| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
3- but
not 1-adrenergically mediated in rat brown fat
cells, even after cold acclimation
The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, S-106 91 Stockholm, Sweden
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
To examine
if acclimation of rats to cold led to alterations in the coupling
between different 
-receptor subtypes and thermogenesis in brown fat
cells, we investigated the adrenergic response patterns in brown fat
cells isolated from warm-acclimated (28°C) and cold-acclimated (4°C) rats. In the cells from warm-acclimated rats, the relative affinities (EC50) for different
agonists (isoprenaline, BRL-37344, norepinephrine, CGP-12177,
dobutamine, and salbutamol) were those expected from their interaction
with a 3-receptor. The response to norepinephrine was competitively inhibited by propranolol with a
pA2
of 
6, implying interaction at the
3-receptor. No evidence for a
1-receptor-mediated response to
the 1-selective agonist dobutamine could be obtained; the low-affinity response observed was
most likely through the
3-receptor. The
1-antagonist ICI-89406 could
not inhibit a specific fraction of the thermogenic response to
norepinephrine. Thus
3-receptors were the only
-receptors involved in the control of thermogenesis in brown fat
cells from warm-acclimated rats. A modified method of preparation was
developed to isolate functional cells from cold-acclimated animals.
Also in these cells, the -receptor coupled to thermogenesis was the 3-receptor, although the
response was desensitized with an approximately sevenfold shift in
EC50 values. The
pA2
for propranolol inhibition of norepinephrine-induced thermogenesis was
also 6 here, and that for ICI-89406 was 5.5, also implying interaction
at the 3-receptor. Thus
acclimation to cold did not alter the -adrenergic receptor subtype
(3) involved in the control of thermogenesis.
norepinephrine; BRL-37344; CGP-12177; dobutamine; salbutamol
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
THERMOGENESIS IN BROWN FAT cells is physiologically
stimulated by norepinephrine released from the sympathetic nervous
system. Cellularly, the thermogenic signal is mainly mediated through -adrenergic receptors, adenylyl cyclase stimulation, and an increase in cytosolic cAMP levels (for general reviews, see Refs. 8, 19, 27, and
35; for description of an 
1-mediated enhancement of the
-response, see Ref. 39). Adipose tissues in most mammals are endowed
with 3-adrenergic receptors as
well as with the more ubiquitous
1-receptors (reviewed in Ref.
21), and therefore either or both -receptor subtype(s) could
theoretically couple the norepinephrine signal to thermogenesis in
brown fat cells.
We demonstrated earlier that in isolated hamster brown fat cells, it is
exclusively through the
3-receptors that the
-adrenergically mediated thermogenesis is stimulated (40). This
clear conclusion may be contrasted with implications from studies of
adrenergic stimulation of adenylyl cyclase activity in rat brown
adipose tissue membrane preparations. In such preparations, the two
-adrenergic receptors, 1 and
3, are both able to couple to
adenylyl cyclase (9, 14, 17), but it is not known to what extent these
two -receptors are able to couple further to thermogenesis in this species (33). Furthermore, the aforementioned conclusions concerning an
exclusive 3-coupling to
thermogenesis in hamsters were obtained with cells that were isolated
from animals living at normal ambient temperatures, i.e., at
temperatures at which brown adipose tissue heat production is not or
only slightly activated. It has been observed that cold exposure and
acclimation to cold, processes that activate brown adipose tissue, may
also lead to alterations in -receptor gene expression and in
-receptor level in the tissue (5, 15, 28) as well as in the levels
of the transducing G proteins
(Gs and
Gi) (31, 32) and of the
mediating adenylyl cyclases (13). Thus the generality of the earlier
conclusion concerning the 3
(rather than 1)-control of thermogenesis in hamster
brown fat cells may be questioned, both with regard to species
specificity and with regard to the relevance of the physiological state
of the animal from which the cells have been prepared.
It would therefore be of interest to clarify the type of -receptor
involved in the control of thermogenesis in brown fat cells isolated
from cold-acclimated rats. However, difficulties in preparation have
limited the number of studies of such cells, and in the few studies
presented, the isolation procedure has included steps that in
themselves may alter the adrenergic responsiveness, such as a
reacclimation or a preincubation period (20, 26). We therefore modified
the cell isolation procedure to allow us to obtain brown fat cells from
cold-acclimated rats and thus to examine the -receptor subtype
involvement also in cells obtained from this physiologically important condition.
We conclude that within the resolution of the experiment in the brown
fat cells from the warm-acclimated rats we were only able to find
evidence for thermogenic coupling via other -receptors than
3-receptors. Acclimation to
cold led to a general desensitization, but there was also no evidence
in these cells that the thermogenic response was mediated via
-receptors other than
3-receptors. Thus also in rat
brown fat cells, 3- but not 1-adrenergic
receptors are coupled to thermogenesis, and this conclusion is valid
even when the animal is in the physiologically recruited state.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Animals. Male or female Sprague-Dawley rats (B&K Universal, Sweden) weighing 150 g at the beginning of the acclimation period were used. The rats were divided into two groups and were placed singly at 4 ± 1°C (cold acclimated) or 28 ± 1°C (warm acclimated) in Macrolon 3 plastic cages with B&K wood chippings as bedding, for 4 wk under artificial lighting (6 h light and 18 h darkness). Both groups ate a rat and mouse standard diet (Solna Foderaffär, Stockholm, Sweden) and had unlimited access to water and food.
Isolation of brown adipocytes. The isolation was performed principally as described earlier (40). Some modifications of the isolation procedure were beneficial for the success of the preparation of cells from cold-acclimated rats.
Each preparation was performed with one cold-acclimated rat or three warm-acclimated rats, on alternating days. The rats were killed by CO2 immediately after transfer from their acclimation condition and then decapitated. The interscapular, axillary, and cervical depots of brown adipose tissue were dissected out; the brown adipose tissue had a deep brown color in cold-acclimated rats (in which ~2 g were obtained per rat) and a light brown color in warm-acclimated rats (0.7 g). The tissue was carefully cleaned of adhering white adipose tissue and muscle and added to a plastic vial with 4 ml modified Krebs-Ringer phosphate (KRP) buffer (see Buffers) containing 0.22 mg/ml collagenase and incubated for 9 min at 32°C (for tissue from cold-acclimated rats) and at 36°C (for warm acclimated). Then 7 ml of modified KRP buffer was added, and the incubations were vortexed at moderate speed for 15 s. The mixture in the vial was then filtered through silk cloth (Joymar Scientific, Hicksville, NY). The filtrate (which contained mainly broken fat cells, fat droplets, and red blood cells) was discarded, and the tissue was extensively minced with scissors and placed in a vial with 3 ml modified KRP buffer containing 0.29 mg/ml collagenase and incubated for 15 min in a shaking water bath with 80 strokes/min. The vial was removed from the bath every 4 min, vortexed moderately for 10 s, gassed with normal air (to prevent possible hypoxia during digestion) and replaced in the bath. Seven milliliters of modified KRP buffer (kept at room temperature) was added immediately after the 15-min digestion. The vial was vortexed for 20 s, and the contents were filtered through silk cloth and centrifuged for 5 min at 64 g (800 rpm). The infranatant was removed by a plastic tube, and 8 ml modified KRP buffer was added to the supernatant layer of cells. The cell suspension was kept at room temperature. The tissue pieces on the silk cloth were then reincubated as described, with 3 ml of modified KRP buffer. At least nine digestions like this were needed to finish the preparation of brown adipocytes. The second and third digestion times were 12 and 10 min, respectively, and the others were 8 min for each digestion. The cells from all digestions were collected and centrifuged, ~10 ml modified KRP buffer was added, and the cells were centrifuged for 3 min at 40 g (600 rpm) at least two times (to wash out collagenase and collect cells). The cells were counted in a Bürker chamber and kept at room temperature, ready for use in the experiment. The whole procedure lasted ~4 h (longer time tended to significantly decrease yield) and resulted in 1 × 106 to 10 × 106 cells per preparation.Measurement of thermogenesis. The rate of thermogenesis was indirectly assessed by measuring the rate of oxygen consumption at 37°C with a Yellow Springs Instrument 4004 Clark-type oxygen probe, as previously described (40). We added 35,000-80,000 cells to Krebs-Ringer bicarbonate buffer (see Buffers) in the oxygen chamber to give a final volume of 1.1 ml (buffer was equilibrated with 5% CO2 in air before addition of cells). After the addition of the cell suspension to the oxygen chamber, the chamber was closed with a cover lid and the cells were incubated for 3-4 min. After this time, different additions were made with a Hamilton syringe through a small hole in the cover lid of the chamber. Dose-response curves were made by successive addition of increasing concentrations of agonists; the response to each successive addition was recorded for 2.5-6 min until a stable response was reached.
Buffers.
For the isolation procedure, we found it helpful to use a somewhat
modified KRP buffer. The modified KRP buffer had the following composition (in mM): 156.4 Na+
(vs. normal 148), 6.9 K+, 1.8 Ca2+ (vs. normal 1.5), 1.4 Mg2+, 128 Cl
(vs. normal 119), 1.4 SO42,
5.6 H2PO4,
16.7 HPO42,
10 glucose, and 10 fructose. Crude BSA (4%) was also included. The pH
was adjusted with Tris base to 7.4. The buffer was pregassed with
normal air.
Chemicals.
Crude and fatty acid-free BSA (fraction V) were purchased from
Boehringer Mannheim (Indianapolis, Indiana). Collagenase (type II,
clostridiopeptidase A, EC 3.4.24.3),
L-norepinephrine bitartrate [(
)-arterenol],
D,L-propranolol, and
isoprenaline (L-isoproterenol D-bitartrate) were obtained from
Sigma (St. Louis, MO). Dobutamine (Dobutrex) was obtained from Lilly
(Indianapolis, Indiana), and salbutamol (Ventoline) was obtained from
Glaxo (Research Triangle Park, NC). BRL-37344 was a gift from
SmithKline Beecham (King of Prussia, PA). CGP-12177 was a gift from
Ciba-Geigy (Basel, Switzerland), and ICI-89406 was a gift from
ICI/Zeneca (Wilmington, DE). All adrenergic agents were freshly
dissolved in water, except for ICI-89406, which was dissolved and
diluted in ethanol:H2O (1:1).
Data analysis and statistics. For calculations, an oxygen content of 434 nmol O/ml distilled water at 37°C was used. The data obtained were analyzed with the reiterative general curve-fitting program of the KaleidaGraph data analysis/graphics application for Macintosh, for best fit to simple Michaelis-Menten kinetics, as detailed in the legends of the relevant figures.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Nature of -adrenergic receptor mediating
thermogenesis in brown fat cells isolated from warm-acclimated rats.
To observe the possible effect of acclimation to cold on the adrenergic
receptors coupled to thermogenesis in isolated brown fat cells, it was
first necessary to establish the adrenergic response pattern of brown
fat cells isolated from warm-acclimated rats.
1-receptors (dobutamine),
2-receptors (salbutamol), and
3-receptors (BRL-37344 and
CGP-12177), as well as a general -agonist (isoprenaline) and a
general adrenergic agonist, i.e., the endogenous agonist
norepinephrine. As seen, a dose-dependent thermogenic response was
obtained with each of these and the parameters describing the
thermogenic effect of each agonist are compiled in Table
1.
|
|
-adrenergic receptor involved in the control of
thermogenesis, one of two points of view may be used: in the global
view, the thermogenic response is considered to be mediated via only a
single receptor; in the complex view, the response observed may involve
contributions from different receptors. We first analyzed the data
according to the global view and then examined to what extent this view
was consistent with the details of the data.
In the global view, the pharmacological profile of the thermogenic
response of the brown fat cells should correlate fully with the
pharmacological profile of one of the known -receptors: 1,
2, or
3. Unbiased pharmacological
profiles of these receptors have been obtained in studies of cells in
which these receptors have been ectopically expressed, notably Chinese
hamster ovary (CHO) cells (10, 25, 34). We have therefore analyzed
(Fig. 2) to what extent the
pharmacological profile of the thermogenic response pattern observed
here in isolated brown fat cells corresponds to the pharmacological
profile earlier obtained for each of the -receptors in the
ectopically expressed system.
|
1- (Fig.
2A) or
2-receptor (Fig.
2B). However, the correlation
between the thermogenic response of the isolated brown fat cells and
the activity of the ectopically expressed
3-receptor was good (Fig. 2C); i.e., it would seem that,
within experimental error, the pharmacological profile of the receptor
mediating thermogenesis and that of the
3-receptor were identical. In
other words, the data are compatible with the response being mediated
(only) through the 3-receptor.
Concerning the individual agonists, some remarks may be made. For
CGP-12177 (Fig. 2C), the relative
affinity is exactly as predicted, but CGP-12177 is only a partial
thermogenic agonist (Fig. 1A and
Table 1); however, this is fully in accordance with it being only a
partial agonist in the ectopic
3-expression system (25). For
salbutamol (Fig. 2C), the relative
affinity is also as predicted. Data on its intrinsic activity on the
3-receptor are not available,
but it is not unlikely that it is only a partial agonist on this
receptor (because low affinity in general is correlated with partial
agonist properties; see Ref. 22), which would be in accordance with
salbutamol being only a partial thermogenic agonist (Fig.
1A and Table 1). For the
1-selective agonist dobutamine, no data from studies on ectopically expressed
3-receptors are available.
However, it is likely that its affinity for the
3-receptor is similar to that
of the 1-selective agonist
prenalterol (34), and, if this is the case, it would fit exactly on the
line in Fig. 2C. Again, the low
affinity would correlate with its only partial agonist activity (Fig.
1A and Table 1).
Thus, on the basis of the global analysis of the relative apparent
affinities of different agonists for stimulation of thermogenesis in
isolated rat brown fat cells, it would seem that the pharmacological profile is that to be expected if the global response is mediated via a
3-receptor. This thus also
means that the stimulatory effects of so-called selective
1- and
2-agonists are understandable as a consequence of these agonists interacting with the
3-receptor.
Antagonist analysis of receptor mediating response to endogenous
agonist norepinephrine.
Although the above data are compatible with the thermogenic response to
different agonists being mediated via the
3-receptor, it is also
necessary to examine whether the thermogenic response to the endogenous
agonist norepinephrine in itself was mediated via the
3-receptor. For this analysis,
we used the differential affinity of the antagonist propranolol for
1/2-receptors
(pA2 of 8-9; see Ref. 38) and for
3-receptors
(pA2
of 6.3 for inhibition of adrenergic stimulation of adenylyl cyclase in
3-CHO cells; see Ref. 25). We
therefore examined the ability of different concentrations of
propranolol to shift the dose-response curve for norepinephrine. The
resulting dose-response curves are shown in Fig.
3A. From
these, the Schild plot in Fig. 3C was
constructed. As seen, the presence of propranolol led to a shift in
dose-response curves corresponding to a
pA2
for propranolol of ~6, i.e., far from the specified
1/2-value
but exactly the value expected for propranolol interacting with
norepinephrine at the
3-receptor. The slope was close
to unity, implying that no other receptor types were involved and that
the interaction was fully competitive.
|
Can evidence for participation of
1-receptors in thermogenic
response be found?
As pointed out, the above analyses were all based on the global view,
i.e., that only one -receptor type is coupled to thermogenesis in
these cells. From the aforementioned data, there is no doubt that such
a receptor would be the
3-receptor. Because, however, there has been some interest in examining whether a
1-component exists in the
thermogenic response, we have examined the brown fat cells in more
detail for such a response.
1-mediated response could be
elicited by the 1-selective
agonist dobutamine. On the basis of studies in other tissues, the
EC50 for dobutamine on the
1-receptor would be expected to
be ~100 nM (1, 37), and a biphasic response, corresponding first to
the interaction of dobutamine with the
1-receptor and then, at higher
concentration, with the
3-receptor, would therefore be
expected if 1-receptors existed
that were coupled to thermogenesis. However, this was not the case
(Fig.
4A):
there was clearly no stimulation of thermogenesis at low dobutamine
concentrations. Rather, the points adhered closely to a monophasic
interaction with a receptor with an apparent affinity of 
1 µM,
likely corresponding to interaction with the
3-receptor.
|
1-antagonist ICI-89406. If a
certain fraction of the thermogenic response to norepinephrine was
mediated via 1-receptors, it
would be anticipated that a part of the dose-response curve for
norepinephrine would be markedly shifted to the right by increasing
concentrations of ICI-89406. However, as is evident from Fig.
5A, no
systematic shifts were observed in any part of the dose-response curve;
in fact, no dose-dependent effect of ICI-89406 on
norepinephrine-induced thermogenesis was observable. Thus we found no
evidence that a fraction of the norepinephrine-induced thermogenesis
was mediated via 1-receptors.
Concerning the interaction of ICI-89406 with the
3-receptor, it is clear that a
pA2
value could not be obtained, because this antagonist was of
insufficient affinity to influence norepinephrine action in these
cells.
|
-adrenergic response was solely mediated via the
3-receptor.
Effect of acclimation to cold on -receptors involved
in stimulation of thermogenesis in isolated brown fat cells.
To investigate whether, e.g., alterations of -receptor gene
expression due to cold exposure and cold acclimation influenced the
thermogenic response pattern of isolated brown fat cells, we developed
an improved method for isolation of such cells from cold-acclimated
rats (to enable comparison, this method was also the one used for
preparation of the brown fat cells from warm-acclimated rats). The
cells from cold-acclimated rats were more fragile than cells from
warm-acclimated or control rats, but a full analysis of the
-receptor response pattern was possible.
3-receptors. There were,
however, two notable differences in the responses. One was that the
maximal increase in respiratory rate values were generally only about
one-half of those in cells from warm-acclimated rats (Table 1). A
similar phenomenon was observed earlier with brown fat cells from
hamsters (36). It cannot be completely excluded that this apparent
decreased responsiveness is secondary to problems in cell counting,
etc. (but see DISCUSSION); however, more unequivocally, the EC50
values were all desensitized by a factor of ~7 (except apparently for
CGP-12177), and this effect would not be influenced by differences in,
e.g., cell counting.
As a consequence of this desensitization, the values for the responses
of the brown fat cells from cold-acclimated rats are principally found
as a parallel-shifted line on the figure that correlates thermogenic
responsiveness in brown fat cells with adenylyl cyclase responsiveness
in 3-CHO cells (Fig.
2C).
Thus the pharmacological profile data are compatible with the
-receptor responsible for the thermogenic response being the 3-receptor, also in brown fat
cells from cold-acclimated rats.
To ascertain that the global response to norepinephrine was indeed
mediated via 3-receptors in
brown fat cells from cold-acclimated rats, the
pA2
of propranolol for inhibition of norepinephrine-induced thermogenesis
was also established in these cells (Fig. 3,
B and C). As seen in the Schild plot (Fig.
3C), the lines for cells from
warm-acclimated and cold-acclimated rats fully coincided, establishing
that the same receptor mediated the global response in both acclimation
situations and that, because the
pA2
was 6, it was the
3-receptor.
In the brown fat cells isolated from cold-acclimated rats, we also
investigated whether we could observe any indications of a contribution
from 1-receptors. We thus also
examined the response of the cells to the
1-selective agonist dobutamine
(Fig. 4B). No clear response at low
dobutamine concentrations could be observed, but a response at very
high concentrations was seen, probably again corresponding to
dobutamine interacting with the
3-receptor. However, due to the
general desensitization, a saturated response could not be obtained
with the dobutamine concentrations used.
Finally, we also examined in these cells whether the
1-selective antagonist
ICI-89406 led to a shift in the dose-response curve for a specific part
of the response to norepinephrine. This was again not the case (Fig.
5B), implying that no discernible part of the thermogenic response to norepinephrine was mediated via
1-receptors. However, in
contrast to the case in cells from warm-acclimated rats (Fig.
5A), a clear dose-dependent shift in the global dose-response curve was observable here. Thus a Schild plot
could be constructed (Fig. 5C),
allowing for determination of the
pA2
of ICI-89406 on the thermogenic response to norepinephrine. The
pA2
was only 5.5, very far from the expected
pA2
for ICI-89406 interacting with the
1-receptor [8.2 is the
pKi of ICI-89406 for the 1-adrenoceptor in brown
adipose tissue (23), and, because the
pA2
and pKi should be
identical for antagonists, 8.2 is also the expected
pA2
value for ICI-89406 on
1-receptors in this
tissue]. Thus also the competitive effect of the
"selective" 1-antagonist ICI-89406 is most easily understood as an interaction of this antagonist with the 3-receptor
(although an independently established pA2
for ICI-89406 on ectopically expressed
3-receptor has not been
published). The slope of the Schild plot was also close to unity here,
implying competitive interaction with only one receptor type. The
reason that a competitive effect of ICI-89406 could be observed in
brown fat cells from cold-acclimated rats but not in those from
warm-acclimated rats is evidently that the latter cells were
desensitized to norepinephrine (cf., Fig. 1 and Table 1). Thus the
concentrations of ICI-89406 used were able to counteract the weakened
norepinephrine effect in the cells from cold-acclimated rats.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In the present investigation, we examined whether acclimation to cold,
a condition known to affect adrenergic receptor gene expression and
receptor complement in brown adipose tissue, leads to alterations in
the degree of involvement of the different -adrenergic receptors in
the acute control of thermogenesis. On the basis of several criteria
(correlation with affinities of ectopically expressed -receptors,
effects of antagonists), we first established that the -adrenergic
receptor coupled to thermogenesis in brown fat cells isolated from
warm-acclimated rats was exclusively the 3-receptor. We further found
that brown fat cells isolated from cold-acclimated rats had a
desensitized response to adrenergic stimulation but that the receptor
subtype involved was still exclusively the
3-receptor. Thus, although most
earlier investigations into adrenergic control of thermogenesis,
especially in rat cell systems, have used cells isolated from animals
living under conditions in which brown adipose tissue was not acutely
or chronically stimulated, it would seem that the basic mediation of
the response to sympathetic stimulation is unaltered in animals in
which the tissue has been under constant adrenergic stimulation during
the chronic demand for heat.
Is there a 1-component in
the thermogenic response in rat brown fat cells?
Although it was as agents selectively stimulating thermogenesis in
intact animals and lipolysis in isolated brown fat cells from rats that
the first 3-selective agents
were identified (2), the question of the relative participation of the
different -receptors, especially
1 versus
3, in the thermogenic response
to adrenergic stimulation has not been resolved in this species. We
have earlier demonstrated that in isolated brown fat cells from
hamsters, the only -receptor coupled to thermogenesis is the
3-receptor (40). In contrast,
in the rat, several types of experimental results have been advocated
as implying that a significant proportion of thermogenesis in this
species is mediated via
1-receptors. Because the
implications of these results contrast to the conclusions from the
present experiments, both with cells from warm-acclimated and from
cold-acclimated rats, we will briefly discuss these earlier observations.
3-receptor, the
1-receptor is expressed in
brown adipose tissue, both at the mRNA level and as evidenced by ligand
binding studies (15, 24, 28, 30). Furthermore, in studies of membrane
homogenates from the tissue, it has been established that both
1- and
3-receptors couple to adenylyl
cyclase (9, 14, 17). Because thermogenesis is mainly induced via an
increase in cAMP levels (27), it could be assumed that both
1- and
3-receptors would be involved
in the control of thermogenesis. This would, however, only be the case
if the 1- and
3-receptors coupled to adenylyl
cyclase were both present in the relevant cell types, and this is not
necessarily the case. Thus the present results indicating no influence
of 1-receptors on thermogenesis
would be compatible with the homogenate adenylyl cyclase data (9, 14,
17), provided that in the population of fully differentiated,
thermogenically competent brown fat cells only
3-coupled adenylyl cyclase was
found (as is indeed the case in brown fat cells from hamster; see Ref.
39) and the 1-coupled adenylyl
cyclase were to be found in another cell population in the tissue.
Indeed, we have demonstrated that only the
1-receptor is coupled to
stimulation of proliferation in young, thermogenically incompetent
brown fat cells (6), at least in the closely related mouse species.
Thus, whereas the 1-receptors
are therefore important for tissue function in a broader sense, we do
not think that the presence of adenylyl cyclase-coupled 1-receptors in the tissue
necessarily indicates that the
1-receptors influence
thermogenesis in the thermogenically competent brown fat cells.
It has also been discussed that
3-receptors should be much less
sensitive to norepinephrine than are
1-receptors, and thus 1-receptors should be
responsible for stimulation of thermogenesis under conditions of mild
to modest physiological stimulation (3, 12, 16). However, in a direct
comparison, the apparent affinity (for stimulation of adenylyl cyclase
in -receptor-expressing CHO cells) has been reported to be 0.8 nM
for norepinephrine stimulation of
1-receptors, 6.3 nM for
stimulation of 3-receptors, and
36 nM for 2-receptors (34).
Thus there is no particular low functional affinity of the
3-receptor for norepinephrine.
In this context, it has also been commented that stimulation of
thermogenesis with a low concentration of norepinephrine is more easily
inhibited by a 1-antagonist
than stimulation with a high concentration of norepinephrine (3). This
phenomenon is principally to be expected in general in any competitive
inhibition (and can indeed also be seen in Fig.
5B). Thus more detailed studies are
required to show that the inhibition of thermogenesis at low norepinephrine concentrations indeed occurs due to interaction with the
1-receptor. At least in hamster
brown fat cells, the thermogenic response to low concentrations of
norepinephrine still showed a fully
3-adrenergic character (40).
Correspondingly, the dose-response curves for norepinephrine in the
presence of 1-antagonists
presented here (Figs. 3 and 5) do not demonstrate the skewed right-hand
shift that would be expected if the most sensitive part of the
dose-response curve were mediated via
1-receptors.
Thus the data we present here do not lend support to the idea that a
significant fraction of thermogenesis in rat brown fat cells is
mediated via 1-receptors either
in cells from warm-acclimated rats or in cells from cold-acclimated
rats. We cannot, of course, exclude with absolute certainty that a very
limited fraction of the response is
1-adrenergic; this fraction
would, however, have to be smaller than the resolution of the data
presented here, i.e., <20%. Such a fraction may therefore be said to
be of minor thermoregulatory interest, even if it were to exist.
Effect of acclimation to cold on adrenergic receptor involvement and sensitivity. It has been a general concern that most studies investigating the control of thermogenesis in brown fat cells have used cells isolated from animals in which the tissue is not in a recruited state. The results obtained may therefore have been considered to be unrepresentative for the function of the cells in physiologically more relevant contexts. In the present investigation, we have thus endeavored to develop the method of cell isolation to allow for preparation of brown fat cells directly from cold-acclimated rats. Such brown fat cells have not been available earlier for study. Only cells from other species (36), cells isolated from rats after short-time reacclimation to warm, or cells studied after prolonged preincubation have been available (20, 26).
The present investigation demonstrates that the results obtained with cells from warm-acclimated animals are fairly representative also of cells isolated from cold-acclimated animals. The response is qualitatively identical, in that in both cases the thermogenic response was solely mediated via the3-receptor. Quantitatively, the
cells from cold-acclimated rats proved to be desensitized, i.e., they
demonstrated higher EC50 values
than those from warm-acclimated rats (similar to what is the case in
hamsters; see Ref. 36). The
3-receptor in itself cannot
desensitize via classical mechanisms (18). Instead, alterations in
receptor gene expression (5, 15) could possibly explain the
desensitization. Activation of postreceptor desensitizing mechanisms,
as observed in hamster cells (31, 36) may, however, be a more likely explanation.
Cold acclimation also led to a decrease in the total level of
norepinephrine-induced thermogenesis per cell; a similar phenomenon has
again been seen in cells from hamsters (36) (whereas cells from
cold-acclimated guinea pigs have been reported to show an increased
level of norepinephrine-induced thermogenesis; see Ref. 29). The
decrease in thermogenic response may initially be considered somewhat
surprising, because the thermogenic capacity of the entire brown
adipose tissue complement increases dramatically during cold
acclimation (11). However, the apparent discrepancy between these
observations may be explained by the fact that the total number of
cells capable of thermogenesis in the tissue is very much increased
during cold acclimation (7), which much more than compensates for the
decrease in the level of norepinephrine-induced thermogenesis of the
individual cell. A possible cellular explanation for the decrease in
the level of norepinephrine-induced thermogenesis per cell would again
be the activation of postreceptor mechanisms, notably of cAMP
phosphodiesterase activity (36), during cold acclimation. At least in
the hamster cells, this activity increases to the extent that
saturating levels of cAMP are not attained, leading to a limitation in
the level of thermogenesis induced. This should probably be considered
a physiologically induced functional desensitization process.
In conclusion, we demonstrated here that in brown fat cells isolated
from warm-acclimated or cold-acclimated rats, the -thermogenic response is fully mediated via the
3-receptor. Considering that the -response in hamster cells is also fully
3-adrenergic (40), it is
tempting to suggest that this is a general conclusion and that the
3-receptor has specific
properties that are advantageous for thermogenesis. However, the brown
adipose tissue of guinea pigs does not express the
3-receptor (4), and it is
therefore principally possible to have physiologically functional
systems relying on 1-receptors
for the control of thermogenesis. The advantages of developing the
3-receptor for the control of
adipose tissue function in certain animals thus remain obscure.
| |
ACKNOWLEDGEMENTS |
|---|
This investigation was supported by the Swedish Natural Science Research Council.
| |
FOOTNOTES |
|---|
Address reprint requests to J. Nedergaard.
Received 2 December 1997; accepted in final form 5 August 1998.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1.
Abou-Mohamed, G.,
R. W. Caldwell,
G. O. Carrier,
M. M. Elmazar,
and
R. R. Tuttle.
Interactions of a novel catecholamine, GP-2-128, with adrenoceptors.
J. Cardiovasc. Pharmacol.
23:
492-500,
1994[Medline].
2.
Arch, J. A. T.,
A. T. Ainsworth,
M. A. Cawthorne,
V. Piercy,
M. V. Sennitt,
V. E. Thody,
C. Wilson,
and
S. Wilson.
Atypical -adrenoceptor on brown adipocytes as target for anti-obesity drugs.
Nature
309:
163-165,
1984[Medline].
3.
Atgié, C.,
F. D'Allaire,
and
L. J. Bukowiecki.
Role of 1- and 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1136-C1142,
1997.
4.
Atgié, C.,
G. Tavernier,
F. D'Allaire,
T. Bengtsson,
L. Marti,
C. Carpéné,
M. Lafontan,
L. J. Bukowiecki,
and
D. Langin.
3-Adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function.
Am. J. Physiol.
271 (Regulatory Integrative Comp. Physiol. 40):
R1729-R1738,
1996
5.
Bengtsson, T.,
K. Redegren,
A. D. Strosberg,
J. Nedergaard,
and
B. Cannon.
Down-regulation of 3-adrenoreceptor gene expression in brown fat cells is transient and recovery is dependent upon a short-lived protein factor.
J. Biol. Chem.
271:
33366-33375,
1996
6.
Bronnikov, G.,
J. Houstek,
and
J. Nedergaard.
-Adrenergic, cAMP-mediated stimulation of proliferation of brown fat cells in primary culture. Mediation via 1 but not via 3 receptors.
J. Biol. Chem.
267:
2006-2013,
1992
7.
Bukowiecki, L. J.,
A. J. Collet,
N. Follea,
G. Guay,
and
L. Jahjah.
Brown adipose tissue hyperplasia: a fundamental mechanism of adaptation to cold and hyperphagia.
Am. J. Physiol.
242 (Endocrinol. Metab. 5):
E353-E359,
1982
8.
Cannon, B.,
and
J. Nedergaard.
The biochemistry of an inefficient tissue: brown adipose tissue.
Essays Biochem.
20:
110-164,
1985[Medline].
9.
Chaudhry, A.,
K. N. Lahners,
and
J. G. Granneman.
Perinatal changes in the coupling of 1- and 3-adrenergic receptors to brown fat adenylyl cyclase.
J. Pharmacol. Exp. Ther.
261:
633-637,
1992
10.
Emorine, L. J.,
S. Marullo,
M.-M. Briend-Sutren,
G. Patey,
K. Tate,
C. Delavier-Klutchko,
and
A. D. Strosberg.
Molecular characterization of the human 3-adrenergic receptor.
Science
245:
1118-1121,
1989
11.
Foster, D. O.,
and
M. L. Frydman.
Nonshivering thermogenesis in the rat. II. Measurements of blood flow with microspheres point to brown adipose tissue as the dominant site of the calorigenesis induced by noradrenaline.
Can. J. Physiol. Pharmacol.
56:
110-122,
1978[Medline].
12.
Galitsky, J.,
C. Carpéné,
A. Bousquet-Melou,
M. Berlan,
and
M. Lafontan.
Differential activation of 1-, 2- and 3-adrenoceptors by catecholamines in white and brown adipocytes.
Fundam. Clin. Pharmacol.
9:
324-331,
1995[Medline].
13.
Granneman, J. G.
Expression of adenylyl cyclase subtypes in brown adipose tissue: neural regulation of type III.
Endocrinology
136:
2007-2012,
1995[Abstract].
14.
Granneman, J. G.
Norepinephrine and BRL 37344 stimulate adenylate cyclase by different receptors in rat brown adipose tissue.
J. Pharmacol. Exp. Ther.
254:
508-513,
1990
15.
Granneman, J. G.,
and
K. N. Lahners.
Differential adrenergic regulation of 1- and 3-adrenoceptor messenger ribonucleic acids in adipose tissues.
Endocrinology
130:
109-114,
1992[Abstract].
16.
Granneman, J. G.,
K. N. Lahners,
and
A. Chaudhry.
Molecular cloning and expression of the rat 3-adrenergic receptor.
Mol. Pharmacol.
40:
895-899,
1991[Abstract].
17.
Granneman, J. G.,
and
C. J. Whitty.
CGP 12177A modulates brown fat adenylate cyclase activity by interacting with two distinct receptor sites.
J. Pharmacol. Exp. Ther.
256:
421-425,
1991
18.
Hausdorff, W. P.,
M. G. Caron,
and
R. J. Lefkowitz.
Turning off the signal: desensitization of -adrenergic receptor function.
FASEB J.
4:
2881-2889,
1990[Abstract].
19.
Himms-Hagen, J.
Brown adipose tissue thermogenesis and obesity.
Prog. Lipid Res.
28:
67-115,
1989[Medline].
20.
Kuroshima, A.,
and
T. Yahata.
Thermogenic responses of brown adipocytes to noradrenaline and glucagon in heat-acclimated and cold-acclimated rats.
Jpn. J. Physiol.
29:
683-690,
1979[Medline].
21.
Lafontan, M.,
and
M. Berlan.
Fat cell adrenergic receptors and the control of white and brown fat cell function.
J. Lipid Res.
34:
1057-1091,
1993[Abstract].
22.
Leff, P.
The two-state model of receptor activation.
Trends Pharmacol. Sci.
16:
89-97,
1995[Medline].
23.
Levin, B. E.,
and
A. C. Sullivan.
Beta-1 receptor is the predominant beta-adrenoceptor on rat brown adipose tissue.
J. Pharmacol. Exp. Ther.
236:
681-688,
1986
24.
Muzzin, P.,
J.-P. Revelli,
F. Kuhne,
J. D. Gocayne,
W. R. McCombie,
J. C. Venter,
J.-P. Giacobino,
and
C. M. Fraser.
An adipose tissue-specific -adrenergic receptor. Molecular cloning and down-regulation in obesity.
J. Biol. Chem.
266:
24053-24058,
1991
25.
Nahmias, C.,
N. Blin,
J.-M. Elalouf,
M. G. Mattei,
A. D. Strosberg,
and
L. J. Emorine.
Molecular characterization of the mouse 3-adrenergic receptor: relationship with the atypical receptor of adipocytes.
EMBO J.
10:
3721-3727,
1991[Medline].
26.
Nedergaard, J.
Catecholamine sensitivity in brown fat cells from cold-acclimated hamsters and rats.
Am. J. Physiol.
242 (Cell Physiol. 11):
C250-C257,
1982
27.
Nedergaard, J.,
and
O. Lindberg.
The brown fat cell.
Int. Rev. Cytol.
74:
187-286,
1982[Medline].
28.
Raasmaja, A. 1- and
-adrenergic receptors in brown adipose tissue and the adrenergic
regulation of thyroxine 5'-deiodinase. Acta Physiol. Scand.
139, Suppl. 590: 1-61, 1990.
29.
Rafael, J.,
W. Fesser,
and
D. Nicholls.
Cold adaption in guinea pig at level of isolated brown adipocyte.
Am. J. Physiol.
250 (Cell Physiol. 19):
C228-C235,
1986
30.
Svoboda, P.,
J. Svartengren,
M. Snochowski,
J. Houstek,
and
B. Cannon.
High number of high-affinity binding sites for ()-(3H)dihydroalprenolol on isolated hamster brown-fat cells. A study of the -adrenergic receptors.
Eur. J. Biochem.
102:
203-210,
1979[Medline].
31.
Svoboda, P.,
L. Unelius,
B. Cannon,
and
J. Nedergaard.
Attenuation of Gs-protein coupling efficiency in brown-adipose-tissue plasma membranes from cold-acclimated hamsters.
Biochem. J.
295:
655-661,
1993.
32.
Svoboda, P.,
L. Unelius,
A. Dicker,
B. Cannon,
G. Milligan,
and
J. Nedergaard.
Cold-induced reduction in Gi proteins in brown adipose tissue. Effects of cellular hypersensitization to noradrenaline caused by pertussis-toxin treatment.
Biochem. J.
314:
761-768,
1996.
33.
Tanaka, E.,
A. Yamakawa,
M. Yamamura,
N. el Borai,
K. Ito,
S. Nakano,
and
H. Nakazawa.
Regulation of heat production of brown adipocytes via typical and atypical -receptors in the rat.
Jpn. J. Physiol.
45:
1043-1051,
1995[Medline].
34.
Tate, K. M.,
M.-M. Briend-Sutren,
L. J. Emorine,
C. Delavier-Klutchko,
S. Marullo,
and
A. D. Strosberg.
Expression of three human -adrenergic-receptor subtypes in transfected chinese hamster ovary cells.
Eur. J. Biochem.
196:
357-361,
1991[Medline].
35.
Trayhurn, P.,
and
D. G. Nicholls.
Brown Adipose Tissue. London: Arnold, 1986.
36.
Unelius, L.,
G. Bronnikov,
N. Mohell,
and
J. Nedergaard.
Physiological desensitization of 3-adrenergic responsiveness in brown fat cells: involvement of a postreceptor mechanism.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1340-C1348,
1993
37.
Vigholt Sorensen, E.,
and
F. Nielsen-Kudsk.
Myocardial pharmacodynamics of dopamine, dobutamine, amrinone and isoprenaline compared in the isolated rabbit heart.
Eur. J. Pharmacol.
124:
51-57,
1986[Medline].
38.
Wilson, C.,
S. Wilson,
V. Piercy,
M. V. Sennitt,
and
J. R. Arch.
The rat lipolytic beta-adrenoceptor: studies using novel beta-adrenoceptor agonists.
Eur. J. Pharmacol.
100:
309-319,
1984[Medline].
39.
Zhao, J.,
B. Cannon,
and
J. Nedergaard.
1-Adrenergic stimulation potentiates the thermogenic action of 3-adrenoceptor-generated cAMP in brown fat cells.
J. Biol. Chem.
272:
32847-32856,
1997
40.
Zhao, J.,
L. Unelius,
T. Bengtsson,
B. Cannon,
and
J. Nedergaard.
Coexisting -adrenoceptor subtypes: significance for thermogenic process in brown fat cells.
Am. J. Physiol.
267 (Cell Physiol. 36):
C969-C979,
1994
This article has been cited by other articles:
|
|
 |
|
  M. Sun, C. J. Lee, and H.-S. Shin Reduced nicotinic receptor function in sympathetic ganglia is responsible for the hypothermia in the acetylcholinesterase knockout mouse J. Physiol., February 1, 2007; 578(3): 751 - 764. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Shabalina, C. Wiklund, T. Bengtsson, A. Jacobsson, B. Cannon, and J. Nedergaard Uncoupling protein-1: involvement in a novel pathway for beta -adrenergic, cAMP-mediated intestinal relaxation Am J Physiol Gastrointest Liver Physiol, November 1, 2002; 283(5): G1107 - G1116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Martinez-deMena, A. Hernandez, and M.-J. Obregon Triiodothyronine is required for the stimulation of type II 5'-deiodinase mRNA in rat brown adipocytes Am J Physiol Endocrinol Metab, May 1, 2002; 282(5): E1119 - E1127. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Nisoli, L. Briscini, A. Giordano, C. Tonello, S. M. Wiesbrock, K. T. Uysal, S. Cinti, M. O. Carruba, and G. S. Hotamisligil Tumor necrosis factor alpha mediates apoptosis of brown adipocytes and defective brown adipocyte function in obesity PNAS, July 5, 2000; 97(14): 8033 - 8038. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Matthias, K. B. E. Ohlson, J. M. Fredriksson, A. Jacobsson, J. Nedergaard, and B. Cannon Thermogenic Responses in Brown Fat Cells Are Fully UCP1-dependent. UCP2 OR UCP3 DO NOT SUBSTITUTE FOR UCP1 IN ADRENERGICALLY OR FATTY ACID-INDUCED THERMOGENESIS J. Biol. Chem., August 11, 2000; 275(33): 25073 - 25081. [Abstract] [Full Text] |
), BRL-37344 (
),
norepinephrine (
), CGP-12177 (
), dobutamine (
), and salbutamol
(
) (salbutamol was only tested in cells from warm-acclimated rats),
as described in MATERIALS AND METHODS.
Results are means + SE of 4 determinations on 4 different cell
preparations (where no error bars are shown, SE was smaller than size
of symbol). For this and the following figures, curves were drawn by
reiterative computerized fitting of mean values to a simple
Michaelis-Menten equation of the type V(A) = basal + 
Vmax · {[A]/([A] + EC50)}, where
[A] indicates agonist concentration, basal indicates basal
respiratory rate,
