Role of
1- and
3-adrenoceptors in the
regulation of lipolysis and thermogenesis in rat brown
adipocytes
Claude
Atgié,
François
D'Allaire, and
Ludwik J.
Bukowiecki
Department of Physiology, Faculty of Medicine, Laval University,
Quebec, Quebec, Canada G1K 7P4
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ABSTRACT |
To evaluate the physiological functions of
1-,
2-, and
3-adrenoceptors (ARs) in brown
adipose tissue, the lipolytic and respiratory effects of various
adrenergic agonists and antagonists were studied in rat brown
adipocytes. The
-agonists stimulated both lipolysis and respiration
(8-10 times above basal levels), with the following order of
potency (concentration eliciting 50% of maximum response):
CL-316243 (
3) > BRL-37344
(
3) > isoproterenol (mainly
1/
2) > norepinephrine (NE; mainly
1/
2) > epinephrine (mainly
1/
2)
dobutamine (
1)
procaterol (
2). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (
1 antagonist) revealed that
more than one type of receptor mediates NE action. It is concluded from
our results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through
1-ARs,
2) NE, at higher levels, stimulates
lipolysis and respiration via both
1- and
3-ARs,
3)
2-ARs play only a minor role,
and 4)
3-ARs may represent the
physiological receptors for the high NE concentrations in the synaptic
cleft, where the high-affinity
1-ARs are presumably
desensitized. It is also suggested that lipolysis represents the
flux-generating step regulating mitochondrial respiration.
brown adipose tissue; brown fat; respiration;
2-adrenoceptors; norepinephrine; epinephrine; sympathetic nervous system
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INTRODUCTION |
IN RECENT YEARS, atypical or
3-adrenoceptors (ARs) have been
identified in a variety of tissues, and particularly in brown adipose
tissue (BAT) (2, 3, 29). It has been well established that
1-,
2-, and
3-ARs coexist in BAT and that
they are all positively coupled to the adenylyl cyclase system (7, 17, 23, 24). However, the relative contribution of the three receptor subtypes in mediating the metabolic effects of norepinephrine (NE), the
physiological effector of thermogenesis in BAT, remains to be defined.
It is also known that
-ARs play only a minor role in mediating BAT
metabolic functions (lipolysis, thermogenesis, and growth), at least in
the rat (10, 20). It is known that NE binds a small number of
1/
2-ARs
(~104/cell) with high affinity
(nanomolar level) and a large number of
3-ARs
(~105/cell) with low affinity
(micromolar level) (17, 33). In vivo, the concentration of circulating
NE is very low (1-25 nM) (18, 19), and it is likely that at such
low levels NE mainly binds the high-affinity receptors (17, 30).
However, in contrast to epinephrine, NE is a neurohormone, and its
concentration in the synaptic cleft may be sufficiently high to locally
affect the low-affinity
3-ARs.
Furthermore, the contribution of the three
-ARs to the regulation of
the various metabolic processes occurring in adipocytes (lipolysis,
lipogenesis, thermogenesis, cell proliferation, differentiation, and so
forth) may vary under different physiological conditions (cold
acclimation, the age of the animals, diet composition, and so on)
(26).
The principal goal of the present studies was to examine the relative
participation of the three
-receptor subtypes in the control of
lipolysis and respiration by NE in isolated rat brown adipocytes. For
this purpose, we compared the effects of NE (a mixed agonist) on
lipolysis and respiration with those of various selective agonists. In
addition, the effects of selective
-antagonists on NE-stimulated
adipocytes were also investigated. Furthermore, parallel measurements
of the effects of selective adrenergic agents on lipolysis and
respiration enabled us to test the hypothesis that the rate of
respiration in brown adipocytes is essentially controlled by the rate
of lipolysis, i.e., that both phenomena are tightly coupled.
Results from these studies indicate that NE, at concentrations usually
found in the circulation (1-25 nM), controls both lipolysis and
respiration mainly via
1-ARs,
whereas, at much higher levels presumably occurring in the synaptic
cleft after sympathetic stimulation (by cold exposure, diet, stress,
and so forth), NE regulates these metabolic processes via both
1- and
3-adrenergic pathways.
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METHODS |
Brown adipocyte isolation.
Female Sprague-Dawley rats weighing 250-300 g were kept at
27°C for at least 2 wk with a photoperiod of 12:12 light-dark and fed Purina Chow ad libitum. Brown adipocytes were isolated, essentially as previously described (9, 10, 17), from pooled interscapular BAT from
2-3 rats. In brief, cleaned pieces of tissue (0.9 g) were
incubated for 15 min at 37°C in 2.5 ml of Krebs-Ringer-bicarbonate buffer containing 2.7 mM glucose, 1% albumin, and 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (KRB buffer; final pH 7.4), in the presence of collagenase (10 mg/ml) under an atmosphere of 95%
O2-5%
CO2. At the end of the digestion
period, the cells were filtered through a nylon filter (500 µm),
diluted in 10 ml of KRB buffer, and centrifuged (80 g) at room temperature for 5 min.
The floating cells were filtered again through a filter (200 µm) and
centrifuged following the same procedure. Finally, the isolated
adipocytes were washed twice with 3 ml of KRB buffer and counted in a
hemocytometer after trypan blue staining.
Oxygen consumption measurements.
Oxygen uptake of brown adipocyte suspensions (1-4 × 105 cells/ml in KRB buffer
containing 4% albumin) was measured polarographically at 37°C in a
water-jacketed Perspex chamber equipped with a Clark-style oxygen
electrode, as previously described (9, 10).
Determination of the lipolytic rates.
For the determination of lipolytic rates, the washed adipocytes were
first diluted to a concentration of 2-3 × 106 cells/ml in KRB buffer and
then preincubated for 15 min at 37°C with gentle shaking (40 cycles/min) (9, 10). At the end of the preincubation period, the cells
were washed twice with freshly bubbled buffer at 37°C. The
adipocytes were then diluted at a concentration of 1.5 × 105 cells/ml in KRB and incubated
for 45 min at 37°C. The lipolytic rates were estimated by measuring
extracellular glycerol release, as previously described (9, 10).
Drugs and chemicals.
(
)-NE bitartrate, (
)-epinephrine bitartrate, bovine serum
albumin (fraction V), and collagenase (type II) were obtained from
Sigma (St. Louis, MO). (
)-Propranolol, (
)-isoproterenol, and (±) dobutamine hydrochloride were purchased from RBI
Biochemicals (Natick, MA). CL-316243 {disodium
(R,R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]propyl]-1,3-benzodioxole-2,2-dicarboxylate} and BRL-37344
{4-[-[2-hydroxy-(3-chloro-phenyl)ethyl]-amino]-phenoxyacetate} were kindly provided by Dr. T. H. Claus (American Cyanamid, Lederle Laboratories, Pearl River, NY) (8) and by Dr. M. A. Cawthorne (SmithKline-Beecham Pharmaceuticals, Epsom, UK), respectively. The
following compounds were provided as generous gifts: ICI-89406 [1-(2-cyanophenoxy)-3-
-(3-phenylureido)
ethylamino-2-propanol] from ICI Pharmaceuticals (Mississauga, ON,
Canada) and CGP-20712A [(±)-(2-(3-carbamoyl-4-trifluormethyl-2-imidazolyl)-phenoxy)-2-propanolmethanesulphonate] from Ciba-Geigy (Mississauga, ON, Canada). Procaterol
[OPC-2009; 5-(1-hydroxy-2-isopropylaminobutyl)-8-hydrocarbostyril
hydrochloride hemihydrate] was kindly provided by Otsuka
Pharmaceuticals (Tokushima, Japan).
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RESULTS |
Effects of NE and CL-316243 on the kinetics of brown adipocyte
respiration.
The goal of the experiment described in Fig.
1 was to compare the respiratory effects of
NE, the physiological effector of thermogenesis, with those induced by
CL-316243, a new agonist that has a high affinity and selectivity for
3-ARs (8). As previously
reported, NE (100 nM) maximally stimulated brown adipocyte oxygen
consumption, 10 times above basal values, within 3 min (Refs. 9, 10;
see also Fig. 1). These effects were entirely mimicked by CL-316243,
added at a 10 times lower concentration than NE (10 nM). When maximal
respiration had stabilized for a few minutes, propranolol (a
-antagonist with a high affinity for
1/
2-ARs
and a much lower affinity for
3-ARs) was injected in the
respiratory chamber. In <3 min, propranolol (1 µM) inhibited 80-90% of the respiration stimulated by 10 times lower
concentrations of NE (100 nM). However, under the same conditions,
propranolol (1 µM) failed to affect respiration stimulated by 100 times lower concentrations of CL-316243 (10 nM). A 1,000 times higher
concentration of propranolol (10 µM) still failed to affect CL-316243
(10 nM)-stimulated respiration, whereas it totally inhibited NE (100 nM)-induced respiration. Propranolol concentration had to be increased
to 100 µM to observe a significant inhibition of CL-316243-induced respiration. Similar results were observed using other
3-agonists such as BRL-37344
(5). The observation that CL-316243-stimulated respiration is resistant
to inhibition by propranolol, whereas NE-induced respiration is very
sensitive, provided a first indication that NE and CL-316243 act via
different receptors.

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Fig. 1.
Modulation of brown adipocyte respiration by norepinephrine (NE; a
nonselective -agonist), CL-316243 (a selective
3-agonist), and propranolol (a
nonselective -antagonist). Adipocytes were isolated and incubated in
Krebs-Ringer-bicarbonate buffer, as described in
METHODS. Oxygen consumption was
measured with an oxygen electrode. Arrows indicate times at which drugs
were injected into respiratory chamber. Concentrations refer to final
concentrations in respiratory chamber. Values are means ± SE of
4-5 separate experiments.
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Dose-response curves of agonist-stimulated respiration.
The dose-response curves for the respiratory effects of selective
1 (dobutamine),
2 (procaterol), and
3 (CL-316243) agonists are
compared with those of the nonselective agonists NE and isoproterenol in Fig. 2. With the exception of
procaterol, all the agonists tested stimulated respiration at rates
similar to those elicited by NE. Other adrenergic agents, such as
BRL-37344 (
3-agonist), CGP-12177 (a
1/
2-antagonist
but also a
3-agonist at higher concentrations; Refs. 2, 3, 32), and epinephrine (mainly
1/
2-agonist),
also maximally stimulated respiration (Table
1). NE enhanced respiration with a potency
that was intermediate between that of selective
1- and
3-agonists: CL-316243
[
3; concentration eliciting 50% of maximum response
(EC50) = 1.3 nM ] > BRL-37344 (
3;
EC50 = 2.3 nM) > isoproterenol
(mainly
1/
2;
EC50 = 5.2 nM) > NE (mainly
1/
2;
EC50 = 25 nM) > epinephrine (mainly
1/
2; EC50 = 40 nM)
dobutamine (
1;
EC50 = 468 nM)
procaterol
(
2; EC50 = 12.9 µM).

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Fig. 2.
Dose-response curves of effects of adrenergic agonists on brown
adipocyte respiration. Experimental conditions were same as for Fig. 1.
Agonists were CL-316243 (CL; selective
3), isoproterenol (ISO;
nonselective ), NE (nonselective ), dobutamine (DOB; selective 1), and procaterol (PROC;
selective 2). Corresponding
values for concentrations eliciting 50% maximum response were
determined by computer analysis (SigmaPlot program) and are given in
Table 1. Values are means ± SE of 4-5 separate experiments.
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Table 1.
Comparison of responsiveness and sensitivity of brown adipocytes for
respiratory and lipolytic effects of adrenergic agonists
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The metabolic relationship between activation of lipolysis and
respiration.
In most cells, substrate supply does not normally control respiration,
except in brown adipocytes where it is generally postulated that fatty
acids released after activation of lipolysis enhance respiration by
binding the mitochondrial uncoupling protein and increasing the
permeability of the inner mitochondrial membrane to protons (for a
review see Ref. 26). If the stimulation of respiration were a simple
consequence of the activation of lipolysis by the hormone-sensitive
lipase, then it would be expected that all agents activating lipolysis
would also activate respiration with similar potencies (9, 38). The
release of new selective
-agonists and antagonists over recent years
enabled us to test this hypothesis in more detail. Using the same cell
preparations and the same agonists as for respiration, we carried out a
series of dose-response experiments to measure the effects of these
agents on the lipolytic rates (glycerol release from the cells; Fig. 3). The responsiveness and sensitivity
(EC50) of brown adipocytes for
the respiratory and lipolytic effects of the various agonists tested in
Figs. 2 and 3 are compared in Table 1. With the exception of the
2-agonist procaterol, all
agents tested maximally stimulated both lipolysis and respiration.
Remarkably, all the agonists enhanced lipolysis and respiration with a
similar order of potency (EC50): CL-316243
BRL-37344 > isoproterenol > NE > epinephrine > dobutamine
procaterol. With the exception of
CGP-12177 and procaterol (for possible explanations, see
DISCUSSION), the
EC50 values for lipolysis were all
two to four times higher than the corresponding
EC50 values for respiration (Table
1), indicating that a relatively small stimulation of lipolysis is
sufficient to maximally drive respiration (9, 38). Thus these data
support the concept that lipolysis represents the flux-generating step
controlling respiration.

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Fig. 3.
Dose-response curves of effects of adrenergic agonists on brown
adipocyte lipolysis. Lipolysis was estimated by measuring glycerol
released from brown adipocytes incubated as described in
METHODS. For other details see Fig.
2.
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Inhibition of the metabolic effects of NE by
1-adrenergic antagonists. To
determine the concentrations of NE that are required to stimulate brown
adipocyte thermogenesis via
1-
or
3-ARs, a series of
dose-response experiments was carried out, investigating the inhibitory
effects of the
1-antagonist
ICI-89406 (Ref. 23, and see Figs. 6B
and 7B) on NE-stimulated respiration
(Fig. 4) and lipolysis (Fig.
5). Increasing concentrations of
ICI-89406 (from 10
9 to
10
5 M) shifted NE
dose-response curves to the right, both for respiration and lipolysis.
Notably, all dose-response curves for lipolysis were shifted to higher
concentrations in comparison with the corresponding curves for
respiration. This observation is consistent with the view that
lipolysis drives respiration and that a relatively small stimulation of
lipolysis is sufficient to maximally activate respiration (9, 10).

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Fig. 4.
Dose-response curves for stimulatory effects of NE on brown adipocyte
respiration measured in presence of increasing concentrations of
ICI-89406 (from 10 9 to
10 5 M). Values are means of
2-3 experiments expressed as %maximal stimulation observed in
absence of antagonist. Inset: Schild
plot transformation (4, 36) of same data. Slope of linear regression line was significantly different from 1 and negative ( 0.22). pA2 value (intercept with
x-axis) was 8.1. DR, dose-ratio
value.
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Fig. 5.
Dose-response curves for stimulatory effects of NE on brown adipocyte
lipolysis measured in presence of increasing concentrations of
ICI-89406 (from 10 9 to
10 5 M). Values are means of
4-5 experiments expressed as %maximal stimulation observed in
absence of antagonist. Inset: Schild
plot transformation (4, 36) of same data. Slope of linear regression line was significantly different from 1 and negative ( 0.15). pA2 value was 7.2.
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To better analyze the antagonist effects of ICI-89406, the respiratory
and lipolytic data in Figs. 4 and 5 were plotted as percent of maximal
stimulation against increasing concentrations of the
1-antagonist (Figs.
6 and 7). It can be seen that the
1-antagonist inhibited by
~50% the stimulatory effects of NE, added at a concentration of 100 nM (Fig. 6), which is the minimal concentration required to maximally
stimulate respiration in the absence of antagonists (Fig. 4). However,
the effects of lower NE concentrations (25 nM), which do not maximally
activate thermogenesis, were totally inhibited by ICI-89406. In
contrast, the
1-antagonist barely inhibited cellular respiration in the presence of supramaximal concentrations of NE (1 µM; Figs. 4 and 6). Similar results were obtained when the effects of ICI-89406 on NE-induced lipolysis were
measured (Figs. 5 and 7), providing further evidence that lipolysis and
respiration represent metabolic processes that are tightly coupled.
In consideration of the observation that ICI-89406 competitively
inhibited the respiratory and lipolytic effects of NE (the dose-response curves in Figs. 4 and 5 were parallel), Schild plot transformations of the data in Figs. 4 and 5 were calculated (see Figs.
4 and 5, insets). The slopes of
regression lines for respiration (Fig. 4) and lipolysis (Fig. 5) were
both significantly different from one, suggesting that more than one
type of receptor is involved (28).

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Fig. 6.
Inhibition of NE-stimulated respiration by ICI-89406. Data were taken
from experiment of Fig. 4 but were plotted as %respiration induced by
NE in absence of ICI-89406. Note that ICI-89406 markedly inhibits
respiration stimulated by low NE concentrations, whereas it barely
affects respiration stimulated by high NE levels.
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Fig. 7.
Inhibition of NE-stimulated lipolysis by ICI-89406. Data were taken
from the experiment of Fig. 5 but were plotted as %lipolysis induced
by NE in absence of ICI-89406. Note that ICI-89406 markedly inhibits
lipolysis stimulated by low NE concentrations, whereas it barely
affects lipolysis stimulated by high NE levels.
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DISCUSSION |
Several lines of evidence indicate that BAT lipolysis and thermogenesis
are principally mediated by
1-
and
3-adrenergic pathways and
that
2-ARs play only a minor
role in controlling these metabolic processes. Dose-response
experiments revealed that
1-
and
3-adrenergic agonists were
very efficient for maximally stimulating brown adipocyte lipolysis and
thermogenesis, whereas high concentrations of selective
2-ARs were required to
partially stimulate brown adipocyte metabolism. These results are in
agreement with our previous binding studies, which were performed with
intact brown adipocytes rather than with membrane preparations, as it is usually done (17). In these studies, intact cells were used to avoid
contamination by other membranes originating from the various cell
types present in BAT. Indeed, typical brown adipocytes represent only
40% of the total cell population present in BAT. Endothelial cells
forming a dense network of capillaries irrigating BAT represent another
50% of the total cells, with the remaining 10% being constituted of
pericytes, interstitial precursor stem cells, brown preadipocytes,
protoadipocytes, mast cells, and so forth (12, 20-22). That
approach allowed us to demonstrate that the majority of
2-ARs detected in
unfractionated membranes by various groups, including ourselves, mainly
originate from cells other than typical brown adipocytes, presumably
endothelial cells that have been shown to contain
2- (and
1-) ARs (1, 17, 40). These
studies also revealed that isolated brown adipocytes contained ~10
times more
3- than
1-ARs. However, the apparent paradox of
3-ARs is that their
affinity for NE, the physiological effector of thermogenesis, is
extremely low, above the micromolar range of concentrations. Plasma
levels of NE vary from ~1 nM in undisturbed rats living at 25°C,
to 3-4 nM at 5°C, to a maximum of 20-25 nM after exposure
to extreme cold temperatures (
15°C) or decapitation (18,
30). At these concentrations, NE barely binds
3-ARs in brown adipocytes
[negative log of inhibition constant Ki
(pKi) = 4.2], but it occupies
1-ARs that have an affinity constant in the nanomolar range
(pKi = 9.3) (17).
Nevertheless, receptor occupancy merely represents the first step of a
series of metabolic events leading to increased thermogenesis. The
magnitude of the physiological response depends on a series of factors, such as the tightness of coupling between receptor occupancy and the
adenylate cyclase system (Gs and
Gi proteins), the system of
protein kinases, the hormone-sensitive lipase, the amount of mitochondria per cell, the concentration of mitochondrial uncoupling protein, and so forth (15, 23-26, 29). Thus, although binding studies are useful for characterizing receptor properties, they must be
completed by metabolic studies to determine the specific functions of
the receptors mediating the physiological effect of NE or other
agonists.
Inhibition studies using the selective
1-antagonist ICI-89406 provided
good evidence that more than one
-receptor subtype was mediating the
metabolic effects of NE. These studies were based on the rationale that
if NE-stimulated respiration or lipolysis were exclusively controlled
by
1-ARs, then one would expect
that an excess of ICI-89406 would totally, or nearly totally, inhibit the metabolic effects of NE. As a matter of fact, ICI-89406 failed to
totally inhibit the stimulatory effects of NE (Figs. 6 and 7),
particularly at NE concentrations that maximally stimulate lipolysis or
respiration (Figs. 2 and 3). Furthermore, Schild transformation (see
Refs. 4 and 36) of competitive inhibition experiments analyzing the
effects of increasing concentrations of ICI-89406 on NE-stimulated
lipolysis or respiration (Figs. 4 and 5) revealed that the slopes of
the regression lines were significantly different from one,
demonstrating that more than one receptor is involved in NE action. In
addition, the corresponding pA2 values for
inhibition by ICI-89406 were relatively low: 8.1 for respiration and
7.2 for lipolysis. Taken as a whole, these results indicate that NE
stimulates lipolysis or respiration by activating both
1- and
3-ARs, although maximal
lipolysis of respiration can be reached by stimulating either
1- or
3-receptors with dobutamine or
CL-316243, respectively (Figs. 2 and 3). The fact that the
pA2 value for respiration is
higher (8.1) than the pA2 value
for lipolysis (7.2) may be interpreted as indicating that lipolysis is
more controlled by the low-affinity
3-ARs than respiration. Similar
observations were observed with another
1-selective antagonist,
CGP-20712A (not shown). The present data also agree with a recent study
showing that ICI-89406 only partially inhibits respiration stimulated
by high (1 µM) NE concentrations in hamster brown adipocytes (39).
However, that study did not report the effects of ICI-89406 on lower NE
concentrations (nanomolar level).
The dose-response experiments comparing the effects of various agonists
on lipolysis and respiration (Figs. 2 and 3 and Table 1) show that the
agonists enhanced lipolysis and respiration with a similar order of
potency:
3-agonists (CL-316243
and BRL-37344) > mixed agonists (isoproterenol, NE, epinephrine) >
1-agonist (dobutamine)
2-agonist (procaterol). In
general, with the exception of CGP-12177 and procaterol, all
EC50 values for lipolysis were two
to four times higher than the corresponding
EC50 values for respiration (Table
1), a finding suggesting that lipolysis and respiration are tightly
coupled phenomena. The apparent discrepancy for procaterol probably
results from the fact that this agent was unable to maximally stimulate
lipolysis and respiration even when added at very high concentrations,
possibly because of the scarcity of
2-ARs in isolated brown
adipocytes (17). On the other hand, CGP-12177 acts as a
1-antagonist at low
concentrations and as a
3-agonist at higher
concentrations (for references, see Ref. 32). Although it is
interesting to observe that this agent was able to maximally stimulate
both lipolysis and respiration, the
EC50 values of CGP-12177
dose-response curves are difficult to interpret, due to the fact that
this agent displays antagonist and agonist properties. Nevertheless,
this latter observation demonstrates that when
1-ARs are blocked by CGP-12177,
maximal respiration and lipolysis can be reached via
3-adrenergic pathways.
A tight coupling between lipolysis and respiration is supported by
other observations. 1) Adenosine (1 µM), an inhibitor of lipolysis and respiration in brown adipocytes,
shifts the dose-response curve for the stimulation of lipolysis and
respiration by NE to higher concentrations by the same order of
magnitude (~10 times) (38). 2)
Propranolol inhibits NE-stimulated respiration as rapidly as it
inhibits lipolysis (in <3 min; see Fig. 1 and Refs. 9, 10).
3) Long-chain fatty acids mimic the
respiratory effects of NE even when endogenous adenosine
3',5'-cyclic monophosphate production and lipolysis are
blocked by an excess of propranolol (9).
4) Specific inhibitors of long-chain
fatty acid oxidation (methyl palmoxirate) rapidly inhibit mitochondrial
respiration (31). These observations together with the present results
suggest that
1- and
3-adrenergic agonists,
similarly to NE, increase mitochondrial respiration because they
increase fatty acid supply to the mitochondria.
Perspective.
What is the physiological function of
3-ARs? This question has been
raised many times and still remains unsolved (25, 37). As pointed out
in the introduction, the central problem in defining a physiological
role for
3-ARs is that their
affinity for NE, the physiological mediator of thermogenesis, is very
low (pKi = 4.2) (17).
Nevertheless, they are ~10 times more numerous (105 receptors/cell) than the
1-ARs that possess a very high
affinity for NE, in the nanomolar range of concentrations. Therefore,
one important question that remains to be solved concerns the
physiological concentrations of NE required to elicit metabolic
responses in BAT. It is known that cold exposure increases plasma NE
levels from ~1 nM (basal values in conscious undisturbed rats at room temperature) up to 20-25 nM, depending on the temperature of
exposure (18, 30). However, it is likely that much higher
concentrations of NE occur between the sympathetic nerve varicosities
and brown adipocyte plasma membranes, particularly after intensive
stress. In contrast to white adipose tissue, BAT is densely innervated with sympathetic nerves that run not only along the capillaries but
also between the individual adipocytes (16). Indirect evidence suggests
that concentrations of NE as high as 100 nM may occur in the synaptic
cleft. Depocas et al. (18, 19) have elegantly demonstrated that infused
NE must reach a plasma concentration of ~100 nM to maximally activate
nonshivering thermogenesis in rats maintained at room temperature.
Remarkably, 100 nM is also the NE concentration that is required to
maximally stimulate thermogenesis in isolated brown adipocytes (Fig. 4)
(9, 10). The data of Fig. 4 clearly demonstrate that the respiratory
effects of 100 nM NE can only be partially (
50%) decreased by
the selective
1-antagonist
ICI-89406, whereas the effects of lower NE concentrations (which do not
maximally activate thermogenesis; 25 nM) can be nearly totally
inhibited by this agent. These data, combined with our binding data
(17), strongly suggest that NE stimulates both
1- and
3-adrenergic pathways when it
maximally activates brown adipocyte thermogenesis.
On the other hand, it is known that
1-ARs may be desensitized or
downregulated by chronic cold exposure (11) or prolonged exposure to
-agonists (13, 23). In contrast,
3-ARs are particularly resistant to catecholamine-induced desensitization (13, 23, 34, 35). It
should also be pointed out that the affinity of
3-ARs for NE is of the same
order of magnitude as the affinity of receptors for neurotransmitters
such as acetylcholine (10-100 µM) (14). Thus receptors for
neurotransmitters appear to have a relatively low affinity for their
physiological agonists (in the micromolar range), possibly because the
concentration of the agonists in the synaptic cleft may reach very high
concentrations.
All these observations suggest that
3-ARs represent the
physiological receptors for NE secreted from sympathetic nerve endings when the concentration of the neurohormone in the synaptic cleft is
high and/or when the high-affinity
1-ARs are desensitized by
prolonged sympathetic stimulation. The main role of the
high-affinity
1-ARs would be to
mediate the effects of circulating NE (<25 nM; partial activation of
lipolysis and thermogenesis, regulation of blood flow and cell
proliferation), whereas the principal function of
3-ARs would be to transmit the
effects of NE released from sympathetic nerves when thermogenesis is
maximally activated by intensive stress. In this context, it has
recently been demonstrated that "cross talk" exists between
1- and
3-AR gene expression, the
evidence being that
1-AR mRNA
(but interestingly not
2AR mRNA) upregulates in BAT and white adipose tissue of mice lacking
3-ARs (37). This may explain
the preponderant role of
1-ARs in mediating the metabolic effects of NE in species lacking
3-ARs, such as the guinea pig
(6, 27).
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ACKNOWLEDGEMENTS |
This work was supported by the Medical Research Council of Canada.
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FOOTNOTES |
Address for reprint requests: L. J. Bukowiecki, Dept. of Physiology,
Faculty of Medicine, Laval University, Québec, PQ, Canada G1K
7P4.
Received 9 October 1996; accepted in final form 5 June 1997.
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