Vol. 276, Issue 1, R143-R151, January 1999
Cold exposure regulates the norepinephrine uptake transporter
in rat brown adipose tissue
Victoria L.
King,
Linda P.
Dwoskin, and
Lisa A.
Cassis
Division of Pharmaceutical Sciences, College of
Pharmacy, University of Kentucky, Lexington, Kentucky
40536-0082
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ABSTRACT |
The neuronal
uptake of norepinephrine (NE) in sympathetically innervated tissues is
mediated by a high-affinity NE uptake transporter (NET). Rat
interscapular brown adipose tissue (ISBAT) is densely innervated by the
sympathetic nervous system for the control of cold- and diet-induced
thermogenesis. To determine if cold exposure regulates the NET, kinetic
parameters for [3H]NE
uptake and
[3H]nisoxetine (Nis)
binding were determined in ISBAT from 7-day cold-exposed (CE) and
control rats. Uptake of
[3H]NE in ISBAT slices
was of high affinity (1.6 µM). After 7 days of cold
exposure the affinity for
[3H]NE uptake was not
altered; however, the uptake capacity was decreased (38%) in ISBAT
slices from CE rats. Kinetic parameters for
[3H]Nis binding
demonstrated a single high-affinity site in ISBAT from CE and control
rats with similar affinity. The density of [3H]Nis sites in ISBAT
was decreased (38%) following cold exposure. A time course (2 h-7
days) for cold exposure demonstrated downregulation of
[3H]Nis binding
density by day 3, which remained
through day 7. The affinity for
[3H]Nis binding was
transiently decreased at 2 h of cold exposure. Similarly, ISBAT NE
content was decreased at 2 h of cold exposure. Pair feeding CE rats
to food intake of controls normalized plasma NE content; however,
[3H]Nis binding
density in ISBAT remained decreased in pair-fed rats. These results
demonstrate that the ISBAT NET is downregulated following cold
exposure. Reductions in ISBAT NE content precede alterations in NET
density; however, plasma NE content is not related to regulation of the NET.
interscapular brown adipose tissue; nisoxetine
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INTRODUCTION |
THE NEURONAL UPTAKE of norepinephrine (NE) in
sympathetically innervated tissues is mediated by a high-affinity
(range 0.2-0.4 µM) NE transporter (NET) located on presynaptic
sympathetic terminals in the central and peripheral nervous system
(17). The NET regulates synaptic NE concentrations in sympathetically
innervated tissues and serves as a conservatory mechanism for recycling
neurotransmitter. Previous investigators have demonstrated regulation
of the NET in response to agents modulating NE substrate content.
Specifically, following reserpine treatment and depletion of
catecholamine stores, decreases were observed in steady-state mRNA
levels for NET in adrenal medulla and locus coeruleus (10, 11).
Moreover, reserpine treatment resulted in a decrease in the number of
NET binding sites in cerebral cortex and locus coeruleus (10, 11, 21). In vivo treatment with monoamine oxidase inhibitors, which increased tissue NE content, resulted in an increase in the number of NET sites
in cerebral cortex (21). Short- and long-term inhibition of NET by in
vivo desipramine treatment resulted in an increase in mRNA expression
for the NET in locus coeruleus (33). Collectively, results from
previous studies suggest that treatment with compounds that increase NE
content positively regulate mRNA expression and protein density of the
NET; conversely, treatment with compounds that deplete NE stores
negatively regulate the NET.
The activity of the sympathetic nervous system is inversely related to
environmental temperature, with an increase in sympathetic activity
following cold exposure. Initial increases in sympathetic nerve
activity following cold exposure (
1 wk) result in generalized vasoconstriction and skeletal muscle shivering to maintain body temperature (20). During prolonged cold exposure, increases in
sympathetic drive result in the long-term maintenance of body temperature through nonshivering thermogenesis. The organ most strongly
stimulated by sympathetic activity following chronic cold exposure is
brown adipose tissue. Rat interscapular brown adipose tissue (ISBAT) is
a well-defined anatomic tissue, receiving bilateral sympathetic
innervation via five intercostal nerves with innervation to the
vasculature and individual brown adipocytes (13). Sympathetic
innervation of brown adipose tissue is comparable to other densely
innervated peripheral tissues (30). Activation of the sympathetic
nervous system enables heat production in ISBAT through nonshivering
thermogenesis (14). After different periods of cold exposure, an
increase in the firing rate of sympathetic nerves to ISBAT results in
catecholamine-mediated stimulation of brown adipocyte lipolysis,
providing the fatty acid fuel for nonshivering thermogenesis (19).
Evidence supporting an increase in sympathetic activity in ISBAT
following cold exposure includes increased tyrosine hydroxylase
activity (32), increased NE turnover (23, 39), and increased in vivo
responsiveness to NE (19). Together, results from previous studies
demonstrate that cold exposure represents a state of enhanced
sympathetic neurotransmission to ISBAT to maintain body temperature
through nonshivering thermogenesis.
In this study ISBAT was used as a model system for examination of
substrate-mediated regulation of the NET. A variety of evidence demonstrates cold-induced elevations in sympathetic neurotransmission of ISBAT; however, the consequences of sympathetic activation on the
NET have not been extensively examined. We hypothesized that increases
in sympathetic neurotransmission following cold exposure positively
regulate the ISBAT NET. In the present study the kinetic parameters for
[3H]NE uptake in ISBAT
slices from control and cold-exposed (CE) rats were defined. Moreover,
radioligand binding analysis of the NET was characterized in ISBAT
membranes from control rats using the selective inhibitor
[3H]nisoxetine (Nis).
Time course studies examined
[3H]Nis binding in
ISBAT membranes from control and CE rats (2 h-7 days of cold exposure).
Additionally, the effect of cold-induced increases in food intake on
the kinetic parameters for
[3H]Nis binding were determined.
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METHODS |
Animals.
Male Sprague-Dawley rats (250-300 g; Harlan Sprague Dawley,
Cumberland, IN) were used in all experiments. On arrival, rats were
housed at a constant temperature of 24°C with a 12:12-h light-dark schedule and ad libitum access to food and water. For the reserpine study control rats received a single injection of reserpine (2.5 mg/kg
ip; Sigma Chemicals, St. Louis, MO) 4 h before examination of
[3H]NE uptake in ISBAT
slices. For cold exposure studies rats were housed in individual cages
for up to 7 days in an animal hibernarium maintained at a constant
temperature of 4°C. Control rats were moved to individual cages for
the duration of time corresponding to cold exposure. For the time
course study body weight was measured before moving rats to the animal
hibernarium and on the final day of the study. The experimental design
assigned one rat from each group (control, 2 h, and 1, 3, 5, and 7 days) to be studied each day for 5 consecutive days. For the
pair-feeding study, body weight, food intake, and water intake were
measured in control and CE rats (7 days) every day at 10 AM. CE rats
were pair fed based on the average food intake of control rats over the
preceding 24-h period; water intake was not limited. All rats were
killed by decapitation.
Kinetic analysis of [3H]NE
uptake in ISBAT slices.
ISBAT was removed from rats, and 500 µm ISBAT slices (24 slices
10-15 mg) were made from vertically oriented tissue cut
sequentially across the entire fat pad using a McIlwain tissue chopper
(7). ISBAT slices were preincubated for 30 min at 37°C in a Dubnoff metabolic shaking incubator in oxygenated (95%
O2-5%
CO2) Krebs bicarbonate buffer
(40 ml) containing (in mM) 108 NaCl, 5.5 dextrose, 14.9 NaHCO3, 0.11 ascorbic acid, 4.7 KCl, 1.18 KH2PO4,
1.2 MgSO4, 1.3 CaCl2, and 0.004 EDTA, pH 7.4. Pargyline (4 µM, Sigma) and metanephrine HCl (1 µM, Sigma) were
added to the Krebs buffer to inhibit monoamine oxidase and
extraneuronal uptake of NE, respectively. For measurement of
[3H]NE uptake
kinetics, individual ISBAT slices were transferred to conical vials
containing buffer (400 µl) with a fixed concentration of
L-[7-3H]NE
(10.5 Ci/mmol specific activity; New England Nuclear, Boston, MA) (6 nM) and increasing concentrations of unlabeled NE (10 nM-8 µM, 8 points; Sigma) in duplicate and incubated for 10 min at 37°C.
Nonspecific uptake was determined by incubating ISBAT slices in
duplicate with [3H]NE
(6 nM) in the presence of the neuronal uptake inhibitor, nomifensine
maleate (10 µM; RBI, Natick, MA). The incubation was terminated via vacuum filtration over glass-fiber filters (no. 30;
Schleicher & Schuell, Keene, NH) using a Millipore 12-well manifold
followed by washing filters three times with 10 ml of ice-cold buffer.
ISBAT slices retained on filters were removed, weighed, and solubilized
in 500 µl TS-2 (Research Products, Mt. Prospect, IL) by incubation at
50°C overnight. Scintillation cocktail (10 ml, 3a70B; Research
Products) was added to the solubilized slices, and tissue
3H was measured by liquid
scintillation spectrometry using a Packard Tri-Carb Liquid
Scintillation Analyzer with an efficiency for 3H counting of 65%. Saturation
isotherms for specific
[3H]NE uptake in ISBAT
slices were constructed using GraphPad Prism 2 software, and the
affinity (Km)
and maximal velocity
(Vmax) were
derived using nonlinear regression analysis.
Kinetic analysis of [3H]Nis
binding in ISBAT membranes.
ISBAT was removed from rats, placed in 5 ml ice-cold buffer (in mM, 50 Tris, 120 NaCl, 5 KCl, pH 7.4), and homogenized three times for 10 s
with a polytron (Kinematica). Homogenates were diluted to a total
volume of 30 ml with the ice-cold membrane buffer and centrifuged in a
supraspeed centrifuge (Sorvall RC28S; F-28/36 rotor) at 1,100 g for 15 min at 4°C. The
supernatant was resuspended with 30 ml of ice-cold buffer and
centrifuged at 40,000 g for 10 min at
4°C. The centrifugation procedure was repeated, and the final
membrane pellet (1-2.5 mg protein/ml buffer) was resuspended in
ice-cold binding buffer (in mM, 50 Tris, 300 NaCl, 5 KCl, pH 7.4).
Protein concentration was determined spectrophotometrically using
Coomassie blue dye with bovine serum albumin as the standard (2).
Saturation binding isotherms were performed by incubating duplicate
aliquots of ISBAT membrane (70 µg) with increasing concentrations of
N-[methyl-3H]Nis
(5.9 Ci/mmol specific activity; New England Nuclear; 0.1-20 nM, 8 points, 50 µl) and binding buffer (250 µl final volume) for 30 min
at 22°C. Nonspecific binding was determined at each concentration
of [3H]Nis by the
addition of mazindol (2 µM; RBI) and fluoxetine (1 µM; Lilly,
Indianapolis, IN) to eliminate NE, dopamine, and serotonin uptake
ligand binding sites (34, 35). Binding was terminated by filtration
over presoaked (0.3% polyethleneimine) glass microfiber filters (no.
32; Schleicher & Schuell) using a Brandel cell harvester. Radioactivity
retained on the filters was measured by liquid scintillation spectrometry. Saturation isotherms for specific
[3H]Nis binding to
ISBAT membranes were constructed using GraphPad Prism 2 software. For
determination of dissociation constant
(Kd) and
maximal binding density (Bmax)
data were analyzed by nonlinear regression analysis using LIGAND.
Measurement of plasma and ISBAT catecholamine content.
The HPLC system consisted of a System Gold model 116 pump (Beckman,
Fullerton, CA), a model 7725 injection valve fitted with a 50-µl
sample loop (Rheodyne, Cotati, CA), a Coulochem model 5100A
electrochemical detector (ESA, Bedford, MA), a model 5011 analytic cell
(ESA), and a catecholamine HR-80 reverse-phase column (ESA) packed with
3 µm spherical silica bonded with octadecylsilane with a graphite
guard filter. HPLC chromatograms were displayed on an
omniscribe chart recorder (Houston Instruments, Houston, TX). The
mobile phase consisted of (in mM) 70 citric acid anhydrous, 0.16 EDTA,
100 1-octane sulfonic acetate trihydrate, 11 NaCl, and 2.5% (vol/vol)
methanol, pH 4.0. The HPLC column was equilibrated with mobile phase
for 12 h before use, and separation was achieved at a flow rate of 1.0 ml/min.
Free catecholamines were extracted from ISBAT or plasma as follows:
50-60 mg ISBAT was homogenized on ice in 1 ml of 0.4 N perchloric
acid containing 400 pg of the internal standard
[dihyroxybenzylamine hydrobromide (DHBA), Sigma] and 200 µl of a 20-ml solution of 0.5% EDTA with 500 mg sodium
metabisulfate. Homogenized samples were centrifuged at 12,365 g for 10 min, and the supernatant was transferred to a tube containing 25 mg of activated alumina
(Bioanalytical Systems, West Lafayette, IN). For plasma extraction, 500 µl of plasma were added to 0.4 N Tris (containing inhibitors and
internal standard) before addition of 25 mg of activated alumina. On
addition of activated alumina the pH was adjusted to 8.6 by the
addition of 3 M Tris · HCl buffer (pH 10.9). The
samples were vortexed for 10 min, interrupting every 2 min to allow the
alumina to settle. The alumina mixture was washed three times with 3 ml
of a 1:1 dilution of Tris · HCl buffer (1.5 M
Tris · HCl, 0.5 mM EDTA, and 0.4 mM sodium
metabisulfate, pH 8.7) and water. The supernatant was removed and the
alumina slurry was transferred to microfilter tubes (0.45 µm nylon;
Alltech, Deerfield, IL) and centrifuged in a tabletop centrifuge at
3,500 rpm for 1 min. The supernatant from this centrifugation was
discarded. Catecholamines were eluted from the alumina utilizing a
two-step procedure consisting of the addition of 100 µl of 0.15 N
perchloric acid, vortexing for 10 s, and centrifugation at 3,500 rpm
for 1 min (total elution volume of 200 µl). The eluent (50 µl) was
injected onto the HPLC for catecholamine analysis. A set of
catecholamine standards (50-900 pg NE and epinephrine) were used
to determine plasma catecholamine content with the DHBA content in
extracts used to correct for recovery.
Statistics.
Results from all studies are presented as means ± SE. For
kinetic parameters derived from
[3H]NE uptake studies
in ISBAT slices from CE and control rats, a Student's unpaired
t-test was used to test for
significant difference. For tissue NE content and kinetic parameters
derived from [3H]Nis
binding in the CE time course study, a one-way ANOVA (time as a between
group factor) was used followed by Tukey's multiple comparison test
for post hoc analysis of time effect. For plasma and tissue NE content
and kinetic parameters derived from
[3H]Nis binding in
ISBAT membranes from pair-feeding studies, a two-way ANOVA (CE,
pair-feeding as between group factors) was used followed by Tukey's
multiple comparison test for post hoc analysis of treatment effect.
Statistical significance was accepted at
P < 0.05.
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RESULTS |
Characterization of [3H]NE
uptake and [3H]Nis binding in
control rat ISBAT.
Preliminary experiments established that
[3H]NE uptake in
vertically oriented slices of ISBAT across the entire pad was of similar magnitude, indicating sufficient homogeneity of innervation for
uniform analysis of neuronal uptake in ISBAT slices (data not shown).
Nonspecific uptake of
[3H]NE in ISBAT slices
represented <24% of total
[3H]NE uptake. Initial
experiments demonstrated that the time course for specific
[3H]NE uptake in ISBAT
slices from control and CE rats was linear over the 90-min time course
examined (data not shown). An incubation time of 10 min was chosen from
the linear part of the curve representative of the initial velocity and
used in subsequent
[3H]NE uptake
experiments to determine saturation kinetics of the ISBAT NET.
Saturation isotherms for
[3H]NE (6 nM) uptake
in ISBAT slices were performed to determine the affinity
(Km) and
capacity (Vmax) for the NET in control rats.
[3H]NE uptake in ISBAT
slices was of high affinity
(Km 1.62 ± 0.35 µM) and low capacity
(Vmax 392 ± 46 fmol · mg wet wt
tissue
1 · min1;
Fig. 1 and Table 1).
Previous investigators have demonstrated that accurate examination of
the initial velocity for neuronal uptake requires elimination of
vesicular stores of catecholamines (1). To determine the effect of
vesicular catecholamine stores on the kinetic analysis of
[3H]NE uptake, control
rats were treated with reserpine 4 h before removal of ISBAT. This
treatment regimen for reserpine has been previously demonstrated to
result in significant depletion of endogenous catecholamine stores in
tail arteries from spontaneously hypertensive and Wistar-Kyoto rats
(8). Reserpine pretreatment did not alter the kinetic parameters for
[3H]NE uptake in ISBAT
slices (Fig. 2 and Table 1).
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Fig. 1.
Saturation isotherm for specific
[3H]norepinephrine
(NE) uptake in interscapular brown adipose tissue (ISBAT) slices from
control and cold-exposed (CE) rats. ISBAT slices from control and CE
rats were prepared as described in
METHODS for examination of
[3H]NE uptake.
Nonspecific uptake was determined by incubation of slices from control
and CE rats in presence of nomifensine. The affinity
(Km) for
[3H]NE uptake was not
different between ISBAT slices from control and CE rats. Maximal
velocity (Vmax)
for [3H]NE uptake was
significantly (P < 0.05) decreased
in ISBAT slices from CE rats compared with control. Data are means ± SE of control and CE rats (n = 10 rats/group).
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Fig. 2.
Saturation isotherm for
[3H]NE uptake in ISBAT
slices from control and reserpine-treated rats. ISBAT slices were
prepared from control and reserpine-treated rats (2.5 mg/kg ip, 4 h
before euthanasia) as described in
METHODS for examination of
[3H]NE uptake.
Nonspecific uptake was determined by incubation of slices from control
and reserpine-treated rats in the presence of nomifensine.
Km and
Vmax for
[3H]NE uptake were not
different between ISBAT slices from control and reserpine-treated rats.
Data are means ± SE of control and reserpine-treated rats
(n = 5 rats/group).
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For radioligand binding studies, the selective noradrenergic inhibitor
[3H]Nis was used as a
ligand for the NET binding site (35). Initial experiments determined
[3H]Nis (5 nM) binding
over a range of ISBAT membrane protein concentrations for 60 min at
22°C (Fig.
3A).
[3H]Nis binding was
linear over the examined range of ISBAT membrane protein
concentrations. Subsequent experiments were performed using 70 µg of
ISBAT membrane protein at each concentration of [3H]Nis. To determine
the time course for equilibrium binding, ISBAT membrane protein was
incubated with a fixed concentration (5 nM) of
[3H]Nis at 22°C
for various times (Fig. 3B).
Specific [3H]Nis
binding plateaued at 10 min and remained unchanged over 60 min. From
these data an incubation time of 30 min at 22°C was chosen for all
saturation binding isotherms.
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Fig. 3.
Membrane protein (A) and time course
(B) for
[3H]nisoxetine (Nis)
binding in ISBAT membranes from control rats. ISBAT membranes were
prepared as described in METHODS.
A: increasing concentrations of ISBAT
membrane protein were incubated with
[3H]Nis (5 nM) for 60 min. B: 100 µg of ISBAT membrane
protein were incubated with
[3H]Nis for various
times at 22°C.
[3H]Nis binding was
linear over protein concentration range examined and reached
equilibrium within 15 min of incubation. Protein concentration of 70 µg with incubation time of 30 min was used for all subsequent binding
isotherms. Data are means ± SE (n = 3 rats).
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Regulation of [3H]NE uptake
and [3H]Nis binding following 7 days of cold exposure.
Saturation isotherms for
[3H]NE uptake in ISBAT
slices were performed to determine the affinity
(Km) and
capacity (Vmax)
for the NET following 7 days of cold exposure. The affinity for
[3H]NE uptake was not
different in ISBAT slices from control and CE rats (Table 1 and Fig.
1). However, the
Vmax for
[3H]NE uptake was
decreased (38%) in ISBAT slices from CE rats compared with control
(Table 1 and Fig. 1).
To determine if decreases in the capacity for
[3H]NE uptake in ISBAT
slices from CE rats were the result of a diminished number of NET
sites, saturation isotherms for
[3H]Nis binding were
performed in ISBAT membranes from control and 7-day CE rats.
[3H]Nis bound with
high affinity to a single class of binding sites (Hill coefficient 1.06 ± 0.05; 0.95-1.17 95% confidence interval) in ISBAT membranes
from control and CE rats. The affinity of
[3H]Nis binding in
ISBAT was not altered in CE rats compared with control; however, the
density (pmol/mg protein) of
[3H]Nis binding sites
was decreased (38%) in ISBAT membranes from CE rats compared with
control (see Fig 5A).
Time course for cold-induced regulation of the NET.
Control rats gained weight over the 7-day time course (Table
2). Body weight decreased from 2 h to 3 days of cold exposure, followed by a rebound in body weight of CE rats
from 5 to 7 days (Table 2). ISBAT mass (expressed as a percentage of
body weight) increased following cold exposure (Table 2). At 2 h of
cold exposure ISBAT NE content decreased followed by a gradual return
to levels not significantly different from control at 7 days of cold
exposure (Fig. 4).
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Fig. 4.
NE content in ISBAT from control and CE rats. NE was extracted from
ISBAT of control and CE rats (2 h-7 days) as described in
METHODS. NE content was analyzed using
HPLC with electrochemical detection. ISBAT NE content was decreased
from 2 h to 5 days of cold exposure (*). At 7 days of cold exposure
ISBAT NE content was not significantly different from control. Data are
means ± SE from control and CE rats
(n = 5 rats/group).
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To determine the time course for cold-induced regulation of NET
density, saturation isotherms for
[3H]Nis binding were
performed in ISBAT membranes from control and CE rats (2 h-7 days).
After 2 h of cold exposure the
Kd for
[3H]Nis binding was
increased in ISBAT membranes from CE rats compared with control (Table
3 and Fig. 5,
A and
B). On longer cold exposure (
3
days) the Kd for
[3H]Nis binding
returned to levels not significantly different from control. The
density of [3H]Nis
binding decreased over time following cold exposure (Table 3 and Fig.
5, A and
B).
[3H]Nis binding
density (pmol/mg protein) was significantly decreased compared with
control at 3 days of cold exposure, with a maximum of 40% reduction in
binding density at 7 days (Table 3 and Fig. 5,
A and
B). When normalized to total ISBAT
protein content (pmol/mg protein),
[3H]Nis binding
density represents the specific activity of the NET protein. When
[3H]Nis binding
density was normalized to account for the increased mass of tissue
following cold exposure (see Table 2), the absolute number (fmol/g
tissue wet wt) of
[3H]Nis binding sites
in the entire ISBAT was significantly decreased over time following
cold exposure (Table 3).
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Table 3.
Time-dependent alterations in kinetic parameters for
[3H]Nis binding in ISBAT membranes from CE and control
rats
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Fig. 5.
Saturation isotherm (A) and
Scatchard plot (B) for
[3H]Nis binding in
ISBAT membranes from control and CE rats. ISBAT membranes from control
and CE rats (2 h-7 days) were incubated with increasing concentrations
of [3H]Nis
(0.1-20.0 nM) for 30 min at 22°C. Dissociation constant
(Kd) for
[3H]Nis binding was
similar in control and CE rats; however, maximal binding
(Bmax) for
[3H]Nis binding in
ISBAT was significantly (P < 0.05)
decreased from 2 h to 7 days of cold exposure. Data are mean ± SE
from control and CE rats (n = 5 rats/group).
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Effect of pair feeding on cold-induced regulation of NET.
Food and water intake increased ~30% following 7 days of cold
exposure (Fig. 6,
A and
B). Pair-fed rats maintained at
4°C were restricted to the average food intake (27.3 ± 0.9 g)
of ambient temperature control rats. Pair-feeding of CE rats eliminated
the increase in water intake (Fig.
6B). Body weight decreased in CE rats compared with controls, with pair-fed rats losing weight over the
7-day study (Fig. 6C).
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Fig. 6.
Food intake (A), water intake
(B), and body weight
(C) in control, CE, and CE pair-fed
rats. Food intake, water intake, and body weight were measured in
individual rats from each group (n = 6 rats/group) at 10 AM each day over 7-day period. Control and CE rats
had unlimited access to food; however, food intake of CE pair-fed rats
was limited to average food intake of control rats for the prior 24-h
period. Food intake increased at 1 day of cold exposure and remained
elevated for 7 days. Water intake increased at day
3 of cold exposure and remained elevated. Pair-feeding
CE rats did not prevent increases in water intake. Data are means ± SE from control and CE rats (n = 6 rats/group).
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When normalized to body weight, ISBAT mass increased (~43 and 38%,
respectively) in CE and CE pair-fed rats compared with control;
however, ISBAT tissue weight did not increase in pair-fed CE rats.
Plasma NE content was increased in CE rats compared with control
(control 4.10 ± 0.72, CE 8.90 ± 0.65 ng/ml,
P < 0.05); however, plasma NE
content in pair-fed CE rats (3.11 ± 0.78 ng/ml) was not different
from control.
To determine the effect of cold-induced increases in food and water
intake on the regulation of the NET, saturation isotherms for
[3H]Nis binding were
performed in ISBAT membranes prepared from control, CE, and
pair-fed CE rats. The affinity for
[3H]Nis binding
was similar in ISBAT membranes from all three groups of rats (Table
4 and Fig. 7,
A and
B).
[3H]Nis binding
density (pmol/mg protein) was decreased in ISBAT from CE (37%) and
pair-fed CE rats (40%) compared with control (Table 4 and Fig. 7,
A and
B).
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Table 4.
Effect of pair feeding on kinetic parameters for [3H]Nis
binding in ISBAT membranes from control, CE, and pair-fed CE rats
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Fig. 7.
Saturation isotherm (A) and
Scatchard plot (B) for
[3H]Nis binding in
ISBAT membranes from control, CE, and CE pair-fed rats. ISBAT membranes
were prepared as described in METHODS
and incubated with increasing concentrations of
[3H]Nis (0.1-20.0
nM) for 30 min at 22°C.
Kd for
[3H]Nis binding in
ISBAT was similar in control and CE rats. In contrast,
Bmax for
[3H]Nis binding was
significantly (P < 0.05) decreased
in ISBAT membranes from both CE and CE pair-fed rats compared with
control. Data are means ± SE from control, CE, and CE pair-fed rats
(n = 6 rats/group).
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DISCUSSION |
Results from this study do not support the hypothesis that elevated
sympathetic neurotransmission in ISBAT following cold exposure
positively regulates the NET. In contrast, following 7 days of cold
exposure at 4°C, the specific activity of the NET protein (by
binding density) and the functional capacity of
[3H]NE uptake were
decreased in ISBAT. Decreases in uptake and binding density for the NET
in ISBAT were evident despite elevated plasma NE content. Moreover,
normalization of plasma NE content by pair feeding CE rats did not
influence cold-induced downregulation of NET binding density. Results
from time course studies demonstrated that reductions in ISBAT NE
content preceded downregulation of the NET. Collectively, these results
demonstrate that cold exposure downregulates the NET in ISBAT.
Moreover, results from this study suggest that cold-induced
downregulation of the ISBAT NET occurs subsequent to enhanced turnover
of ISBAT sympathetic activity.
The description and initial characterization of the NET occurred in the
early 1960s (16), with cloning of the transporter for NE uptake in 1991 (24). Since the initial description of the NET, research has addressed
the kinetics of transport and definition of the substrate binding site
(4). However, there have been relatively few studies specifically
addressing regulation of the NET. Previous studies demonstrated that
acute and chronic treatment with tricyclic antidepressants increased
mRNA for the NET in rat locus coeruleus (33). Other regulators of the
NET that have been examined in rats include reserpine (decrease in uptake sites and mRNA expression; 10, 21) and monoamine oxidase inhibitors (increase in uptake sites; 10). Collectively, results from
previous studies suggest that the NET is positively regulated by NE
substrate levels.
The activity of the NET in brown adipose tissue has not been examined
extensively. To kinetically characterize the uptake of
[3H]NE in ISBAT
several conditions pertaining to assessment of transporter activity
were optimized, including examination of substrate uptake during the
initial velocity of the transporter, inhibition of substrate metabolism
during the experimental incubation, and removal of the influences of
vesicular and extraneuronal uptake of substrate in the tissue
preparation. Kinetic analysis of
[3H]NE uptake in ISBAT
slices demonstrated a single site transporter with an affinity in the
range of that previously described for the NET (17, 24). The maximal
functional capacity
(Vmax) for [3H]NE uptake in ISBAT
slices was comparable to brain regions with dense noradrenergic
innervation (9, 31). Moreover, results from this study are in agreement
with previous studies reporting high catecholamine concentrations and
extensive catecholamine histofluorescence in ISBAT as evidence for the
density of sympathetic innervation (30).
Past findings have established that in animals chronically exposed to
cold, shivering thermogenesis is replaced by nonshivering thermogenesis
(15). Brown adipose tissue has been demonstrated to be a major site of
nonshivering thermogenesis following cold exposure, accounting for an
increase in heat production and resultant metabolic rate (29). Previous
studies have demonstrated that the majority of the adaptive changes in
essential components of ISBAT occur following 1 wk of cold exposure
(26). However, as early as 1 h following cold exposure there is an
increase in [3H]GDP
binding to ISBAT mitochondria, indicative of increased thermogenic activity (22). Additional changes that have been observed in CE rats
include increases in ISBAT weight (5, 6, 36), protein content (7),
uncoupling protein (15), lipoprotein lipase (27), plasma NE content
(25), and food intake (18). In agreement with previous studies, results
from the present study demonstrate that cold exposure resulted in
increased food intake, increased ISBAT mass, and elevated plasma NE content.
Previous estimations of turnover of NE as an index of sympathetic
activity demonstrated a rapid activation of sympathetic activity within
4 h of cold exposure that was sustained for 10 days (38). In agreement
with previous results, in the present study ISBAT NE content decreased
following acute cold exposure, suggesting increased turnover of ISBAT
catecholamines (26). Moreover, in agreement with previous studies (25),
plasma NE content increased following 7 days of cold exposure.
Collectively, these results demonstrate activation of the sympathetic
nervous system following cold exposure.
Although it is well recognized that increases in sympathetic nervous
system activity account for the majority of stimulated thermogenesis in
response to cold, there have been few studies examining indexes of
sympathetic neuroeffector function at the level of the sympathetic
nerve terminal in brown adipose tissue. Moreover, the ramifications of
cold-induced stimulation of sympathetic neurotransmission to ISBAT on
regulation of the NET have not been extensively investigated. Previous
studies in our laboratory have demonstrated a decrease in
[3H]NE uptake in ISBAT
slices following 7 days of cold exposure; however, kinetic analysis of
the NET was not performed (7). Results from this study confirm and
extend previous results, demonstrating a decrease in the maximum
capacity for [3H]NE
uptake in ISBAT slices following 7 days of cold exposure. However,
these results do not support previous observations of substrate-mediated positive regulation of the ISBAT NET (10, 11, 21).
Potential mechanisms contributing to a lack of substrate-induced positive regulation of the ISBAT NET in the present study include cold-induced alterations in a variety of humoral factors, differences between peripheral and central sympathetic neurons, and the degree of
cold-induced activation of sympathetic neurotransmission.
The highly selective radioligand for the NET binding site,
[3H]Nis, was used in
these studies to determine if reductions in the functional capacity for
[3H]NE uptake in ISBAT
slices from CE rats were the result of a decreased density of NET
binding sites. Previous investigators have demonstrated a 400- and
1,000-fold greater affinity of
[3H]Nis for the NET
compared with the dopamine and serotonin transporters, respectively
(34, 37). Moreover, compounds that fail to inhibit the binding of
[3H]Nis also have
limited ability to inhibit
[3H]NE uptake (34).
Radioligand binding analysis using
[3H]Nis in ISBAT
membranes was saturable, protein-dependent, and demonstrated a single,
high-affinity binding site with pharmacological characteristics similar
to those previously identified (34). In agreement with the observed
reductions (38%) in maximal functional capacity
(Vmax) for
[3H]NE uptake, the
density of [3H]Nis
binding sites was reduced by 38% following 7 days of cold exposure.
Time-course studies demonstrated that cold-induced downregulation of
[3H]Nis binding
density occurred between 1 and 3 days of cold exposure and was preceded
by reductions in ISBAT catecholamine content. To determine the role of
tissue hypertrophy and hyperplasia in the observed regulation of the
NET following cold exposure,
[3H]Nis binding
density was normalized by multiplying the density of
[3H]Nis sites by the
total tissue wet weight. This normalization allowed for calculation of
the total number of NET binding sites in the entire ISBAT organ.
Reductions in [3H]Nis
binding density in response to cold exposure were evident by specific
activity (pmol/mg protein) and absolute number (pmol/g wet wt).
Moreover, correlation regression analysis of
[3H]Nis binding
density by ISBAT mass was not significant
(r2 = 0.029).
Collectively, these results demonstrate that reductions in
[3H]Nis binding
density following cold exposure are independent of tissue hypertrophy.
Alternatively, the time course for cold-induced alterations in ISBAT NE
content and [3H]Nis
binding density suggest substrate-induced downregulation of the ISBAT
NET following cold exposure.
Interestingly, in the present study the affinity for
[3H]Nis binding in
ISBAT decreased following acute (2 h) cold exposure. Reductions in the
affinity for [3H]Nis
binding following 2 h of cold exposure occurred independently of
alterations in ISBAT NET density. These results demonstrate differential regulation of the NET following acute and chronic cold
exposure, beginning with a transient reduction in binding affinity
followed by downregulation of the number of NET sites on chronic cold
exposure. Although results from the present study do not conclusively
identify NE as the cold-induced modulator of the ISBAT NET, the time
course for ISBAT NE content and regulation of
[3H]Nis binding
kinetic parameters
(Kd,
Bmax) following cold exposure support further study of substrate-mediated regulation of the ISBAT NET.
It is generally accepted that elevations in food intake, as observed in
the present study, result in stimulation of sympathetic nervous system
activity (38). Conversely, previous studies have demonstrated that food
restriction lowered turnover of NE in ISBAT (3, 39). Interestingly, in
this study elevations in plasma NE content were normalized by pair
feeding CE rats to food intake levels of controls. However,
cold-induced reductions in
[3H]Nis binding
density remained evident despite restricted food intake. Previous
studies have demonstrated that pair feeding CE rats to food intake
levels of controls limited cold-induced hypertrophy of ISBAT (18). In
agreement with these results, in the present study ISBAT tissue weight
was not increased in CE rats that were pair fed. Importantly,
reductions in ISBAT NET density remained evident in CE rats that were
pair fed, demonstrating that reductions in ISBAT NET density were
independent of changes in tissue mass. Collectively, these results
demonstrate that reductions in the density of ISBAT NET sites are
independent of cold-induced elevations in food intake, plasma NE
content, and tissue mass.
In summary, results from this study demonstrate time-dependent
regulation of the ISBAT NET following cold exposure. Acute cold
exposure decreased the affinity of the ISBAT NET, whereas chronic cold
exposure decreased the density of NET binding sites. Cold-induced
alterations in the NET occurred independent of tissue mass, plasma NE
content, and food intake. In contrast, cold-induced alterations in the
ISBAT NET occurred coincident with (acute) or before (chronic) changes
in ISBAT NE content. These results suggest that in the cold exposure
model of heightened sympathetic neurotransmission, the functional
activity of the ISBAT NET is reduced in a substrate-related manner,
potentially contributing to the maintenance of stimulated thermogenesis.
Perspectives
Our results imply that the synaptic concentration of neurotransmitter
substrate, i.e., NE, inversely regulates the density of the neuronal
uptake transporter on peripheral sympathetic nerve terminals. In
response to cold exposure and enhanced sympathetic drive, reductions in
the density of uptake transporter sites on ISBAT sympathetic nerve
terminals would limit removal of released NE, resulting in maximal
stimulation at postsynaptic receptor sites. Importantly, the observed
downregulation in NET density in brown adipose tissue would be
anticipated to contribute to the maintenance of thermogenesis. We
speculate that substrate-mediated regulation of the NET may extrapolate
to other peripheral tissues innervated by the sympathetic nervous
system. The broad implication of this finding is that
pathophysiological states associated with altered synaptic
concentrations of NE would exhibit substrate-dependent regulation of
NET density. For example, in a disease state such as congestive heart
failure with documented increases in sympathetic drive, we hypothesize
that downregulation of NET density on cardiac sympathetic nerve
terminals would potentially elevate synaptic NE concentrations and
alter postsynaptic receptor responsiveness. Finally, we hypothesize
that substrate-dependent regulation of the NET on peripheral
sympathetic terminals may contribute to regional heterogeneity in
peripheral sympathetic activation.
| |
ACKNOWLEDGEMENTS |
This work was supported by the National Heart, Lung, and Blood
Institute Grant HL-52934.
| |
FOOTNOTES |
We acknowledge the technical assistance of Michael J. Fettinger and the
use of the animal hibernarium at the Veterans Administration Hospital
Research Facility.
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. §1734 solely to indicate this fact.
Address for reprint requests: L. A. Cassis, Rm. 401B, College of
Pharmacy, Div. of Pharmaceutical Sciences, Univ. of Kentucky,
Lexington, KY 40536-0082.
Received 10 July 1998; accepted in final form 10 September 1998.
| |
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