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CNRS-UPR 0415 and Université Paris VII, Institut Cochin de Génétique Moléculaire, F-75014 Paris, France
Address all correspondence and requests for reprints to: Dr. Ralf Jockers, Laboratoire d’Immuno-Pharmacologie Moléculaire, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, F-75014 Paris, France. E-mail: jockers{at}icgm.cochin.inserm.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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80% of the total). However, ß3AR was poorly coupled
to the adenylyl cyclase pathway in PAZ6 cells, contributing to only
10% of the isoproterenol-induced accumulation of cAMP, whereas 20%
and 70% of the signal depended on ß1- and
ß2-subtypes, respectively. Upon isoproterenol
stimulation, ß1- and ß2AR down-regulated
with a half-life of about 3 h and the ß3AR with a
half-life of 30–40 h. Long term stimulation with both saturating
(micromolar) and nonsaturating (nanomolar) concentrations of
ß-adrenergic agonists caused a complete desensitization of the
ß-adrenergic response at the adenylyl cyclase level and loss of
stimulated protein kinase A activity and CREB phosphorylation. These
results suggest that cAMP-dependent processes will be desensitized upon
permanent treatment with ß3AR agonists. Further studies
should establish whether the ß3AR is coupled to other
signaling pathways in human brown adipocytes and whether these may
contribute to BAT hypertrophy and/or thermogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2AR that block lipolysis as a consequence of its
inhibitory effect on adenylyl cyclase (2). In brown adipose tissue
(BAT), the activation of ßAR promotes both lipolysis and
thermogenesis (3). The synthesis of UCP1, the major uncoupling protein
controlling thermogenesis in BAT, is stimulated by cAMP. The three ßAR subtypes are coexpressed in adipose tissues. It has been proposed that the selective activation of the ß3AR in either WAT or BAT could be beneficial for the treatment of obesity (4, 5, 6, 7, 8, 9). This hypothesis is based on several unique properties of the ß3AR. Expression of ß3AR is restricted to a small number of tissues, including BAT and WAT (2), whereas ß1- and ß2AR are found in most tissues; ß3AR-selective agonists would thus cause fewer side-effects than compounds also active on the other subtypes. In several animal models, ß3AR plays a predominant role in the control of lipolysis and thermogenesis compared with the other two subtypes (2, 10). Finally, studies conducted on transfected cell lines and animal models have shown that the ß3AR seems to be resistant to receptor desensitization, a phenomenon of attenuation of receptor responsiveness, which markedly hampers ß1- and ß2AR-dependent signals upon sustained receptor activation (11, 12, 13, 14, 15).
Previous studies showed that about 40% of ß-adrenergically stimulated energy expenditure in humans is maintained when ß1- and ß2AR are specifically blocked (16). Also supporting the involvement of the ß3AR in human fat metabolism regulation are genetic studies showing that a functional mutation of the ß3AR may be associated with obesity (9, 17). This W64R mutation was shown to markedly decrease the ability of the ß3AR to activate adenylyl cyclase (18). However, other experiments do not support the hypothesis that the ß3AR are good targets of antiobesity treatments. For example, the contribution of the ß3AR to the ß-adrenergic response of WAT is probably minor in humans compared with that in rodents (1, 19, 20). In addition, mechanisms of desensitization affecting the signaling cascade downstream the activated ß3AR may occur in some cells (21, 22, 23).
To what extent the ß3AR contributes to the physiological ß-adrenergic response in human BAT or whether ß3AR signaling is impaired in adipose tissues upon sustained stimulation has not been addressed to date because appropriate models of human adipose tissues were missing. We have addressed these questions in the present study using human PAZ6 adipocytes as a model. PAZ6 is an immortalized human brown preadipocyte cell line that, upon differentiation, maintains in culture the morphology of a mature adipocyte and expresses the brown adipocyte-specific marker gene UCP1 (24). Our results indicate that PAZ6 adipocytes are an appropriate model to investigate ß-adrenergic regulation in human brown fat cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Media
Medium 1 consisted of DMEM-Ham’s F-12 (1:1, vol/vol)
supplemented with 15 mM HEPES, penicillin (100 U/ml),
streptomycin (0.1 mg/ml), and 10% FCS. Medium 2 was medium 1
supplemented as previously described (24). Medium 3 consisted of DMEM
supplemented with HEPES (15 mM), penicillin (100 U/ml),
streptomycin (0.1 mg/ml), 1% FCS, and 2% BSA (fatty acid free).
Culture of PAZ6 cell line
Immortalization of the human brown preadipocyte cell line has
been described previously (24). Preadipocytes were cultivated in medium
1 in an atmosphere of 92.5% air-7.5% CO2. The medium was
changed every other day. For differentiation, cells were seeded at
10,000/cm2 and cultivated for 3 days in medium 1, then
medium was changed for medium 2. IBMX (0.25 mM) and
ascorbic acid (0.1 mM) were added during the first 4 days
of differentiation. After 14 days, PAZ6 adipocytes were washed with
PBS, and medium 1 supplemented with 1 nM
T3 was added 24 h before the
experiments.
Isolation of mature human brown adipocytes and primary
adipocytes
Peritumoral BAT was obtained from an adult patient during the
surgical procedure for a pheochromocytoma. Brown adipocytes were
isolated by collagenase digestion according to the method described by
Hauner (25). Tissue was maintained at room temperature in PBS
containing 20 mg/ml BSA until transfer to the laboratory. The tissue
was then repeatedly rinsed in PBS, cut into small pieces, and digested
in Krebs-Ringer buffer containing 1 mg/ml collagenase (type II) and 20
mg/ml BSA (4 ml buffer/g tissue) at 37 C for 60 min in a shaking water
bath at 100 strokes/min. After filtration through a nylon screen (pore
size, 190 µm), the cell suspension was centrifuged at 50 x
g for 5 min. Floating cells were separated from the
infranatant. Floating cells were washed once with Krebs-Ringer buffer
containing BSA and then three times in medium 3. Cells were finally
resuspended in medium 3 at about 105 cells/ml and incubated
in an atmosphere of 92.5% air-7.5% CO2 for various times.
These cells were termed mature adipocytes in the text. The infranatant
was recentrifuged at 200 x g for 10 min. The
supernatant was discarded, and the cell pellet was washed once with
Krebs-Ringer buffer containing BSA and once with medium DMEM-Ham’s
F-12 (1:1, vol/vol). Red blood cells were eliminated by treatment with
Gey’s buffer. Cells were seeded into 35-mm culture dishes at a density
of 35,000/cm2 in 2 ml medium 1. Cultures were maintained at
37 C for 24 h in an atmosphere of 92.5% air-7.5%
CO2. After attachment, cells were washed with PBS, and
medium 1 supplemented with 33 µM biotin and 17
µM pantothenate was added. The medium was changed every
other day. Once confluent, cells were differentiated for 12 days as
described for the PAZ6 cell line. These cells were termed primary
adipocytes.
RT-PCR
Total RNA was extracted, and RT-PCR reactions performed as
described previously (24). The sequences for the sense and antisense
oligonucleotides for the ß3AR were
5'-CCCAATACCGCCAACAGT-3' and 5'-CGACCCACACCA GGACCACAG-3', respectively
(annealing temperature, 62 C).
Stimulation of ßARs and membrane preparation
PAZ6 adipocytes. Cells were incubated in medium 1
supplemented with 1 nM T3 for 24
h and then pretreated, or not, with different ligands in the presence
of 10 µM ascorbic acid for various periods of time at 37
C in the same medium. Culture dishes were placed on ice and washed
twice with ice-cold PBS, and the cells were detached mechanically in
ice-cold buffer containing 5 mM Tris, 2 mM EDTA
(pH 7.4), 5 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin,
and 10 mg/liter benzamidine. Cell suspensions were homogenized with a
Polytron homogenizer (Ultra-Turrax T25, Janke & Undel) three times for
5 sec each time at maximal setting. The lysate was centrifuged at
450 x g for 5 min at 4 C. The supernatant was
centrifuged at 43,000 x g for 20 min at 4 C, and the
pellet was resuspended in 75 mM Tris (pH 7.4), 5
mM MgCl2, 2 mM EDTA, and protease
inhibitors (as above) and immediately used for radioligand binding
experiments. Protein concentrations were determined by the method of
Bradford (26) with the Bio-Rad protein assay system using BSA as
standard.
Mature adipocytes. Cells were incubated in the presence of 10 µM norepinephrine in medium 3 containing 10 µM ascorbic acid or the vehicle alone for the indicated periods of time. Floating cells were separated from the infranatant and resuspended in 10 ml/106 cells of ice-cold buffer containing 5 mM Tris, 2 mM EDTA (pH 7.4), 5 mg/liter soybean trypsin inhibitor, 5 mg/liter leupeptin, and 10 mg/liter benzamidine. Cell suspensions were homogenized with a Polytron homogenizer (Janke & Kunkel Ultra-Turrax T25) three times for 5 sec each time at maximal setting. Lysates were centrifuged at 43,000 x g for 20 min at 4 C, and the pellet was washed once in the same buffer. The final pellet was resuspended in 75 mM Tris (pH 7.4), 5 mM MgCl2, 2 mM EDTA, and protease inhibitors (as above) and immediately used in radioligand binding experiments.
Radioligand binding assays
Cell membranes (20–50 µg protein) were incubated in a final
volume of 0.25 ml in 75 mM Tris (pH 7.4), 5 mM
MgCl2, 2 mM EDTA containing 50 mg/100 ml BSA, 1
µM desipramine, and [125I]CYP as
radioligand (50–1800 pM). Specific binding was defined as
binding displaced by 50 µM bupranolol. Assays were
carried out for 90 min at 25 C and were terminated by rapid filtration
through Whatman GF/C glass-fiber filters (Whatman, Clifton, NJ)
previously soaked in PBS containing 0.3% polyethyleneimine (to reduce
nonspecific binding). To determine the receptor half-life, data were
fitted using an exponential decay equation (y =
Ae(-Bx) + E). In this equation, A
represents the span, B the rate constant, and E the plateau.
Determination of intracellular cAMP levels
PAZ6 adipocytes. Differentiated PAZ6 cells were incubated in
medium 1 supplemented with 1 nM T3
for 24 h and then pretreated, or not, with different ligands in
the presence of 10 µM ascorbic acid for 3 h to 4
days at 37 C in the same medium. During sustained stimulation, the
ligand-containing medium was replaced at least once a day. Finally,
cells were treated with or without the indicated ligands for 15 min in
the same medium containing 1 mM IBMX. The incubation buffer
was discarded, and cells were lysed in 1 M NaOH for at
least 20 min at 37 C. The lysate was neutralized with 1 M
acetic acid and centrifuged in a microfuge at maximum speed for 10 min.
The supernatant was used for cAMP determination using a
[3H]cAMP assay system (Amersham Life Science, Arlington
Heights, IL).
Primary adipocytes. Precursor cells were differentiated into adipocytes in 24-well culture dishes as described above, then pretreated and stimulated as described for PAZ6 adipocytes. The stimulation buffer was discarded, and cells were lysed with ethanol (65%; cooled to -20 C) for 10 min on ice. The lysate was centrifuged for 10 min in a microfuge at the maximal setting at 4 C. The supernatant was concentrated in a Speed-Vac (Savant, Farmingdale, NY), and the pellet was diluted in 10 mM Tris, pH 7.4, and 1 mM EDTA. cAMP concentrations were determined using an enzyme immunoassay system (Amersham Life Science).
Mature adipocytes. Mature adipocytes were isolated as described and pretreated, or not, with different ligands in the presence of 10 µM ascorbic acid for the indicated times in medium 3 (105 cells/2 ml) at 37 C in an atmosphere of 92.5% air-7.5% CO2. Cells were stimulated, or not, with different ligands for 15 min in a volume of 0.25 ml. Cells were placed on ice, and 0.5 ml ethanol (-20 C) was added. After 10 min, the precipitate was centrifuged for 60 min in a microfuge at maximal setting at 4 C. cAMP concentrations were measured as described above.
Protein kinase A (PKA) assay
PAZ6 adipocytes were pretreated and stimulated to activate
adenylyl cyclase as described above. Stimulated cells were washed
rapidly with ice-cold PBS containing 0.5 mM IBMX and
detached mechanically in lysis buffer containing 20 mM
Tris-HCl (pH 7.4), 30 mM NaCl, 5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 10
mM NaF, 0.5 mM IBMX, 1 mM
dithiothreitol (DTT), 5 mg/liter soybean trypsin inhibitor, 5 mg/liter
leupeptin, 10 mg/liter benzamidine, and 10% glycerol. Cell suspensions
were homogenized as described above. The lysate was centrifuged in a
microfuge at maximal setting for 45 min at 4 C. PKA activity was
measured in supernatants using a colorimetric PKA assay kit (SpinZyme
format, Pierce Chemical Co., Rockford, IL). Reactions were carried out
at 30 C for 60 min. Maximal PKA stimulation in the sample was
determined in the presence of 0.1 mM exogenous cAMP and
calculated as the difference between activity in the presence and
absence of the PKA-specific inhibitor H89 (3 µM).
CREB phosphorylation
PAZ6 adipocytes were pretreated and stimulated as described for
the determination of intracellular cAMP levels. Stimulated cells were
rapidly washed with ice-cold PBS containing 0.5 mM IBMX and
detached mechanically in 0.5 ml cell lysis buffer containing 20
mM HEPES (pH 7.8), 20 mM NaCl, 50
mM NaF, 3 mM DTT, 30 nM okadaic
acid, 300 mM phenylmethylsulfonylfluoride, 0.5% Nonidet
P-40 (vol/vol), and 1 mg/ml each of pepstatin, antipain, leupeptin, and
aprotinin. Cell extracts were centrifuged for 2 min at 6,000 x
g. The pellet was resuspended in 100 µl nuclear lysis
buffer containing 20 mM HEPES (pH 7.8), 420 mM
NaCl, 50 mM NaF, 1.5 mM MgCl2, 0.2
mM EDTA, 25% glycerol (vol/vol), 3 mM DTT, 30
nM okadaic acid, 300 mM PMSF, 0.5% Nonidet
P-40 (vol/vol), and 1 mg/ml each of pepstatin, antipain, leupeptin, and
aprotinin. After 15 min on ice, the extracts were centrifuged at
10,000 x g for 2 min. The supernatants were collected,
and protein concentration was determined using Bradford reagent. One
hundred micrograms of proteins were separated by SDS-PAGE (10% gels)
and transferred on to nitrocellulose membranes. Immunoblot analysis was
carried out with an anti-phosphorylated CREB antibody (Upstate
Biotechnology, Lake Placid, NY; dilution, 1:2000). To quantify the
total amount of CREB present in the extracts, the antiphospho-CREB
antibody was stripped off, and membranes were reprobed with anti-CREB
antibody (Upstate Biotechnology; dilution, 1:1000).
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Results |
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The brown phenotype of the isolated BAT was confirmed by the detection
of the brown adipocyte-specific marker gene UCP1 by RT-PCR (data not
shown). Expression of the ß3AR was also shown in these
cells by RT-PCR (Fig. 1
). The relative
proportions of expressed ßAR subtypes were determined in membranes
prepared from isolated mature adipocytes by radioligand binding
experiments using [125I]CYP as radioligand. At a
concentration of 380 pM [125I]CYP, which
saturates ß1- and ß2AR, but not
ß3AR, 52 ± 10 fmol ß-adrenergic binding sites/mg
protein were detected. At a concentration of 1850 pM
[125I]CYP, which saturates all three ßAR, 224 ±
79 fmol binding sites/mg protein were detected. These results indicate
that the ß3AR is the most abundant subtype in mature
human brown adipocytes.
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). Saturation binding experiments with
[125I]CYP revealed a predominant low affinity receptor
population (24). The Kd value of 520 pM was
characteristic for ß3AR. To quantify ß1-
and ß2AR-binding sites in these cells, we conducted
competition binding experiments in the presence of the
ß1AR-selective ligand CGP20712A and the
ß2AR-selective ligand ICI118551. A
[125I]CYP concentration of 50 pM was used in
these studies to minimize interference with the predominant low
affinity ß3AR population (Table 1). Competition curves for both ligands
were biphasic. The Ki values of high affinity binding sites
were in good agreement with values reported for ß1- and
ß2AR in transfected cell lines (see reference values in
Table 1) (5, 28). A difference in affinity of 4 orders of magnitude
between high and low affinity sites was observed for both ligands.
Using a concentration of 200 pM [125I]CYP,
which saturates ß1- and ß2AR, and 0.1
µM CGP20712A or 0.1 µM ICI118551, to
displace the radioligand from all ß1- or
ß2AR binding sites, we determined that the densities of
ß1AR and ß2AR subtypes were, respectively,
5 ± 3 and 15 ± 5 fmol/mg protein. At a concentration of 1.5
nM [125I]CYP, corresponding to 3 times the
calculated Kd for ß3AR in PAZ6 adipocytes
(24), 82 ± 15 fmol ß3AR/mg protein were detected in
the presence of 0.1 µM CGP20712A and ICI118551 (n =
7). Thus, as in mature brown adipocytes, PAZ6 cells express a majority
of ß3AR-binding sites, accounting for 75–80% of the
total ßAR.
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). A strong increase
in cAMP concentration was promoted by the natural ligand
norepinephrine, at a concentration that activates all three ßARs.
Isoproterenol, a specific ßAR agonist, promoted a similar cAMP
response, confirming the presence of functionally coupled ßARs in
these cells. In cells pretreated with norepinephrine for up to 7
h, cAMP levels were significantly lower than those measured after a
first short stimulation. Restimulation of the same cells with forskolin
promoted a maximal cAMP response, indicating that adenylyl cyclase
itself was still functional despite the sustained stimulation of ßAR
in culture. No detectable accumulation of cAMP over basal levels,
however, was observed in adipocytes using CGP12177A, a
ß3AR-specific partial agonist, which at the same time
antagonizes ß1- and ß2AR.
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). These cells tested positive for the expression of the
BAT-specific marker UCP1 (data not shown). Short term stimulation with
either norepinephrine or isoproterenol of primary adipocytes caused an
increase in intracellular cAMP levels, whereas CGP12177A had no
measurable effect. After a 24-h pretreatment with norepinephrine,
intracellular cAMP levels were not significantly different from basal
levels in primary adipocytes. In conclusion, the ß-adrenergic
response is desensitized in isolated adipocytes and the
ß3AR is not productively coupled to adenylyl cyclase.
In PAZ6 adipocytes, norepinephrine and isoproterenol promoted a 10-fold
increase in cAMP concentrations above basal values, which was
comparable to the effect obtained by the direct stimulation of adenylyl
cyclase with forskolin (Fig. 3). The
dose-response curve with isoproterenol showed an EC50 of
approximately 80 nM (Fig. 4A). The same concentration was used in
competition experiments in the presence of increasing amounts of ßAR
antagonists (Table 2). Inhibition by the
ßAR-nonselective antagonist bupranolol was monophasic, complete, and
of high affinity. Inhibition by CGP20712A (ß1AR
selective) and ICI118551 (ß2AR selective) was biphasic.
High affinity IC50 values were in the nanomolar range and
were separated by 3 orders of magnitude from the IC50 of
low affinity binding sites. ß1AR and ß2AR
contributed, respectively, to 20 ± 6% and 70 ± 22%
(n = 3) of the isoproterenol-promoted cAMP accumulation in PAZ6
adipocytes (Table 2). Short term incubation with the
ß3AR-selective partial agonist CGP12177A caused a small,
but significant, and dose-dependent increase in cAMP levels up to
1.91 ± 0.18 times the basal value (n = 10; P
< 0.05), with an EC50 of approximately 600 nM
(Figs. 3 and 4B). This value is in good agreement with values reported
in Chinese hamster ovary cells transfected with the human
ß3AR gene (29). The CGP12177A-mediated adenylyl cyclase
stimulation was completely blocked by the nonselective ß-adrenergic
antagonist, bupranolol (50 µM), but was not blocked by
the selective ß1- and ß2AR blockers
CGP20712A and ICI118551 (100 nM), confirming that CGP12177A
activates specifically ß3ARs in PAZ6 adipocytes
(1.84 ± 0.33 times the basal value; n = 4; P
= 0.05). As CGP12177A is a partial agonist with an intrinsic activity
of 0.68 for the human ß3AR, we can estimate that this
receptor accounts for approximately 10–15% of the stimulation induced
by isoproterenol in PAZ6 adipocytes.
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). Similar results were obtained after long term
stimulation with isoproterenol. After 3 h of isoproterenol
stimulation, the signal was decreased by more than 70% (2.6-fold over
basal; n = 3; P < 0.05). In conclusion, ßAR
expressed by PAZ6 adipocytes and mature human brown adipocytes display
similar responses to ß-agonists and similar levels of desensitization
after sustained activation.
Long term stimulation down-regulates ßAR in mature brown
adipocytes and PAZ6 adipocytes
Down-regulation of ßARs may participate in the desensitization
of the ß-adrenergic signal after long term exposure to the ligand
(30). The density of ßAR was determined in membranes prepared from
mature brown adipocytes immediately before cultivation and after
19 h of culture in the presence or absence of norepinephrine (Fig. 5A). In the absence of norepinephrine,
the number of ßAR in cultivated cells did not change compared with
the control value, whereas in the presence of norepinephrine, 54% of
total ßARs were down-regulated.
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). The
CGP20712A/ICI118551-sensitive population (ß1AR and
ß2AR) displayed a rapid down-regulation, with a half-life
of approximately 3 h. The CGP20712A/ICI118551-insensitive
population (ß3AR) showed a slower down-regulation, with a
half-life of approximately 30–40 h. These results are in agreement
with previous observations in transfected cell lines indicating that
the ß3AR may be down-regulated in certain cell types and
that down-regulation of the ß3AR is slower than that of
ß1- and ß2AR (12). The data show that
down-regulation of all ßAR subtypes contributes to the
desensitization of the ß-adrenergic response in human brown
adipocytes.
Desensitization of ß-adrenergic response in PAZ6 adipocytes
downstream from adenylyl cyclase
Submaximal activation of adenylyl cyclase was sufficient for full
PKA activation in several cell systems, including rat adipocytes (31, 32). To evaluate whether the desensitization of the
ß-adrenergic/adenylyl cyclase pathway observed at the level of
intracellular cAMP would also affect PKA function, we measured PKA
activity in vitro after short and long term stimulation with
ß-adrenergic ligands in PAZ6 adipocytes (Fig. 6A). Short term stimulation with
norepinephrine and CGP12177A caused 3-fold (P < 0.01)
and 1.6-fold (P = 0.02) increases in PKA activity,
respectively. After 24 h of stimulation with both ligands, PKA
activity returned to the basal level in each case and remained at this
level for the subsequent 3 days of cell stimulation.
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Norepinephrine caused a 2- to 3-fold increase (P <
0.01) in CREB phosphorylation that was comparable to that elicited by
forskolin. After long term stimulation with norepinephrine (up to 4
days), the phosphorylation level of CREB returned to basal values.
These data show that desensitization of the ß-adrenergic-dependent
adenylyl cyclase response affects downstream functional effects of
cAMP.
Desensitization of ß-adrenergic responsiveness in PAZ6 adipocytes
occurs at nanomolar concentrations of agonist
Long-term stimulation of ßAR with micromolar (saturating)
concentrations of agonist completely desensitized the ß-adrenergic
response in PAZ6 adipocytes (see Figs. 3 and 6). We investigated
whether desensitization of the ß-adrenergic response was also induced
by more physiological concentrations of agonists. Dose-response
experiments studying cAMP accumulation promoted by isoproterenol were
performed with untreated PAZ6 adipocytes and cells pretreated for 45
min or 24 h with the same concentration of agonist (Fig. 7). All concentrations of isoproterenol
capable of stimulating cAMP accumulation also caused a partial
desensitization after 45 min and a complete desensitization after
24 h of preincubation. These results demonstrate that even
nanomolar concentrations of agonist are sufficient to promote a
complete desensitization of the ß-adrenergic response after long-term
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human adipocytes were collected from the peritumoral brown fat in a patient undergoing surgery for removal of a pheochromocytoma. This peritumoral fat is a source of organized BAT in adults (27). Brown adipocytes isolated from this tissue expressed mRNAs of UCP1 and the ß3AR. Binding studies with [125I]CYP on these cells showed the presence of two populations of ßAR-binding sites: high affinity binding sites, corresponding to ß1- and ß2AR, and low affinity binding sites, corresponding to the ß3AR. The latter accounted for more than 75% of the total sites. PAZ6 adipocytes showed very similar proportions of high and low affinity binding sites (20% and 80%, respectively). High affinity sites were further characterized in PAZ6 adipocytes with selective antagonists in competition binding experiments; approximately 75% of them corresponded to ß2AR, and 25% of them corresponded to ß1AR. Similar observations were made in human WAT membranes, where ß3AR was the predominant ßAR subtype (R. Jockers and P. de Coppet, unpublished observations), and in human newborn BAT, where substantial quantities of ß3AR protein were reported (33).
Coupling of ßAR to its principal effector, adenylyl cyclase, was investigated in PAZ6 adipocytes and mature brown adipocytes after stimulation with various agonists. Norepinephrine, the natural hormone, was used in most experiments at a concentration sufficient for maximal stimulation of all subtypes (the stimulation observed in the presence of the most powerful agonist, isoproterenol). CGP12177A was used as the most selective marker currently available for human ß3AR activity. Despite the fact that this ligand is only a partial agonist toward the ß3AR, it is a potent antagonist of both ß1- and ß2AR (34). Recent preliminary data obtained in human WAT and heart tissue led the researchers to the assumption that CGP12177A might act on additional targets in these tissues (35, 36).
Mature brown adipocytes were cultivated for more than 12 h before functional assays to allow receptors to recover from any interference caused by the high level of catecholamines and ß-blocking medications present in the donor’s blood. After incubation with norepinephrine or isoproterenol, a large accumulation of cAMP was observed in mature brown adipocytes, whereas CGP12177A did not promote any significant signal. The lack of effect of CGP12177A was not caused by desensitization of the ß3AR pathway in vivo, as this compound was also ineffective in primary adipocytes differentiated in vitro from adipocyte precursor cells, a process that takes approximately 2 weeks after cell isolation. Recently, it was reported that, in human adipocytes differentiated in vitro, transcription of UCP1 mRNA could be induced by ß3-adrenergic agonists and that this phenomenon was cAMP dependent. (37). In this study, the researchers quantified transcriptional effects promoted by ß3-adrenergic agonists using a RT-PCR-based technique. RT-PCR is presumably more sensitive than our assay based on determination of intracellular cAMP. The absence of measurable cAMP accumulation in mature adipocytes and in in vitro differentiated adipocytes incubated with CGP12177A in our study could be due to the small number of cells available for assays. Under our experimental conditions, very low concentrations of cAMP were difficult to measure. Even if the ß3AR can promote some cAMP accumulation in mature brown adipocytes, its coupling to adenylyl cyclase is much less effective than that of the other two subtypes.
ß3AR-dependent cAMP accumulation was also very weak in PAZ6 adipocytes upon CGP12177A stimulation compared with that caused by norepinephrine and isoproterenol. Thus, in both mature human brown adipocytes and PAZ6 adipocytes, ß3AR are poorly coupled to the adenylyl cyclase pathway despite the fact that they represent the predominant ßAR subtype. Our results are consistent with previous studies of WAT from human and primates, indicating that, contrary to what is observed in rodents (2), ß3AR expressed in this tissue have a minor effect on lipolysis (19, 20).
Based on the observation that the ß3AR is not subject to desensitization after exposure to agonists in transfected fibroblasts, it was suggested that ß3AR responsiveness may remain functional while ßAR signals mediated by ß1- and ß2AR subtypes are desensitized (11, 12, 13, 14, 15). In addition, it was reported that long term administration of a selective ß3AR agonist to rodents and dogs may cause hypertrophy of BAT, indicating the existence of a long lasting ß3AR receptivity in vivo (7). However, in both mature human brown adipocytes and PAZ6 adipocytes, a few hours of incubation with 10 µM norepinephrine or CGP12177A caused a significant desensitization of the system; subsequent stimulation with the same agonists promoted a much lower increase in cAMP levels. Freshly isolated mature adipocytes cannot be maintained in culture for more than 24 h. PAZ6 adipocytes and adipocytes differentiated in vitro from isolated adipocyte precursor cells were used to study the effect of longer periods of stimulation with ßAR agonists. After 24-h stimulation, ligands could not cause any increase in cAMP concentrations over basal levels, and upon longer stimulations, desensitization persisted throughout the 4 days of the experiment. Receptor down-regulation is at least in part responsible for desensitization of the ß3AR/adenylyl cyclase pathway in PAZ6 adipocytes. This phenomenon was documented in transfected fibroblasts and mouse BAT (12, 38).
It has been reported that submaximal activation of adenylyl cyclase and a modest increase in cAMP concentrations may be sufficient to fully activate PKA in transfected Chinese hamster fibroblast cells and rat adipocytes (12, 31, 39). We investigated whether this phenomenon would occur in PAZ6 adipocytes. After long term ß-adrenergic stimulation, neither PKA activity nor the phosphorylation of one of its substrates, CREB, was any longer increased compared with basal values in PAZ6 adipocytes. These results indicate that desensitization of the ß-adrenergic response caused by long term stimulation with pharmacological concentrations of agonists affects molecular targets downstream from adenylyl cyclase, such as PKA and CREB.
In the experiments discussed above, pharmacological, saturating concentrations of agonists were used to fully stimulate receptors. Saturating concentrations of agonists are known to activate more desensitization mechanisms than subsaturating concentrations, which do not occupy all receptors. For example, GRKs are known to phosphorylate, and thus desensitize, only receptors occupied by their ligand and not resting receptors (40). We tested whether lower concentrations of ligand, below the activation constant (Kact), might activate the adenylyl cyclase pathway in PAZ6 adipocytes without causing a complete desensitization of the system. Even at low (nanomolar) concentrations of agonist, the ß-adrenergic/adenylyl cyclase pathway was completely desensitized.
In conclusion, we have shown that PAZ6 adipocytes are a good model of human brown adipocytes in terms of functional response to catecholamines, activation of adenylyl cyclase pathway, ßAR subtype composition, and regulation. In both PAZ6 adipocytes and mature human brown adipocytes, ß3AR plays a minor role in activating the adenylyl cyclase pathway. In addition, long term stimulation of the ß3AR failed to maintain the adenylyl cyclase response once ß1AR and ß2AR subtypes were desensitized. From these results, it seems unlikely that long term administration of ß3AR agonists to obese patients would stimulate adenylyl cyclase-dependent thermogenesis in BAT. However, hypertrophy of BAT has been documented in patients with pheochromocytoma and in dogs treated with ß3AR-selective agonists. These observations suggest that ßARs in general and the ß3AR in particular may activate signaling pathways different from the adenylyl cyclase system, which escape desensitization in vivo. It is interesting to note that the ß3AR, the most abundant ßAR subtype in PAZ6 adipocytes and mature brown adipocytes, is poorly coupled to adenylyl cyclase in these cells. It has also been shown that the ß3AR activates enzymes involved in cell proliferation, such as protein kinase B and glycogen-synthase kinase-3, in rat adipocytes in a cAMP-independent manner (41). Further studies will investigate whether the ß3AR is coupled to other pathways in human brown adipose cells and whether these may contribute to promote BAT hypertrophy and consequently increase energy expenditure.
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Received December 2, 1997.
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coupling efficiency in brown-adipose-tissue plasma
membranes from cold-acclimated hamsters. Biochem J 295:655–661
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), by ICI118551 and CGP20712A in
untreated cells. *, P < 0.01 compared with
untreated cells. Data are the mean ± SEM of five
independent experiments carried out in triplicate.
) with different concentrations of isoproterenol and stimulated for
15 min with the same ligand concentration in the presence of 1
mM IBMX. cAMP levels were determined as described in
Materials and Methods. Data are the mean ±
SEM of three independent experiments performed in
duplicate.