
(46). The only definitive clinical marker of severity of sleep
deprivation is an eventual, acute terminal condition of advanced morbidity and hypothermia. We have used this retrospectively in other studies to determine the temporal
course of each animal in proportion to its survival period (9,
22, 27). In the present study, sleep deprivation or control
conditions were maintained for 15–21 days, which brackets
the final quartile of survival defined in previous studies. We
wished to produce sleep deprivation long enough for marked
hypothyroxinemia and its sequelae to become manifest but
short enough to preclude advanced morbidity and hypothermia. To these ends, food intake, body weight, and 24-h body
temperature were monitored daily, as previously described
(9, 27). Special food tubes with a waste receptacle afforded
accurate measurement of food consumption (9). In four pairs
of animals, the deprivation period extended to 20–21 days, by
which time the fourth sleep-deprived rat had reached advanced morbidity. The subsequent two pairs of animals were
studied after 16 days.
Tissue collection and processing. At the end of the experimental periods, rats were deeply anesthetized with injectable
anesthetics and analgesics, and cardiac puncture was performed for exsanguination and blood sampling. The brain
was removed and frozen in ⫺40°C hexane. Blood that had
been drawn into serum separator tubes during the cardiac
puncture procedure was centrifuged, and the serum was
stored at ⫺80°C for assay of T4, T3, and TSH.
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
at basal levels; therefore, TSH is inappropriately low
for the amount of circulating T4 (23). However, stimulation tests of the pituitary-thyroid axis by administration of exogenous thyrotropin-releasing hormone
(TRH) reveal a normal rise in plasma TSH comparable
to control levels, and a subsequent, appropriate rise in
plasma free T4 and free T3 throughout the progressive
course of hypothyroxinemia (23). This pattern is consistent with central hypothyroidism in humans and
experimental animals (47, 52) and points to critical
alterations of the thyroidal axis by central mechanisms
leading to TRH deficiency.
TRH-containing neurons in the paraventricular nucleus (PVN) of the hypothalamus exert control over the
biosynthesis and release of TSH from the pituitary
(reviewed in Refs. 15, 31). Experimental hypothyroidism in rats results in a significant increase in preproTRH mRNA expression and peptide content in the
PVN (35, 53, 57, 63) and hypothalamic TRH release
(50). In the present study, we evaluated TRH transcript expression in the PVN by in situ hybridization in
sleep-deprived and comparison rats. We also evaluated
type II 5⬘-deiodinase (5⬘-DII) mRNA expression in the
arcuate nucleus and median eminence (ARC-ME),
known to be rich in 5⬘-DII and to show increased
expression during thyroidectomy produced by chemicals or surgery (60). The principal finding is that prepro-TRH mRNA levels increase during prolonged sleep
deprivation as an apparently appropriate response to
decreased circulating thyroid hormone concentration,
but expected TSH responses to peripheral hypothyroxinemia and to increased TRH transcript levels do not
occur. 5⬘-DII mRNA expression in the ARC-ME was
slightly but insignificantly increased in sleep-deprived
animals rather than being suppressed, as would be
expected if thyroid hormones were present in sufficient
local excess to exert increased negative feedback on the
PVN, and thereby contribute to downregulation of the
thyroid axis. These findings point to posttranscriptional mechanisms and/or inhibitory factors as responsible for TRH deficiency and resultant hypothyroxinemia induced by sleep deprivation.
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
AJP-Endocrinol Metab • VOL
reflect the relative signal intensity in this nucleus vs. Rt
and LH.
Hormone determinations. Serum from unoperated colony
rats was assayed for quality control and to provide values for
comparison with those of the treatment groups. Serum concentrations of total T4 and total T3 were determined in
single-batch radioimmunoassays using commercial kits (Diagnostic Systems Laboratories, Webster, TX). The coefficient
of variation with an assay was ⬍3% for T4 and ⬍6% for T3.
Serum TSH was determined by immunoassay by AniLytics,
(Gaithersburg, MD). The low and high sensitivities were 0.4
and ⬎50 ng/ml, and the coefficient of variation with the assay
was ⬍4%.
Data analysis. A one-way analysis of variance (ANOVA)
was used to determine a main effect due to treatment. Post
hoc comparisons among the individual groups of sleep-deprived, yoked, and sham controls utilized the Bonferroni
correction for multiple comparisons. For all analyses, statistical significance was considered when P ⬍ 0.05. Variance is given as means ⫾ SD unless specifically noted as
means ⫾ SE.
RESULTS
Physiological variables and plasma concentrations of
TSH, T4, and T3. Sleep-deprived rats were hyperphagic
and lost weight. Daily food consumption averaged
⫹205 ⫾ 21% (range: ⫹187 to ⫹231%) of baseline
amounts on each of the last 3 days of each experiment,
and weight loss was ⫺9.5 ⫾ 4.8% (range: ⫺1.0 to
⫺15.3%) of baseline at the time of tissue collection.
Corresponding food consumption and body weight
changes in yoked controls were ⫹148 ⫾ 25% (range:
⫹114 to ⫹174%) and ⫹3.8 ⫾ 1.5% (range: ⫹1.6 to
⫹5.3%), whereas respective values in sham control rats
were ⫹109 ⫾ 10% (range: ⫹100 to ⫹125%) and ⫹5.0 ⫾
4.0% (range: ⫹1.7 to ⫹10.7%). Core temperature was
assessed for a decline ⬎1°C below baseline during 24 h,
indicative of advanced morbidity, which occurred in
one rat sleep deprived for 21 days.
Total T4 differed significantly among all treatment
groups (F2,15 ⫽ 41.2; P ⬍ 0.0001), being most profoundly reduced in the sleep-deprived group (sleep
deprived vs. yoked, t ⫽ ⫺5.97, P ⬍ 0.02; sleep-deprived
vs. sham, t ⫽ ⫺7.81; P ⬍ 0.02), with an intermediate
reduction in the partially sleep-deprived, yoked group
(yoked vs. sham, t ⫽ ⫺4.42 P ⬍ 0.02) (Fig. 1). Total T3
concentration did not differ among groups (sleep deprived, 1.6 ⫾ 0.2; yoked, 1.5 ⫾ 0.2; sham, 1.6 ⫾ 0.1
nmol/l). Despite marked differences in T4 concentration, TSH concentrations did not significantly differ
among groups (sleep deprived, 2.8 ⫾ 1.5; yoked, 1.9 ⫾
0.9; sham, 1.7 ⫾ 0.3 ng/ml). The T4 and TSH results for
individual sleep-deprived, yoked, and sham treatment
animals are shown in Fig. 1.
Prepro-TRH and 5⬘-DII mRNA expression in the
sleep-deprived brain. Representative autoradiographic
images in Fig. 2 illustrate the distribution of TRH
transcripts in sham control and sleep-deprived rat
brain, showing strong expression in the hypophysiotropic area of the PVN, as well as in the LH and Rt.
Quantitative hybridization results demonstrate that
prepro-TRH mRNA expression in the Rt and LH did
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
In situ hybridization. The prepro-TRH oligonucleotide (5⬘GTCTTTTTCCTCCTCCTCCCTTTTGCCTGGATGCTGGCGTTTTGTGAT-3⬘) was a 48-bp sequence used by other
investigators (64) complementary to a region of the TRH
transcript characterized in rat brain by Lechan et al. (39).
The 5⬘-DII oligonucleotide (5⬘-GCCATCTGAAGGGTGAGCCTCATCAATGTATACCAACAGG-3⬘) was a 40-mer derived
from the sequence published by Croteau et al. (13), complementary to a conserved region of Rattus norvegicus and
Rattus rattus type II iodothyronine deiodinase that also has
90 and 88% homology with human and Rana catesbeiana
sequences, respectively.
Frozen brain sections (16 ␮m) were cut on a cryostat at
⫺18°C, collected on polylysine-coated slides, and processed
for in situ hybridization as previously described (44). For
each animal, a total of 10 sets of slides was collected, each set
consisting of brain sections spaced at 160-␮m intervals. In
the present analyses, slides encompassing the extent of the
hypothalamus were fixed in 4% paraformaldehyde in phosphate-buffered saline, acetylated, glycinated, dehydrated
through graded ethanol, and extracted with chloroform. The
sections were hybridized for 3 h at 37°C in a hybridization
mix containing 20 pmol/ml 35S-labeled oligonucleotide probe
in 2⫻ SSC, 50% formamide, 10% dextran sulfate, 1⫻ Denhardt’s solution, 0.5 mg/ml salmon DNA, 0.25 mg/ml yeast
tRNA, and 100 mM dithiothreitol. After stringency washes in
2⫻ SSC and 50% formamide at 42°C (prepro-TRH) or 37°C
(5⬘-DII), sections were dehydrated through graded ethanol
and exposed against Kodak SB-5 film for 10 days (TRH) or 7
days (5⬘-DII) together with 14C standards (Amersham Life
Sciences, Arlington Heights, IL). Some films were also exposed for longer intervals to obtain 5⬘-DII images for illustration (for example, see Fig. 3A).
Brain sections from six rats per group were hybridized for
prepro-TRH and 5⬘-DII mRNA, with the exception that five
rats comprised the sham group for 5⬘-DII mRNA analysis.
One yoked rat was excluded from quantitative analysis of
prepro-TRH mRNA expression, because the sections that had
been selected and hybridized with those of the other animals
had not captured the appropriate level of hypothalamus.
Another yoked rat was excluded from analysis of 5⬘-DII
mRNA expression because the ARC-ME was damaged during
dissection. The autoradiogram of each hybridized section was
captured as a calibrated image by use of the NIH Image
program, and those demonstrating peak hybridization signal
at approximately the same rostral-caudal brain level were
selected from each animal for quantitative densitometry of
prepro-TRH mRNA in the PVN, the lateral hypothalamus
(LH), and the reticular thalamic nucleus (Rt) and 5⬘-DII in
the ARC-ME. For measurements of the PVN, Rt, and ARCME, the appropriate structure was thrice outlined within the
border of greatest signal contrast from surrounding brain.
For measurements of the LH, which exhibited scattered
points of high signal density (see Fig. 2A), a standard size
oval shape encompassing this structure was used to measure
density within a fixed area across all sections. Bilateral
measurements of PVN, Rt, and LH, minus background, were
averaged to yield one value per structure for each animal.
The investigators independently scanned and measured the
prepro-TRH signal in the PVN, with good agreement (r2 ⫽
0.96). Further evaluations of average hybridization derived
from density measurements in fixed areas encompassing the
PVN in two to three sequential sections yielded no change in
relative signal intensity determined from peak sections, indicating that these provided representative sampling of PVN
for each animal. The data presented and analyzed below
were derived from PVN profiles in peak sections to better
E87
E88
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
Fig. 1. Serum thyroxine (T4; A) and thyroid-stimulating hormone (TSH; B) concentrations during sleep deprivation in
individual animals. Group means (horizontal bars) for plasma T4 concentrations
in sleep-deprived (■), yoked (E), and sham
control (Œ) rats were 15.3 ⫾ 3.6, 25.7 ⫾ 2.2,
and 36.7 ⫾ 5.7 nmol/l, respectively. *Statistical significance between groups is indicated. Group means for serum TSH
were 2.8 ⫾ 1.5, 1.9 ⫾ 0.9, and 1.7 ⫾ 0.3
nmol/l, respectively. Group differences
were highly significant for T4, but not for
TSH, despite low T4 in the partially
(yoked) and totally sleep-deprived groups.
the sham control group, and in all cases individual
values were greater than the average of the sham
control group.
Post hoc evaluation of results for individual animals
as a function of the duration of sleep deprivation
showed that neither of the sleep-deprived rats studied
at 15–16 days showed elevated TRH transcript levels
in PVN, whereas three of the four animals studied at
20–21 days averaged approximately three times the
control hybridization signal (Fig. 4). The one sleepdeprived animal that did not exhibit elevated preproTRH mRNA at 21 days was also the only rat exhibiting
advanced morbidity in this study, characterized by a
24-h drop in core temperature of ⬎1°C. The two highest values for prepro-TRH mRNA expression in yoked
Fig. 2. Prepro-TRH mRNA expression in
brain and the effect of sleep deprivation.
Left: representative autoradiograms illustrate prepro-TRH mRNA distribution in
paraventricular nucleus (PVN), lateral
hypothalamus (LH), and reticular thalamic nucleus (Rt) of a sham control animal (top) and a sleep-deprived animal
(bottom). Illustrated fields are ⬃8.5 mm in
width. Right: quantitative densitometry
demonstrates a significant increase in prepro-TRH hybridization in PVN of sleepdeprived vs. sham control rats (* P ⬍ 0.02).
Error bars, SE.
AJP-Endocrinol Metab • VOL
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
not differ among sleep-deprived, yoked, and sham control groups. Prepro-TRH mRNA expression in the PVN
showed a significant increase in the sleep-deprived
group vs. sham control (P ⬍ 0.02) and a similar trend
that did not reach significance in the yoked group.
Representative autoradiographic images of 5⬘-DII
mRNA expression in the ARC-ME region are shown in
Fig. 3 together with quantitative hybridization results
for the three experimental groups. Although mean
5⬘-DII hybridization tended to be higher in sleep-deprived animals (sleep deprived: 99 ⫾ 24; yoked: 76 ⫾
11; sham control: 82 ⫾ 19 nCi/g), there were no significant differences among the groups. Post hoc review of
5⬘-DII mRNA data showed that two sleep-deprived
animals displayed a signal ⬎2 SD above the average of
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
E89
animals were also observed at 20–21 days. These results demonstrate that upregulation of TRH transcription is a late event in the course of sleep deprivation.
Post hoc evaluation of the relationships between
hormone concentration and transcript levels within
groups did not reveal statistically significant correlations between T4 concentrations and either TSH concentrations or prepro-TRH transcript levels, nor between TSH concentrations and TRH transcript levels,
owing in part to low variability in T4 and TSH concentrations. However, sleep-deprived animals studied after 20–21 days tended to have higher TSH concentrations than animals studied after 15–16 days (Fig. 4).
DISCUSSION
Sleep deprivation resulted in the characteristic spectrum of changes observed in previous studies, including weight loss and dramatically increased food consumption (22, 27) and marked hypothyroxinemia (8,
23) (Fig. 1). The progressive decline in circulating total
and free T4 and T3 observed in previous studies indicates that the mechanisms responsible for decreased
hormone production/release are affected early and persistently. Previous investigations suggested that alterations in central mechanisms regulating TRH synthesis and release may be responsible, since sleepdeprived rats with low thyroid hormone levels and
unchanged TSH respond to exogenous TRH administration with an appropriate burst of TSH release and
thyroid activation throughout the average 21-day experimental period (23). The stimulated TSH release in
sleep-deprived rats is not blunted, which, when it occurs in response to food restriction (49), may be considered part of an adaptive response to lower energy
requirements. The thyroid hormone profile of the sleepdeprived rat is consistent with central hypothyroidism
in humans (47, 52) and resembles TRH deficiency in
rats produced by lesions of the PVN, which results in
Fig. 4. Time course of prepro-TRH mRNA
and TSH responses during sleep deprivation.
Results from individual animals are illustrated for prepro-TRH mRNA expression in
the PVN (A) and for serum TSH concentrations (B). Yoked (E) and sleep-deprived (■)
animals were studied after 15–16 or 20–21
experimental days, when there was marked
hypothyroxinemia in the sleep-deprived animals. The blocked area represents the
mean ⫾ SD of control values, derived from
individual animals indicated at day 0 (Œ).
AJP-Endocrinol Metab • VOL
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
Fig. 3. 5⬘-Deiodinase type II (5⬘-DII) mRNA expression in hypothalamus and the effect of sleep
deprivation. Left: representative autoradiograms
illustrate in situ hybridization of 5⬘-DII mRNA
in the arcuate nucleus-median eminence (ARCME) and periventricular region (PE) of a sham
control animal (top) and a sleep-deprived animal
(bottom). The films were subjected to prolonged
exposure relative to those used for densitometry
to visualize more clearly the structures expressing 5⬘-DII mRNA. Illustrated fields are ⬃4.5 mm
in width. Right: quantitative hybridization results in sleep-deprived, yoked, and sham control
groups. There were no statistically significant
group differences. Error bars, SE.
E90
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
AJP-Endocrinol Metab • VOL
change in serum T3, are associated with three- to
fourfold increases in serum TSH (40). Severalfold increases in TSH occur by 4–6 days after thyroidectomy
treatments (29, 30, 38), before any detectable change in
prepro-TRH mRNA expression (38). As experimental
hypothyroidism is prolonged beyond this initial period,
TSH levels increase progressively to more than 10-fold
euthyroid levels (29, 30, 38). Maintenance of 50% of
normal plasma T4 in thyroidectomized animals by supplementary T4 administration for 14 days is associated
with an eight- to ninefold elevation of plasma TSH
concentration (16). The blunted TSH response in sleepdeprived animals, therefore, is inappropriate, given
both the magnitude and duration of hypothyroxinemia
and the above-demonstrated increase in TRH transcripts.
TRH deficiency produced by PVN lesions likewise
results in a markedly blunted TSH response to hypothyroidism. Serum TSH concentrations in PVN-lesioned animals show only 30% of the increase seen in
thyroidectomized rats with intact PVN (30, 57). This is
associated with a dramatically blunted increase in
TSH subunit mRNA levels in anterior pituitary in
response to hypothyroidism of 2–4 wk duration (57).
The threshold for thyroid hormone feedback onto the
pituitary adenohypophysis is lowered in PVN-lesioned
or deafferented animals (29, 41), such that even small
doses of T4 are effective in completely suppressing the
marginal increases in serum TSH (41). On the basis of
the similarities between sleep-deprived and PVN-lesioned animals, TRH deficiency remains a more compelling explanation for thyroid axis suppression than
does primary TSH deficiency, particularly in light of
the fact that sleep-deprived rats respond to exogenous
TRH administration with an appropriate increase in
serum TSH (23). The present results, demonstrating
appropriate increases in TRH transcript levels in PVN,
direct attention to factors regulating the translation of
prepro-TRH mRNA, processing and release of TRH
peptide, and/or feedback responsiveness of pituitary
thyrotrophs as the foci of altered thyroid hormone
regulation in sleep-deprived animals.
An additional comparison between PVN-lesioned
and sleep-deprived rats concerns the T4-to-T3 conversion enzyme 5⬘-DII. This enzyme is upregulated in
response to decreased circulating T4, with identical
concentration dependence of activities in pituitary, cerebral cortex, and BAT (54, 55). Prolonged experimental hypothyroidism increases pituitary 5⬘-DII activity
to 400–900% of euthyroid levels (13, 48). This response
is not reversed by subsequent PVN lesions, but such
lesions in euthyroid animals reduce plasma T4 and T3
by 60 and 75%, respectively, yet produce only a moderate 50% elevation of pituitary 5⬘-DII at 1 wk (43). A
previous study of rats sleep deprived for 14 days likewise revealed only a modest, statistically insignificant
40% increase in 5⬘-DII activity in the pituitary,
whereas that in BAT was increased 100-fold (6). Supplemental T4 treatment partially reduced the BAT
5⬘-DII activation during sleep deprivation (6), indicating a sensitivity to T4 that would predict a larger than
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
decreased plasma T4 and T3 (43), decreased plasma
TSH response to hypothyroidism (3, 43), but normal
TSH secretion in response to exogenous TRH administration (3).
Prepro-TRH mRNA expression in the PVN showed a
significant increase in the sleep-deprived group, and a
similar trend in the yoked group, relative to the sham
control group. TRH transcript levels in the LH and the
Rt did not differ among groups (Figs. 2 and 4), consistent with preferential upregulation of TRH biosynthesis in the hypophysiotropic pathway (19, 35, 53, 57).
Post hoc evaluation of individual sleep-deprived animals revealed threefold elevations in three of four
animals studied at 20–21 days but not in those studied
after shorter duration despite nearly comparable hypothyroxinemia. Many days also are required to observe increased prepro-TRH mRNA in experimental
models of thyroid hormone depletion. T4 is diminished
after chemical or surgical thyroidectomy to a concentration much lower than that observed in sleep-deprived rats within 3–6 days (29, 30, 38) without an
appreciable increase in TRH transcript levels in the
PVN (38). Thereafter, prepro-TRH mRNA begins to
rise (38), concurrent with TRH depletion in the median
eminence and much of the hypothalamus (63). In thyroidectomized animals, prepro-TRH mRNA levels
reach 155–190% of the euthyroid level by 14 days (38,
42, 63) and 200–250% of euthyroid control by 21 days
or longer (11, 12, 34, 38, 53). These peak changes are
comparable to those observed in this study, and the
lack of accumulation of TRH transcripts in 16-day
sleep-deprived animals may be consistent with the
more gradual progression to a severely hypothyroxinemic state during sleep deprivation (8, 23) than during
experimental hypothyroidism. However, yoked animals tended to show elevated prepro-TRH mRNA expression at 20–21 days despite more moderate decreases in peripheral T4 (Fig. 4) (8, 23), indicating that
the duration of hypothyroxinemia may be as important
a factor as its severity in triggering this response. TRH
transcripts were not elevated in one sleep-deprived rat
studied after 21 days, but this was accompanied by
advanced morbidity. It remains to be determined
whether failure to upregulate TRH expression may
result in a more severe pathological course or, conversely, whether an abrupt decline in prepro-TRH
mRNA expression may be associated with the terminal
phase of sleep deprivation.
A major feature distinguishing thyroidectomy and
sleep deprivation is the TSH response. Sleep-deprived
animals in the present study showed no change, or only
slight increases, in plasma TSH at the end of the
deprivation period. This is consistent with a previous
study that utilized indwelling catheters for repeated
blood sampling that revealed unchanged TSH across
experimental days despite progressive hypothyroxinemia (23). In contrast, TSH levels rise during chemical
thyroidectomy even before serum T4 or T3 shows a
detectable change (40). Modest declines of serum T4 by
11–24% during 4 days of low-dose methylmercaptoimidazole or propylthiouracil treatment, with little or no
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
AJP-Endocrinol Metab • VOL
in increased TRH release through the portal system,
resulting in elevated plasma TSH (15). Under certain
conditions, such as exposure to cold, ascending noradrenergic and/or adrenergic projections to the PVN (reviewed in Refs. 38, 64) appear to override the normal
inhibitory effects of increased T3 and produce increased TRH mRNA expression in the PVN (64). To
assess functional activation in the sleep-deprived rat
brain, a study of metabolic activity revealed that glucose metabolism is decreased in most hypothalamic
structures except the lateral hypothalamic feeding “on”
center (24). Although the PVN had not been explicitly
quantitated in that study, the particularly low metabolic rates of adjacent structures indicate a decreased
metabolic activity that may reflect tonic inhibition in
this region. Another candidate factor, dopamine, exerts
inhibitory control on TRH at two central levels, affecting both hypothalamic TRH release (4) and pituitary
TSH responses to TRH (37). L-Dopa administration to
hypothyroid patients lowers elevated TSH levels without changing the response to TRH (reviewed in Ref.
37). Abundant evidence shows that dopamine acts on
pituitary thyrotrophs (reviewed in Ref. 37). Recent
evidence has also linked dopamine to the regulation of
the expression of 5⬘-DII mRNA in the ARC-ME via a
dopamine- and cAMP-regulated phosphoprotein found
in tanycytes (28), which may be relevant insofar as
5⬘-DII mRNA was not found to be robustly increased in
this region in the present study.
Several additional nonthyroidal factors are implicated in sleep deprivation-induced hypothyroxinemia
by virtue of coexisting metabolic (8, 24, 27) and host
defense abnormalities (21, 26). Neuropeptide Y can
induce food-seeking behavior and hyperphagia (58),
which are prominent features of the sleep deprivation
profile. Neuropeptide Y is contained in neurons innervating TRH cells (59) and appears to block catecholaminergic inputs to the PVN (reviewed in Ref.
38). Endotoxin and cytokine exposure, implicated in
the hypercatabolic and immune-suppressed state of
the sleep-deprived animal (26), produces key characteristics of central hypothyroidism, including low
plasma T4 and T3 but inappropriately normal or decreased TSH levels (33, 36, 62). Conversely, thyroid
axis suppression by glucocorticoids is not a candidate
mechanism, because overactivation of the hypothalamicpituitary-adrenal axis has not been found in this
model (23), and the present results provide direct evidence against glucocorticoid-induced reductions in prepro-TRH mRNA expression in PVN (32).
In summary, sleep deprivation produces the paradoxical combination of peripheral hypercatabolism and
central hypothyroxinemia. Other clinical states to
which sleep deprivation may be typically compared are
associated with suppression of TRH mRNA expression
in the PVN, which appears to explain suppression of
the thyroid axis in these cases. In contrast, increased
prepro-TRH mRNA expression was demonstrated in
the sleep-deprived brain, consistent with compensatory TRH biosynthesis. These data suggest that the
locus of abnormal thyroid hormone regulation lies after
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
observed increase in pituitary 5⬘-DII activity. The attenuated responsiveness of pituitary 5⬘-DII activity
may be indicative of comparable TRH deficiency in
PVN-lesioned and sleep-deprived animals.
Regulation of 5⬘-DII in responses to changing thyroid
hormone status has also been investigated in brain.
Studies in cerebral cortex indicate that, although enzyme activity is primarily responsive to T4, 5⬘-DII
mRNA expression is inversely related to T3 levels (10).
Local production of T3 from T4 via 5⬘-DII in astrocytes
(17) and tanycytes (60) of the ARC-ME has been proposed to play a specific role in signaling the PVN,
which lacks 5⬘-DII (48). We found moderate increases
in 5⬘-DII mRNA in ARC-ME of two sleep-deprived
animals (Fig. 4), consistent with the modest 30–60%
elevations observed after hypothyroxinemia resulting
from fasting (18) or thyroidectomy (18, 60). The greater
difficulty in achieving consistent sample locations in
sections through the ARC-ME renders the quantitation
of 5⬘-DII mRNA expression somewhat less reliable
than the prepro-TRH measurements. Nevertheless,
there was clearly no indication of reduced 5⬘-DII
mRNA in the ARC-ME that might indicate local T3
excess. In comparison, 5⬘-DII mRNA levels in ARC-ME
are decreased in euthyroid animals treated with T4
(18). Feedback action of thyroid hormones exerted at
the level of the hypothalamus is considered an important factor affecting TRH release (51) as well as its
production. Although we did not specifically evaluate
5⬘-DII mRNA levels in the periventricular area (PE),
there was likewise no indication of the severalfold
increase observed in this region that appears uniquely
induced by fasting and associated with enhanced feedback downregulation of prepro-TRH expression in the
PVN by T3 (18) (see Fig. 3).
Recent reports have described acute upregulation of
5⬘-DII activity in several brain regions in response to
diverse stresses, including a model of forced walking
with an eventual period of short-term sleep deprivation
(7, 20). In the latter paradigm, increased activity was
particularly prominent in frontal cortex. Because enzyme activity and mRNA levels can be regulated independently (see above), it is difficult to relate such
observations to the present hybridization results. The
levels of 5⬘-DII mRNA are much lower in cortex than
ARC-ME, and we saw no suggestion of a change in
transcript levels in cortex. It should be noted that
reported increases in enzyme activity were more prominent after 10 h of forced mobility than after the longer
intervals that included sleep deprivation (7), further
indicating that this is an acute response unlikely to be
relevant to the model of prolonged sleep deprivation
employed in the present study.
Besides thyroid hormones, several nonthyroidal factors affect TRH translation, production, and release via
short and long control loops and therefore have the
potential to explain thyroid axis suppression during
sleep deprivation. Hypothetically, sustained wakefulness may lead to decreased neuronal stimulation of the
PVN by other brain regions. Electrical stimulation,
used to mimic neuronal excitation of the PVN, results
E91
E92
PREPRO-TRH
MRNA
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
prepro-TRH transcription, at steps up to and including
release of the mature peptide. Features of the sleepdeprived profile point to nonthyroidal control mechanisms, such as inhibitory neurotransmitters and neuromodulators, including those related to immunity, as
potential candidates mediating central hypothyroxinemia induced by sleep deprivation.
17.
18.
We thank Dr. Qihong Zhou for preparation of frozen sections, and
Jason M. Scott for technical assistance.
Research support was provided by the National Institute of Neurological Disorders and Stroke (NS-38733) and the National Heart,
Lung, and Blood Institute (HL-59271).
19.
REFERENCES
20.
AJP-Endocrinol Metab • VOL
21.
22.
23.
24.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
1. Anonymous. Treatment of Sleep Disorders of Older People. NIH
Consens Dev Conf Consens Statement, 1990, vol. 8, no. 3.
2. Anonymous. Wake up America: a national sleep alert. In:
Executive Summary and Executive Report to the United States
Congress and to the Secretary of the US Department of Health
and Human Services. Washington, DC: National Commission on
Sleep Disorders Research, 1993, p. 1–76.
3. Aizawa T and Greer MA. Delineation of the hypothalamic area
controlling thyrotropin secretion in the rat. Endocrinology 109:
1731–1738, 1981.
4. Andersson K and Eneroth P. Thyroidectomy and central
catecholamine neurons of the male rat. Evidence for the existence of an inhibitory dopaminergic mechanism in the external
layer of the median eminence and for a facilitatory noradrenergic
mechanism in the paraventricular hypothalamic nucleus regulating TSH secretion. Neuroendocrinology 45: 14–27, 1987.
5. Aurell J and Elmqvist D. Sleep in the surgical intensive care
unit: continuous polygraphic recording of sleep in nine patients
receiving postoperative care. Br Med J 290: 1029–1032, 1985.
6. Balzano S, Bergmann BM, Gilliland MA, Silva JE,
Rechtschaffen A, and Refetoff S. Effect of total sleep deprivation on 5⬘-deiodinase activity of rat brown adipose tissue.
Endocrinology 127: 882–890, 1990.
7. Baumgartner A, Hiedra L, Pinna G, Eravci M, Prengel H,
and Meinhold H. Rat brain type II 5⬘-iodothyronine deiodinase
activity is extremely sensitive to stress. J Neurochem 71: 817–
826, 1998.
8. Bergmann BM, Everson CA, Kushida CA, Fang VS, Leitch
CA, Schoeller DA, Refetoff S, and Rechtschaffen A. Sleep
deprivation in the rat. V. Energy use and mediation. Sleep 12:
31–41, 1989.
9. Bergmann BM, Kushida CA, Everson CA, Gilliland MA,
Obermeyer W, and Rechtschaffen A. Sleep deprivation in the
rat. II. Methodology. Sleep 12: 5–12, 1989.
10. Burmeister LA, Pachucki J, and St. Germain DL. Thyroid
hormones inhibit type 2 iodothyronine deiodinase in the rat
cerebral cortex by both pre- and posttranslational mechanisms.
Endocrinology 138: 5231–5237, 1997.
11. Calza L, Aloe L, and Giadino L. Thyroid hormone-induced
plasticity in the adult rat brain. Brain Res Bull 44: 549–557,
1997.
12. Ceccatelli S, Giardino L, and Calza L. Response of hypothalamic peptide mRNAs to thyroidectomy. Neuroendocrinology 56:
694–703, 1992.
13. Croteau W, Davey JC, Galton VA, and St. Germain DL.
Cloning of the mammalian type II iodothyronine deiodinase. A
selenoprotein differentially expressed and regulated in human
and rat brain and other tissues. J Clin Invest 98: 405–417, 1996.
14. Cureton-Lane RA and Fontaine DK. Sleep in the pediatric
ICU: an empirical investigation. Am J Crit Care 6: 56–63, 1997.
15. De Greef WJ, Rondeel JM, van Haasteren GA, Klootwijk
W, and Visser TJ. Regulation of hypothalamic TRH production
and release in the rat. Acta Med Austriaca 19, Suppl 1: 77–79,
1992.
16. DeVito WJ, Connors JM, and Hedge GA. The pituitary TSH
response to TRH is inversely related to the plasma TSH concentration and directly related to the pituitary TSH content during
hypothyroidism in the rat. Acta Endocrinol (Copenh) 114: 27–36,
1987.
Diano S, Naftolin F, Goglia F, Csernus V, and Horvath TL.
Monosynaptic pathway between the arcuate nucleus expressing
glial type II iodothyronine 5⬘-deiodinase mRNA and the median
eminence-projective TRH cells of the rat paraventricular nucleus. J Neuroendocrinol 10: 731–742, 1998.
Diano S, Naftolin F, Goglia F, and Horvath TL. Fastinginduced increase in type II iodothyronine deiodinase activity and
messenger ribonucleic acid levels is not reversed by thyroxine in
the rat hypothalamus. Endocrinology 139: 2879–2884, 1998.
Dyess EM, Segerson TP, Liposits Z, Paull WK, Kaplan MM,
Wu P, Jackson IM, and Lechan RM. Triiodothyronine exerts
direct cell-specific regulation of thyrotropin-releasing hormone
gene expression in the hypothalamic paraventricular nucleus.
Endocrinology 123: 2291–2297, 1988.
Eravci M, Pinna G, Meinhold H, and Baumgartner A.
Effects of pharmacological and nonpharmacological treatments
on thyroid hormone metabolism and concentrations in rat brain.
Endocrinology 141: 1027–1040, 2000.
Everson CA. Sustained sleep deprivation impairs host defense.
Am J Physiol Regulatory Integrative Comp Physiol 265: R1148–
R1154, 1993.
Everson CA, Bergmann BM, and Rechtschaffen A. Sleep
deprivation in the rat. III. Total sleep deprivation. Sleep 12:
13–21, 1989.
Everson CA and Reed HL. Pituitary and peripheral thyroid
hormone responses to thyrotropin-releasing hormone during
sustained sleep deprivation in freely moving rats. Endocrinology
136: 1426–1434, 1995.
Everson CA, Smith CB, and Sokoloff L. Effects of prolonged
sleep deprivation on local rates of cerebral energy metabolism in
freely moving rats. J Neurosci 14: 6769–6778, 1994.
Everson CA and Toth LA. Systemic bacterial invasion induced
by sleep deprivation. Am J Physiol Regulatory Integrative Comp
Physiol 278: R905–R916, 2000.
Everson CA and Wehr TA. Nutritional and metabolic adaptations to prolonged sleep deprivation in the rat. Am J Physiol
Regulatory Integrative Comp Physiol 264: R376–R387, 1993.
Fekete C, Mihaly E, Herscovici S, Salas J, Tu H, Larsen
PR, and Lechan RM. DARPP-32 and CREB are present in type
2 iodothyronine deiodinase-producing tanycytes: implications for
the regulation of type 2 deiodinase activity. Brain Res 862:
154–161, 2000.
Fukuda H and Greer MA. The effect of basal hypothalamic
isolation on pituitary-thyroid activity and the response to propylthiouracil. Endocrinology 100: 911–917, 1977.
Greer MA, Sato N, Wang X, Greer SE, and McAdams S.
Evidence that the major physiological role of TRH in the hypothalamic paraventricular nuclei may be to regulate the set-point
for thyroid hormone negative feedback on the pituitary thyrotroph. Neuroendocrinology 57: 569–575, 1993.
Joseph-Bravo P, Uribe RM, Vargas MA, Perez-Martinez L,
Zoeller T, and Charli JL. Multifactorial modulation of TRH
metabolism. Cell Mol Neurobiol 18: 231–247, 1998.
Kakucska I, Qi Y, and Lechan RM. Changes in adrenal status
affect hypothalamic thyrotropin-releasing hormone gene expression in parallel with corticotropin-releasing hormone. Endocrinology 137: 2795–2802, 1995.
Kakucska I, Romero LI, Clark BD, Rondeel JM, Qi Y, Alex
S, Emerson CH, and Lechan RM. Suppression of thyrotropinreleasing hormone gene expression by interleukin-1-beta in the
rat: implications for nonthyroidal illness. Neuroendocrinology
59: 129–137, 1994.
Kim SY, Post RM, and Rosen JB. Differential regulation of
basal and kindling-induced TRH mRNA expression by thyroid
hormone in the hypothalamic and limbic structures. Neuroendocrinology 63: 297–304, 1996.
Koller KJ, Wolff RS, Warden MK, and Zoeller RT. Thyroid
hormones regulate levels of thyrotropin-releasing-hormone
mRNA in the paraventricular nucleus. Proc Natl Acad Sci USA
84: 7329–7333, 1987.
Kondo K, Harbuz MS, Levy A, and Lightman SL. Inhibition
of the hypothalamic-pituitary-thyroid axis in response to lipo-
PREPRO-TRH
37.
38.
39.
40.
41.
43.
44.
45.
46.
47.
48.
49.
50.
51.
EXPRESSION IN SLEEP-DEPRIVED RAT BRAIN
polysaccharide is independent of changes in circulating corticosteroids. Neuroimmunomodulation 4: 188–194, 1997.
Krulich L. Neurotransmitter control of thyrotropin secretion.
Neuroendocrinology 35: 139–147, 1982.
Lechan RM and Toni R. Thyrotropin-releasing hormone neuronal systems in the central nervous system. In: Endocrinology,
edited by C Nemeroff. Boca Raton, FL: CRC, 1992, p. 279–330.
Lechan RM, Wu P, Jackson IM, Wolf H, Cooperman S,
Mandel G, and Goodman RH. Thyrotropin-releasing hormone
precursor: characterization in rat brain. Science 231: 159–161,
1986.
Mannisto PT, Ranta T, and Leppaluoto J. Effects of methylmercaptoimidazole (MMI), propylthiouracil (PTU), potassium
perchlorate (KClO4) and potassium iodide (KI) on the serum
concentrations of thyrotrophin (TSH) and thyroid hormones in
the rat. Acta Endocrinol (Copenh) 91: 271–281, 1979.
Martin JB, Boshans R, and Reichlin S. Feedback regulation
of TSH secretion in rats with hypothalamic lesions. Endocrinology 87: 1032–1040, 1970.
Muller LM and Kennedy JA. Quantification of rat pre-prothyrotropin releasing hormone (TRH) mRNA by reverse transcription-polymerase chain reaction using external and internal
standardization. J Neurosci Methods 68: 269–274, 1996.
Murakami M, Tanaka K, Greer MA, and Mori M. Anterior
pituitary type II thyroxine 5⬘-deiodinase activity is not affected
by lesions of the hypothalamic paraventricular nucleus which
profoundly depress pituitary thyrotropin secretion. Endocrinology 123: 1676–1681, 1988.
Nowak TS Jr. Localization of 70 kDa stress protein mRNA
induction in gerbil brain after ischemia. J Cereb Blood Flow
Metab 11: 432–439, 1991.
Pilcher JJ, Bergmann BM, Refetoff S, Fang VS, and
Rechtschaffen A. Sleep deprivation in the rat. XIII. The effect
of hypothyroidism on sleep deprivation symptoms. Sleep 14:
201–210, 1991.
Rechtschaffen A, Bergmann BM, Everson CA, Kushida
CA, and Gilliland MA. Sleep deprivation in the rat. X. Integration and discussion of the findings. Sleep 12: 68–87, 1989.
Refetoff S. Thyroid function tests and effects of drugs on thyroid
function. In: Endocrinology, edited by LJ DeGroot. Philadelphia,
PA: Saunders, 1989, p. 590–639.
Riskind PN, Kolodny JM, and Larsen PR. The regional
hypothalamic distribution of type II 5⬘-monodeiodinase in euthyroid and hypothyroid rats. Brain Res 420: 194–198, 1987.
Rodriguez F, Mellado M, Montoya E, and Jolin T. Sensitivity of thyrotropin secretion to TSH-releasing hormone in foodrestricted rats. Acta Endocrinol (Copenh) 124: 194–202, 1991.
Rondeel JM, de Greef WJ, Klootwijk W, and Visser TJ.
Effects of hypothyroidism on hypothalamic release of thyrotropin-releasing hormone in rats. Endocrinology 130: 651–656,
1992.
Rondeel JM, de Greef WJ, van der Schoot P, Karels B,
Klootwijk W, and Visser TJ. Effect of thyroid status and
AJP-Endocrinol Metab • VOL
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
E93
paraventricular area lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood.
Endocrinology 123: 523–527, 1988.
Samuels MH and Ridgway EC. Central hypothyroidism. Endocrinol Metab Clin North Am 21: 903–919, 1992.
Segerson TP, Kauer J, Wolfe HC, Mobtaker H, Wu P,
Jackson IM, and Lechan RM. Thyroid hormone regulates
TRH biosynthesis in the paraventricular nucleus of the rat
hypothalamus. Science 238: 78–80, 1987.
Silva JE and Larsen PR. Interrelationships among thyroxine,
growth hormone, and the sympathetic nervous system in the
regulation of 5⬘-iodothyronine deiodinase in rat brown adipose
tissue. J Clin Invest 77: 1214–1223, 1986.
Silva JE and Leonard JL. Regulation of rat cerebrocortical
and adenohypophyseal type II 5⬘-deiodinase by thyroxine, triiodothyronine, and reverse triiodothyronine. Endocrinology 116:
1627–1635, 1985.
Szymusiak R and Satinoff E. Maximal REM sleep time defines a narrower thermoneutral zone than does minimal metabolic rate. Physiol Behav 26: 687–690, 1981.
Taylor T, Wondisford FE, Blaine T, and Weintraub BD.
The paraventricular nucleus of the hypothalamus has a major
role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion. Endocrinology 126: 317–324, 1990.
Tomaszuk A, Simpson C, and Williams G. Neuropeptide Y,
the hypothalamus and the regulation of energy homeostasis.
Horm Res 46: 53–58, 1996.
Toni R, Jackson IM, and Lechan RM. Neuropeptide-Y-immunoreactive innervation of thyrotropin-releasing hormone-synthesizing neurons in the rat hypothalamic paraventricular nucleus. Endocrinology 126: 2444–2453, 1990.
Tu HM, Kim SW, Salvatore D, Bartha T, Legradi G, Larsen
PR, and Lechan RM. Regional distribution of type 2 thyroxine
deiodinase messenger ribonucleic acid in rat hypothalamus and
pituitary and its regulation by thyroid hormone. Endocrinology
138: 3359–3368, 1997.
Utiger RD. Thyrotropin-releasing hormone and thyrotropin secretion. J Lab Clin Med 109: 327–335, 1987.
Van Haasteren GA, van der Meer MJ, Hermus AR, Linkels
E, Klootwijk W, Kaptein E, van Toor H, Sweep CG, Visser
TJ, and de Greef WJ. Different effects of continuous infusion of
interleukin-1 and interleukin-6 on the hypothalamic-hypophysial-thyroid axis. Endocrinology 135: 1336–1345, 1994.
Yamada M, Satoh T, Monden T, Murakami M, Iriuchijima
T, Wilber JF, and Mori M. Influences of hypothyroidism on
TRH concentrations and preproTRH mRNA levels in rat hypothalamus: a simple and reliable method to detect preproTRH
mRNA level. Neuroendocrinology 55: 317–320, 1992.
Zoeller RT, Kabeer N, and Albers HE. Cold exposure elevates
cellular levels of messenger ribonucleic acid encoding thyrotropin-releasing hormone in paraventricular nucleus despite elevated levels of thyroid hormones. Endocrinology 127: 2955–
2962, 1990.
283 • JULY 2002 •
www.ajpendo.org
Downloaded from http://ajpendo.physiology.org/ by 10.220.33.3 on May 7, 2017
42.
MRNA