From the
Immortalized brown adipocyte cell lines derived from a mouse
hibernoma express all three
The major function of the white adipocyte in mammals is to serve
as a storage depot for excess metabolic energy that can be retrieved
during periods of net caloric insufficiency. In addition to white
adipocytes, brown adipose tissue contains a specialized adipocyte which
stores triglycerides as an energy source dedicated to fueling
nonshivering thermogenesis, a process important for regulating body
temperature and composition (lean versus fat mass)
(1, 2) . Catecholamines play a major role in regulating
both of these processes. In both white and brown adipocytes,
catecholamine activation of
The lack of adequate cell culture models has hindered
progress in examining these biochemical aspects of brown adipocyte
physiology. Several short term brown adipocyte primary culture systems
have been developed
(15, 16, 18, 25, 26, 27) .
However, these cultures show only transient expression of Ucp,
and attempts to establish cell lines from normal stromal vascular cells
derived from BAT have failed. In addition, since some of these cultures
were reported to require glucocorticoids in order to differentiate
(26, 28) , and these steroids by themselves have been
shown to modulate
Recently, immortalized mouse brown adipocyte cell lines have been
established which appear to retain tissue-specific characteristics of
brown adipose tissue, including norepinephrine-induced Ucp expression and cell proliferation
(31) . In these studies
we have characterized the
Reverse
transcriptase-PCR analyses were performed using a GeneAmp RNA PCR Kit
from Perkin-Elmer according to the manufacturer's instructions.
Moloney murine leukemia virus reverse transcriptase was used to
synthesize the cDNA for amplification. The oligonucleotide primers were
designed according to Nahmias et al.(7) . For the
The expression of individual
The functional activity
of the
Since this immortalized brown adipocyte cell line
contains all three
We have characterized
We have also shown in these studies that at least three different
forms of adenylyl cyclase are expressed in white and brown adipocyte
cell lines and tissues. These include adenylyl cyclase types 5 and 6,
and the recently identified type 10 enzyme.
A key
characteristic of these brown adipocyte cell lines is that they have
retained the ability to express the brown adipocyte-specific Ucp in response to
The
It has been
reported that the
The first major physiological
function of brown adipocytes that we have examined with regard to
adrenergic regulation is Ucp induction. Our experiments
demonstrate that norepinephrine at a concentration expected to activate
primarily
From our studies we
conclude that each of the
The dose-response curves shown in Fig. 5
were analyzed by nonlinear regression for best fit to a one- or
two-site model as described previously (11). K
We thank Dr. Thomas Gettys for the gift of antisera to
cAMP, Dr. Thomas Claus for the gift of CL316,243, and Drs. Marc Caron
and Robert Lefkowitz for advice and helpful discussions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-adrenergic receptor subtypes,
including
-adrenergic receptor (AR). In response to
norepinephrine, cAMP production by plasma membranes from four clonal
cell lines was stimulated to levels comparable with brown adipocytes
isolated from interscapular brown adipose tissue (72.8-89.6
versus 97.8 pmol cAMP/min/mg of protein, respectively). All
cell lines responded to the highly selective
-adrenergic receptor agonist CL316,243 by stimulating
adenylyl cyclase activity (3-10-fold over basal).
-,
-, and
-adrenergic receptor mRNA was detected by Northern
blotting and/or reverse transcriptase-polymerase chain reaction.
Competition binding assays with the antagonists CGP20712A and
I-cyanopindolol showed the proportions of
AR and
AR in immortalized cells to be
similar to brown adipocytes from tissue (cells: 35%
AR, 65%
AR; brown adipocytes from
tissue:
AR 41%, 59%
AR). Expression
of brown fat-specific mitochondrial uncoupling protein ( Ucp)
was stimulated by
-adrenergic agonists in two of the four cell
lines. The ability of individual
AR subtypes to regulate Ucp expression was examined with combinations of selective
-adrenergic agonists and antagonists. Expression of Ucp could be induced by any of the
-adrenergic receptor subtypes.
However, the greatest response was obtained by stimulating all three
-adrenergic receptor subtypes simultaneously (100 µM
isoproterenol). Incubation of membranes from cultured cells or brown
adipocytes from tissue with CL316,243 at an optimal concentration (5
µM) did not prevent norepinephrine from further
stimulating adenylyl cyclase activity, suggesting that the combined
activation of
AR/
AR, plus
AR, together produced an additive cAMP response.
Multiple forms of adenylyl cyclase were identified in brown and white
adipocyte cell lines and tissues. Northern blot analysis detected
adenylyl cyclase types 5, 6, and 10. Screening of reverse
transcriptase-PCR products by DNA sequencing confirmed the identities
of these forms and lower levels of additional isoforms, raising the
possibility that
-adrenergic receptor subtypes in adipocytes
couple to distinct adenylyl cyclases. Because these cell lines display
functional and phenotypic similarities to interscapular brown
adipocytes, they will be a useful model to study the regulation of
-adrenergic receptor expression and function, and the control of
Ucp expression and activity.
-adrenergic receptors
(
ARs)
(
)
1 and stimulation of the
intracellular cAMP pathway mediate this metabolic regulation
(3) .
-Agonist stimulation of brown adipocyte thermogenesis
occurs in response to cold exposure
(4) or excess caloric
intake
(5) . Brown adipocytes contain a large number of
mitochondria and express, in response to
-adrenergic stimulation,
a unique mitochondrial proton channel, the ``uncoupling
protein'' ( Ucp)
(6) . This channel uncouples
mitochondrial respiration from oxidative phosphorylation to dissipate
energy as heat instead of synthesizing ATP. All three
AR subtypes,
including the adipocyte-specific
AR, are expressed in
white and brown adipocytes
(7, 8, 9, 10, 11) , and each is
coupled to the stimulation of adenylyl cyclase with the consequent
elevation of intracellular cAMP levels.
-Adrenergic agonists have
been shown to stimulate lipolysis
(5, 12, 13, 14) , Ucp expression
(6, 15, 16) , and proliferation
(17, 18) of brown adipocytes. In particular,
AR and
AR have been implicated in
cell proliferation and Ucp induction, respectively
(15, 18, 19, 20, 21, 22) ,
but the relationships between the expression and function of
AR
subtypes, and their roles in brown adipocytes capable of proliferation
versus those that are developmentally mature and able to
express Ucp, are not understood. Moreover, factors that
modulate
AR expression such as
-adrenergic agonists and
corticosteroids
(23, 24) , which may consequently affect
Ucp expression, have not been investigated in brown adipose
tissue.
AR expression
(23, 29, 30) , cell lines dependent upon these
agents may be unsuitable for studies of
AR function in adipocytes.
ARs and their signal transduction
properties in four of these clonal cell lines by Northern blotting and
-agonist-stimulated adenylyl cyclase activity and compared these
data to results obtained from mouse brown adipocytes derived from
interscapular BAT. Two of the four clones examined express all three
AR subtypes and stimulate Ucp transcription in response
to
-agonists. Thus, these cell lines provide a new tool for
examining the mechanisms involved in
-adrenergic regulation of
brown adipocyte differentiation and Ucp expression, and the
interactions among the three
AR subtypes coupled to the same
second messenger pathway.
Animals
Genetically lean (+/+)
C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor,
ME). They were housed at the Duke University Vivarium at 20-22
°C and fed Purina mouse chow and water ad libitum. Animals
were killed at 11-12 weeks of age by intraperitoneal nembutal
injection (5 mg in 0.1 ml), and interscapular brown adipose tissue was
collected for the isolation of total RNA and preparation of plasma
membranes. All procedures were conducted in accordance with principles
and guidelines established by the National Institutes of Health for the
care and use of laboratory animals.
Cell Culture
Brown adipocyte cell lines were
derived from a mouse brown adipose tumor as described previously
(20, 31) . Fig. 1shows a phase contrast
photograph of clones B3 ( A) and B7 ( B). The cells
were grown in Dulbecco's modified Eagle's medium
supplemented with D-biotin (33 µM), ascorbate
(100 µM), calcium pantothenate (17 µM), fetal
bovine serum (10%), penicillin (10 units/ml), streptomycin (0.1 mg/ml),
and fungizone (0.125 mg/ml) (Life Technologies, Inc.) in a humidified
atmosphere of 5.5% CO, 94.5% air. Cells maintained in this
standard medium contain multilocular lipid droplets, lipid synthesizing
enzymes, and all
AR subtypes. The adipogenic characteristics of
the cells can be enhanced by the addition of insulin (50 nM)
and T3 (0.5 nM)
(31) .
Figure 1:
Photomicrographs of B3 and B7 brown
adipocyte cells, showing the accumulation of lipid droplets and the
morphology of the cells. A, B3 cells; B, B7
cells.
Isolation and Analysis of RNA
Total cellular RNA
was prepared by the cesium chloride gradient method as previously
detailed
(32) . For Northern blot hybridization, RNA was
denatured by the glyoxal procedure, fractionated through 1.2% agarose
gels, and blotted onto Biotrans nylon membranes (ICN, Irvine, CA) as
described previously
(23) . DNA fragments that were used as
hybridization probes were obtained from the following sources. For the
three AR subtypes, fragments specific for each receptor were
prepared by polymerase chain reaction as described previously
(11) . The cDNA fragment for ADD1, a transcription factor
associated with adipocyte differentiation and highly expressed in BAT,
was generously provided by Dr. Bruce M. Spiegelman
(33) . For
the mitochondrial Ucp, a 300-base pair BglI fragment
was used
(34) . The probe for the lipogenic enzyme
glycerol-3-phosphate dehydrogenase was a 0.8-kilobase pair
HindIII fragment from exon 2 of the Gdc-1 gene
(35) .
The cDNA fragment for the ubiquitous glycolytic enzyme glyceraldehyde
3-phosphate dehydrogenase was obtained from Clontech (Palo Alto, CA).
DNA fragments specific for individual isoforms of adenylyl cyclase were
generated by PCR using domain 2 primers as described
(36) .
Radiolabeled probes were prepared by nick translation of the purified
DNA fragments in the presence of
[
-
P]deoxy-CTP to a specific activity of
between 3 and 7
10
dpm/µg DNA. Blots were
hybridized and washed as described
(11, 23) .
AR, the sense strand primer (5`-GCTCCGTGGCCTCACAG-3`)
and antisense primer (5`-CTCGGCATCTGCCCCTA-3`) correspond to
nucleotides 572-588 and nucleotides 1098-1114,
respectively, of the mouse
AR gene, as reported by
Nahmias et al.(7) and van Spronsen et al.(37) . Prior to amplification total cellular RNA from mouse
brown adipocyte cell lines and mouse brown adipose tissue was incubated
in a 20-µl reaction containing 50 mM Tris, 10 mM
MgCl
, and 1 unit of RNase-free DNase (U. S. Biochemical
Corp.) for 30 min at 37 °C. The DNase was denatured by heating to
95 °C for 5 min. The RNA (250 ng) was then annealed to the
antisense primer in a 20-µl reaction containing 5 mM
MgCl
and reverse-transcribed at 42 °C for 15 min,
followed by incubation at 99 °C for 5 min to denature the enzyme.
After addition of the sense strand primer, 2.5 units of AmpliTaq
polymerase and 10
PCR buffer (final concentrations: 10
mM Tris, 50 mM KCl, 1.5 mM
MgCl
), the PCR was initiated (95 °C for 30 s, 58 °C
for 30 s, and 72 °C for 1.5 min; 40 cycles). Amplification
reactions without the initial reverse transcriptase step were included
as controls for all samples. Mouse genomic DNA (500 ng) was amplified
as a positive control, and water was amplified as a control for reagent
contamination. Ten percent of each PCR reaction mixture was applied to
a 3% (w/v) MetaPhor agarose gel (FMC, Rockland, ME) and stained with
ethidium bromide (1 µg/ml). For reverse transcriptase-PCR analysis
of adenylyl cyclase isoforms, primers were prepared to sequences in
domain 2 that are conserved among all the isoforms and amplified as
described
(36) . Amplified products were isolated by gel
electrophoresis and subcloned into PCRII TA cloning vector (Invitrogen;
San Diego, CA). Individual clones were selected, grown, and plasmid DNA
was prepared and sequenced from vector primers (T7 and SP6) using
[
-
P]dATP and Sequenase (U. S. Biochemical
Corp.).
Preparation of Plasma Membranes and Adenylyl Cyclase
Assay
Brown adipocytes were isolated from interscapular brown
adipose tissue by collagenase digestion as described by Rodbell
(38) with slight modifications. The tissue was minced and then
incubated in Dulbecco's modified Eagle's medium
supplemented with 20 mg of bovine serum albumin/ml and 0.24 mg/ml
collagenase type I (Worthington Biochemical, Freehold, NJ) for
30-45 min with shaking at 37 °C. Plasma membranes were
prepared from the purified brown adipocytes and from the brown
adipocyte cell lines as described in earlier studies
(11) .
Adenylyl cyclase activity was assessed in these plasma membrane
preparations by measuring the cAMP formed by radioimmunoassay
(39) using a polyclonal antiserum produced as described
(40) . (-)-Norepinephrine, (-)-isoproterenol,
salbutamol, and (-)-propranolol were obtained from Sigma.
CGP20712A was a gift from Ciba-Geigy (Summit, NJ), BRL37344 was a gift
from Smith-Kline Beecham (Surrey, United Kingdom), and CL316,243 was a
gift from American Cyanamid Company (Pearl River, NY). ICI118,551 was
purchased from Cambridge Research Biochemicals (Wilmington, DE).
Protein concentrations were determined by the method of Bradford
(41) .
Competition Binding Assays to Determine Proportion of
Binding assays were
performed on membranes prepared from purified interscapular brown
adipocytes and brown adipocyte cell lines according to established
methods
(11, 23) . The 25 mM Hepes-buffered
incubation media contained 30 µg of membrane protein, 30
pMAR and
AR
I-cyanopindolol (CYP) (DuPont NEN), 5
µM CL316,243, 10 mM MgCl
, and
indicated concentrations of various unlabeled competing ligands. The
AR-selective agonist CL316,243 was included to block
I-CYP binding to a small, but significant, percentage of
AR (see legend to Fig. 6). Following incubation
at 37 °C for 1 h, the suspensions were filtered through Whatman
GF/C filters and rapidly washed four times with ice-cold 0.5
phosphate-buffered saline. The radioactivity remaining on the filters
was counted.
Figure 6:
Competition binding analysis to determine
proportions of AR versus
AR
in membranes from purified interscapular BA and B7 adipocytes. Plasma
membranes prepared from B7 adipocytes or purified mouse brown
adipocytes were incubated for 1 h at 37 °C with 30 pM
I-CYP and increasing concentrations of unlabeled
(-)-propranolol (
) or CGP20712A (
). Five
µM CL316,243 was also included in all tubes to eliminate
I-CYP binding to a small percentage of
AR (
, inset). Membrane-bound
I-CYP was collected on filters and counted. Data are
expressed as B/ B
, the fraction of total
counts bound in the presence of the competing
ligand.
Data Analysis
Adenylyl cyclase dose-response
curves and competition binding curves were analyzed by least squares
nonlinear regression and assessed for best fit to a 1-component or
2-component model (Graphpad Inplot, San Diego, CA and LIGAND, National
Institutes of Health, Bethesda, MD). Test of the adequacy of the 1-
versus 2-component model was by an F-test. Comparisons between
data sets were made by analysis of variance. A value of p <
0.05 was considered significant.
AR subtypes in four brown
adipocyte cell lines was evaluated by Northern blot hybridization
(Fig. 2). Comparable levels of
AR mRNA
transcripts were detected in all four cell lines (B13 not shown) as
well as from interscapular brown adipose tissue (IBAT). The estimated
size of the
AR transcript (2.6 kb) is similar to
previous reports in a variety of tissues including adipocytes
(11, 30, 42) .
AR mRNA was
expressed in all the cells and brown adipocytes derived from fresh
tissue. The presence of two transcripts for
AR, a
major 2.4-kb and a minor 2.0-kb species, is similar to our earlier
observations
(11, 23) . Since these data are from clonal
cell lines and purified adipocytes isolated from tissues, it is
apparent that the presence of
AR mRNA is not simply
due to contamination from surrounding vasculature and neurons. The
expression of
AR mRNA, the
AR subtype that is
expressed predominantly in adipose tissue
(7, 8, 9, 11, 13) , varied the
most among the cell lines. By Northern blotting we detected
AR mRNA in the B3 and B7 cells, but not in B9 or B13
cells. Fig. 2shows that
AR mRNA is most
abundant in the B7 cell line and, as we observed previously in white
and brown adipose tissue
(11) , consists of three distinct
AR transcripts: a major species of 2.1 kb and minor
transcripts of 2.6 and 3.5 kb. Although we did not detect
AR mRNA in the B9 and B13 cells by Northern blotting,
AR-specific amplification products were identified in
all four cell lines by reverse transcriptase-PCR (Fig. 3). Since
B9 and B13 cells also generated a modest response to a highly selective
AR agonist (see below), these data together suggest
that all four cell lines express
AR, with the highest
levels of functional
AR in B3 and B7 cells.
Figure 2:
Northern blot of brown adipocyte cell
lines (B3, B7, B9) and IBAT. Forty µg of total cellular RNA from
IBAT or adipocytes that had been incubated in the absence (-) or
presence (+) of 10
M norepinephrine
( NE) were fractionated through 1.2% agarose gels and blotted
onto nylon membranes as described under ``Materials and
Methods.'' Blots were hybridized with
-
P-labeled
DNA probes (3.0
10
dpm/ml) for the mouse
-adrenergic receptor subtypes (
AR,
AR, and
AR) and selected adipose
tissue gene products such as uncoupling protein ( Ucp), IBAT
transcription factor ADD1, and glycerol-3-phosphate dehydrogenase
( GPDH). Blots were also hybridized with the ubiquitous
glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase
( GAPDH) as an internal control for the amount of RNA applied
to the gel. Final washing conditions were 0.1
SSC, 0.1% SDS at
55 °C.
Figure 3:
Identification of AR
transcript by reverse transcriptase-PCR. Total cellular RNA (250 ng)
from brown adipocyte lines (B3, B7, B9, B13) and IBAT was annealed to
the antisense primer and reverse transcribed with Moloney murine
leukemia virus reverse transcriptase. The cDNA was amplified by PCR in
the presence of 1.5 mM MgCl
as described under
``Materials and Methods'' ( + lanes). Control
PCR reactions containing 500 ng of mouse genomic DNA ( DNA
lane) or RNA without the initial reverse transcriptase step
( - lanes), were included as positive and negative
controls, respectively. A water blank was also reverse-transcribed and
amplified as a control for reagent contamination
( H
0 lane). The arrow indicates
the predicted fragment size of 542 base pairs. Lane M contains
HaeIII-digested
X174 DNA molecular weight markers
ranging from 1353 to 271 base pairs.
We next
examined these cell lines for the expression of gene products
characteristic of adipocytes and of brown adipocytes in particular. As
reported in the initial characterization of the original brown fat
tumor cells from which these lines were derived
(20, 31) , the brown fat-specific uncoupling protein
( Ucp) was expressed only in response to AR agonists. The
lower portion of Fig. 2shows that Ucp expression was
readily induced in the B7 and B9 cells by 10
M norepinephrine for 4 h ( ``+'' lanes). By contrast, Ucp was undetectable in the B3 or
B13 cells, at least by Northern blot analysis. The level of Ucp expression was greater in the B7 than the B9 cells but was still
significantly less than in IBAT. For example, although equal amounts of
RNA were analyzed for each sample, the IBAT lane was exposed to film
for 8 h and the adipocyte lanes were exposed for
5 days. All four
cell lines also contained abundant levels of mRNA for ADD1, a
transcription factor preferentially expressed in brown adipose tissue
(33) . Expression of the lipogenic enzyme glycerol-3-phosphate
dehydrogenase (GPDH) was also present, with similar levels of GPDH mRNA
in the cell lines and IBAT. Although the expression of these markers
was not significantly changed by the presence of 10
M norepinephrine for 4 h, it is interesting to note that
the expression of the
AR transcript was slightly
elevated by norepinephrine in B7 cells ( n = 2). On the
other hand, norepinephrine also appeared to decrease the expression of
the
- and
AR transcripts in the B3
and B7 adipocytes ( n = 3).
ARs in the brown adipocyte lines was assessed by the
ability of
-agonists to stimulate adenylyl cyclase activity and
compared with that observed in brown adipocytes (BA) purified from IBAT
(Fig. 4). Since preliminary experiments performed in intact cells
and plasma membranes gave essentially identical results, we chose to
use plasma membranes in these experiments for greater sampling
consistency. The stimulation of adenylyl cyclase activity in BA
membranes by norepinephrine, the predominant endogenous ligand for
ARs in brown adipose tissue, was complex and best fit to a
two-site curve. The high-affinity component of the curve, with a
K
of 5.2
10
M, accounted for 15.2% of the adenylyl cyclase activity,
with the remaining 84.8% from a lower affinity component
( K
= 4.2
10
M). Dissection of the norepinephrine dose-response
curves into components which could be attributed to all three
AR
subtypes is difficult, since there is less than a 10-fold difference in
the reported K
for norepinephrine to stimulate
adenylyl cyclase by the individual
ARs
(43) . The brown
adipocyte cell lines responded to norepinephrine with similar maximal
levels of stimulation (ranging from 72.8 to 89.6 pmol of cAMP/mg of
membrane protein/min). Among the four cell lines, the greatest maximal
response was achieved in the B7 and B3 cells and was slightly less than
that in the BA membranes. The K
of the adipocyte
lines ranged from 1.6
10
M to 5.1
10
M. Although the results from B7
cells appeared to fit a two-site model as in the BA from tissue, they
were not statistically significant ( p = 0.075). The
data from cell lines B3, B9, and B13 were best fit to a single
component. Interestingly, the BA consistently displayed a significantly
higher basal adenylyl cyclase activity than the BAT cell lines.
However, although BA was capable of a greater maximal stimulation, the
cell lines actually produced a higher degree of agonist stimulation
over basal activity (B3, 14.4-fold; B7, 17.6-fold; B9, 11.3-fold; B13,
10.4-fold) than the BA membranes (3.8-fold). The reason for this
difference is not immediately clear.
Figure 4:
Norepinephrine-stimulated adenylyl cyclase
activity in membranes from purified interscapular BA and brown
adipocyte cell lines (B3, B7, B9, B13). The assays contained
10-12 µg of adipocyte membrane protein and were incubated at
37 °C for 10 min. The cAMP produced was measured by
radioimmunoassay as described under ``Materials and
Methods.'' The data are expressed as picomoles of cAMP produced
per mg of membrane protein/min of
incubation.
To evaluate more specifically
the functional activity of the AR expressed in the
brown adipocyte cell lines, we used the highly selective
AR agonist CL316,243
(44) to stimulate
adenylyl cyclase and compared the results to parallel experiments
performed in BA. The BA, B3, and B7 dose-response curves displayed good
stimulation (Fig. 5) and were best fit to a single component, as
would be expected for an agonist selective for a single receptor
subtype. The K
of B3 and B7 were comparable with
that of the BA membranes (). Once again, the basal adenylyl
cyclase activity was much higher in the BA membranes than in the cell
lines, whereas the effective -fold stimulation over basal activity was
greater in the cell lines (B3, 13.3-fold; B7, 11.7-fold) than the BA
membranes (3.5-fold). The dose-reponse curves for the B9 and B13 cells
were considerably weaker, only 2.7- and 1.9-fold over basal,
respectively. These results in the B9 and B13 lines are consistent with
the finding that only a small amount of
AR is present
in these cells and indicate that the amount of functional
AR in these cells is proportional to the level of the
AR mRNA.
Figure 5:
Stimulation of adenylyl cyclase activity
by the AR-selective agonist CL316,243 in membranes
from purified interscapular BA and brown adipocyte cell lines (B3, B7,
B9, B13). The assays contained 10-12 µg of adipocyte membrane
protein and were incubated at 37 °C for 10 min. The cAMP produced
was measured by radioimmunoassay as described under ``Materials
and Methods.'' The data are expressed as picomoles of cAMP
produced per mg of membrane protein/min of
incubation.
The B7 cell line is most similar to IBAT
according to the characterization of the cell lines by Northern blot
analysis and adenylyl cyclase responses using AR agonists. Thus,
this cell line was chosen for further characterization. Our data on
functional activity and Northern blots indicate the presence of all
three
AR subtypes. Although a high-affinity radioligand capable of
quantitating
AR in tissue preparations is not
available, we determined the relative proportions of
-
and
AR that comprise the high-affinity component of
adenylyl cyclase stimulation
(11) . For this purpose competition
binding assays were performed using the radioligand antagonist
I-CYP (30 pM) and the
AR-selective antagonist CGP20712A, which displays
1000-fold selectivity for
AR over
AR
(45) . In these experiments, shown in
Fig. 6
, 5 µM CL316,243 was included in all tubes to
eliminate binding of
I-CYP to a small but significant
fraction of
AR sites (see insets to
Fig. 6
). The nonselective
/
AR
antagonist (-)-propranolol inhibited
I-CYP binding
with similar potency in the B7 and BA brown adipocyte plasma membranes
(3.9
10
M and 6.9
10
M, respectively). In both BA and B7
membranes the
AR-selective antagonist CGP20712A
produced distinctly biphasic curves. The high-affinity component of the
curve, defined as
AR, accounted for 35% of the binding
sites in the B7 plasma membranes with an IC
of 8.0
10
M. The remaining sites (65%) comprised a
low affinity component (IC
10.5
10
M); this 1000-fold weaker affinity for CGP20712A
identifies this second component as the
AR
(45) . The relative proportions of
- and
AR and the IC
values for the brown
adipocyte plasma membranes were similar to that of the B7 cells. The
high-affinity
AR component of the curve had an
IC
of 5.9
10
M and
accounted for 41% of the binding sites, and there was a slightly
smaller proportion of
AR in the brown adipocytes (59%)
with an IC
of 15.5
10
M.
AR subtypes, like brown adipocytes derived from
fresh tissue, we investigated the functional significance of these
three receptor subtypes for the regulation of adenylyl cyclase
stimulation and Ucp expression. Cyclic AMP production mediated
through the
AR is distinguishable from that generated
by activation of
- and
AR with the
highly selective
AR agonist CL316,243. We examined
whether stimulation of individual
AR subtypes alone or in
combinations produced additive or nonadditive responses. To determine
the contribution of individual
ARs to total adenylyl cyclase
activity, BA and B7 membranes were stimulated by norepinephrine alone,
CL316,243 alone, or by norepinephrine in the presence of a saturating
concentration (5 µM) of CL316,243. In both BA and the B7
cell line, norepinephrine stimulated adenylyl cyclase activity to a
greater extent than the
AR agonist CL316,243
(Fig. 7). These results are essentially identical to the data in
Figs. 4 and 5. The data indicate that stimulation of all three
AR
subtypes with norepinephrine leads to a greater activation of adenylyl
cyclase activity than stimulation of
AR alone with
CL316,243. Interestingly, in both BA and the B7 cell line, when 5
µM CL316,243 was included in all tubes, thus raising the
base-line activity to the maximal stimulation achieved with CL316,243
alone, the maximal response to norepinephrine under these conditions
was essentially identical to that of norepinephrine alone. These data
suggest that adenylyl cyclase stimulation through multiple
AR
subtypes is additive, in the sense that the same maximum level is
reached by any route. The ability of
AR, together with
AR and
AR, to produce this additive
cAMP response, in both cell lines and brown adipose tissue, suggests
that the contribution of the
AR receptor to adenylyl
cyclase activation is distinct from that attributed to
AR or
AR. Alternatively, the
AR may be less efficiently coupled to this pathway.
Finally, our data show that a significant proportion of the functional
response to norepinephrine in either BA derived from IBAT or B7 cells
can be attributed to
- and/or
AR.
Figure 7:
Subtype-selective stimulation of adenylyl
cyclase in membranes from purified brown adipocytes from IBAT or B7
cells is additive. -Adrenergic agonist stimulated adenylyl cyclase
activity in membranes from purified interscapular BA and B7 adipocytes.
The assays contained 10-12 µg of adipocyte membrane protein
and were incubated at 37 °C for 10 min. The cAMP produced was
measured by radioimmunoassay as described under ``Materials and
Methods.'' The data are expressed as picomoles of cAMP produced
per mg of membrane protein/min of incubation.
, CL316,243;
,
norepinephrine;
, norepinephrine + 5 µM
CL316,243.
One mechanism whereby individual receptor subtypes within the same
cell may modulate the extent of adenylyl cyclase activity is through
their differential coupling to particular forms of adenylyl cyclase.
There are at least 10 distinct genes for adenylyl cyclase; some show
complex regulation by G protein or
subunits, others
are sensitive to intracellular calcium concentrations or calmodulin
(46, 47) . We screened brown and white adipose tissues
and cell lines for the expression of adenylyl cyclases by Northern
blotting (Fig. 8) and reverse transcriptase-PCR coupled with DNA
sequencing. Although it is generally difficult to detect adenylyl
cyclase transcripts by Northern blot we could identify types 5, 6, and
10 in white and brown adipose tissue and in the B7 brown adipocyte cell
line by this method (Fig. 8). Types 6 and 10 were also detected
in differentiated 3T3-F442A white adipocytes. Fig. 8presents two
different exposures of the blots for types 5 and 6. These two isoforms
appear to be more abundant in WAT than in BAT. The sizes of the
transcripts identified for type 5 (
8.3 and 6.9 kb) and type 6
(
6.0 kb) are consistent with previous estimates
(48, 49) . Adenylyl cyclase type 10 (8.3 kb), a newly
identified form that has a wide tissue distribution
(
)
was also identified in both white and brown adipose tissue
and cell lines and appears to be more abundant in BAT than WAT. These
Northern blot results were further confirmed by reverse
transcriptase-PCR analysis of B7 cells and brown adipocytes (BA)
isolated from tissue and sequencing of the PCR products. The majority
of clones analyzed from both B7 and BA encoded type 10 (14 of 18 for
B7; 13 of 14 for BA). The remaining clones were identified as types 3,
6, and 7.
Figure 8:
Expression of adenylyl cyclase isoenzymes
in white and brown adipose tissues and cell lines. Total cellular RNA
was isolated from interscapular BAT and gonadal WAT isolated from
C57BL/6J male mice, B7 brown adipocytes, and differentiated 3T3-F442
cells. Forty µg of RNA were fractionated through 1.2% agarose gels
and blotted onto nylon membranes as described under ``Materials
and Methods.'' Blots were hybridized with
-
P-labeled mouse DNA probes (3.0
10
dpm/ml) for adenylyl cyclase types V, VI, and X. The fragments
correspond to unique regions of each isozyme as detailed in Ref. 36.
Final washing conditions were 0.1
SSC, 0.1% SDS at 55 °C.
For cyclase types V and VI two exposures of the same blot are shown to
clarify the presence of weaker hybridizing
species.
Using a variety of selective agonists and antagonists we
evaluated the efficacy of individual AR subtypes to activate
Ucp transcription. In the first set of experiments
(Fig. 9 A), B7 cells were incubated with norepinephrine
(10
M). A significant stimulation of
Ucp mRNA was observed ( lane 2). This result is
essentially identical to that shown in Fig. 2. The highly
selective
AR agonist CL316,243 was also able to
promote Ucp expression ( lane 3), to a slightly
greater extent than norepinephrine. However, the most robust induction
of Ucp was observed with isoproterenol at a concentration
(10
M) which activates all three
AR
subtypes ( lane 4). Since norepinephrine at a concentration of
10
M would be expected to primarily
activate
AR
(42, 43) , these results
suggest that Ucp expression can be induced through either
- or
AR individually or through the
combined activity of all three subtypes. To explore this issue in
greater detail and examine specifically the ability of
AR and
AR to stimulate Ucp expression, we chose to use selective agonists and antagonists for
these two
AR subtypes. Fig. 9 B shows that
isoproterenol, CL316,243, and norepinephrine at 1 µM could
stimulate Ucp gene expression with about equal potency. The
nonselective
AR-/
AR-antagonist
propranolol significantly reduced the ability of norepinephrine to
induce Ucp, whereas either the
-selective
antagonist CGP20712A or the
AR-antagonist ICI118,551
partially inhibited this response. The
AR-selective
agonist salbutamol was also able to increase Ucp mRNA levels,
and this induction was blocked by ICI118,551. Epinephrine (1
µM) was also able to increase Ucp mRNA levels
(not shown). Moreover, confirming conclusions of other investigators
that Ucp induction is mediated largely by intracellular cAMP
(15, 16) , we found in other experiments that the
membrane-permeable analogue of cAMP, dibutyryl cAMP (1 mM),
and forskolin (100 µM) stimulated Ucp mRNA levels
with equal or greater potency as isoproterenol (2.8- and 7.2-fold over
isoproterenol, respectively). Together these data suggest that both
AR and
AR, in addition to
AR, are capable of mediating Ucp induction in
brown adipocytes.
Figure 9:Ucp mRNA levels in B7 adipocytes
exposed to -adrenergic agonists. The cells were either untreated
or exposed to various
-adrenergic agonists at the indicated
concentrations for 4 h. Forty µg of total cellular RNA from the
adipocytes were fractionated through 1.2% agarose gels and blotted as
described under ``Materials and Methods.'' Blots were
hybridized with
-
P-labeled DNA probes (3.0
10
dpm/ml) for Ucp or the ubiquitous glycolytic
enzyme, glyceraldehyde 3-phosphate dehydrogenase ( G3PDH), to
correct for the amount of RNA applied to the gel. Final washing
conditions were 0.1
SSC, 0.1% SDS at 55 °C. A,
representative Northern blot of B7 cell RNA. Lane 1,
untreated; lane 2, 10
M
norepinephrine; lane 3, 10
M
CL316,243; lane 4, 10
M
isoproterenol. B, analysis of ability of
AR
and
AR activation to induce Ucp expression.
Cells were treated with the indicated agonists and antagonists for 4 h,
and Ucp expression was analyzed by Northern blotting . Iso, isoproterenol (1 µM); CL, CL316,243 (1
µM); NE, norepinephrine (1 µM);
Pro, propranolol (0.1 µM); CGP, CGP20712A (0.5 µM); ICI, ICI118,551 (0.5
µM); Sal, salbutamol (1 µM). The
data are expressed as the percentage of the induction observed with
isoproterenol (1 µM) and are the average of three to four
experiments, except salbutamol, which was performed once.
Asterisk, significantly different from isoproterenol-treated
cells ( p
0.05).
-adrenergic-mediated signal
transduction and Ucp gene expression in four clonal brown
adipocyte cell lines derived from a mouse interscapular brown adipose
tumor. By RNA analysis and functional assays our results demonstrate
that all three
AR subtypes are expressed, similar to brown
adipocytes isolated from rodent and hamster brown adipose tissue. We
found transcripts for
- and
AR to be
present at equivalent levels in all four cell lines, whereas expression
of the
AR was variable. In the B7 and B3 cell lines,
AR transcripts were readily detected by Northern blot
hybridization, whereas reverse transcriptase-PCR was required to detect
AR mRNA in B9 and B13 cells. Consistent with these
findings, stimulation of adenylyl cyclase activity with the
AR-selective agonist CL316,243 produced a robust
response in B3 and B7 cells and a reduced level of stimulation in B9
and B13 cells. We also determined by competition binding that both B7
cells and brown adipocytes from tissue possess comparable proportions
of
AR and
AR. As we and others have
previously discussed
(9, 11, 50) , the lack of a
high-affinity radioligand capable of detecting the
AR
in tissue preparations precludes quantitation of this receptor subtype.
This new member
of the adenylyl cyclase gene family shows a broad mRNA tissue
distribution. Relevant to our studies, type 10 appears to be the most
abundant form present in white and brown adipose tissue, as well as in
the 3T3-F442A and B7 cell lines. It should be noted that while types 5
and 6 are structurally and functionally similar
(36, 46) , the type 10 enzyme represents a new subclass
of adenylyl cyclase, whose regulatory features have not yet been
characterized. Therefore it is possible that differential coupling of
receptors to these adenylyl cyclases will regulate distinct
physiological responses. This is the first description of the adenylyl
cyclases isoforms present in adipocytes, and it will now be important
to examine the biochemical and regulatory significance of these
isoforms in
AR-mediated adipocyte metabolism.
-adrenergic stimulation. They also display
patterns of adipocyte-specific gene expression similar to normal brown
fat. Remarkably, even though the cell lines have been derived from a
hibernoma induced by the ectopic expression of SV40 T-antigen and have
the mark of an immortalized, transformed cell, they nevertheless have
retained the ability to proliferate in response to stimulation by
norepinephrine
(31) . Based upon a number of biochemical and
functional criteria the B7 cell line, in particular, appears to closely
resemble brown adipocytes derived from tissue, thus making it a good
model for in vitro studies. For several years adipocyte cell
lines such as the 3T3-L1, 3T3-F442A, and ob17 preadipocytes have been
useful for investigating the role of hormones on gene expression during
white adipose tissue adipogenesis
(51) . Similarly, these brown
adipocyte cell lines now provide the opportunity to extend the
molecular analysis to genes critical for brown adipocyte physiology in
a manner that is not possible in vivo. An unusual and valuable
feature of these cells that distinguishes them from other white or
brown preadipocyte lines is that they display
``differentiated'' characteristics independent of cell
density and do not require treatment with glucocorticoids or agents
that affect cAMP levels, such as isobutylmethylxanthine, in order to
differentiate. The importance of this difference is that these drugs
have been shown to directly affect expression of the
ARs
(23, 24, 29, 30, 53) .
Therefore, results from such cultures that purport to examine
adrenergic control of adipocyte function must be interpreted with
caution. Also unlike our clonal lines, a brown adipocyte line (HIB 1B)
recently established by the laboratories of Spiegelman and Ricquier
(28, 52) requires a period of differentiation from a
preadipocyte state before expressing either Ucp or
AR. It is interesting that while the adipogenic
morphology of our brown adipocyte clonal lines has been found to be
quite stable
(31) , the highest degree of phenotypic variability
is associated with
AR and Ucp expression, and
these differences do not seem to vary in tandem. For example, the B3
cell line contains readily detectable
AR, but Ucp induction is essentially absent, whereas the converse is true for
the B9 clone. These findings imply that some function besides the
ability of catecholamines to stimulate the cAMP pathway is critical for
Ucp expression. Such elements might include tissue-specific
transcription factors or other downstream effectors that are targets of
the cAMP-dependent protein kinase.
-adrenergic signal
transduction system in adipocytes is complex. The tissue contains three
known
AR subtypes that are each coupled to the stimulation of
adenylyl cyclase. Moreover, as we have shown, there are at least three
adenylyl cyclase isozymes expressed in adipocytes. Activation of the
cAMP pathway, in turn, regulates lipolysis in the white adipocyte.
However, in the brown adipocyte cAMP also regulates oxygen consumption,
Ucp induction, mitochondriogenesis, and cell proliferation:
all processes required for nonshivering thermogenesis. Very little is
understood about the coupling mechanism between the receptors and these
physiological functions. Because of this complexity, it has been
unclear whether the individual
AR subtypes in brown adipocytes
stimulate a common pool of adenylyl cyclase and whether their
synergistic activation is required. For example, we do not know whether
a specific
AR subtype is coupled to a specific adenylyl cyclase to
modulate a specific function. Alternatively, the regulation of
thermogenesis may be too important to be left to only one receptor
subtype, and the signal transduction pathways in brown fat may be very
redundant. In fact, one explanation proposed for the existence of three
AR subtypes in adipocytes is the potential to circumvent the
development of tachyphylaxis, or ``desensitization,'' in
order to maintain thermogenic competence
(54) . Biochemical
evidence suggests that the three
ARs display differences in their
ability to be phosphorylated by regulatory kinases. Phosphorylation is
associated with the rapid phase of receptor desensitization
(55) , and the
AR has been reported to be the
most resistant to this desensitization
(54, 56) .
However, given the fact that several different adenylyl cyclase
isoforms with various types of regulatory modulation
(36, 46) are present in white and brown adipocytes, it is also
possible that each
AR subtype may be coupled to one or more
specific adenylyl cyclase isoforms, leading to compartmentalization of
the functional adrenergic responses. In this regard, we note that
Hollenga et al.(57) reported that the
AR-selective agonist BRL37344 was substantially weaker
than isoproterenol to raise intracellular cAMP levels in rat white
adipocytes, but BRL stimulated lipolysis more efficiently. One
interpretation of their finding is that
AR might
activate some other signal transduction pathway in addition to adenylyl
cyclase. However, it is also possible that the
AR is
coupled to a particular adenylyl cyclase isoform that generates a more
effective, functional, second messenger response. In our studies using
both subtype-selective and nonselective
-agonists, our data are
consistent with a model whereby
AR-stimulated adenylyl
cyclase activity is distinct from that of
- and/or
AR. In both B7 cells and brown adipocytes derived from
mouse tissue, there is a maximum adenylyl cyclase response that can be
reached by stimulating all three subtypes simultaneously, and the
individual subtypes appear to contribute discrete proportions to the
overall response. We do not discount the possibility that the
AR is coupled to more than one signal transduction
system; clearly this issue must be explored in greater detail in future
studies, and the immortalized brown adipocytes that we describe offer
an opportunity to delineate these coupling pathways.
AR regulates brown adipocyte
proliferation while the
AR plays no role in this
process
(18) . However, the experiments that have addressed
these properties in the past have not fully excluded the possibility
that the
AR may have been the only receptor expressed
on the cells under study. Perhaps a cell capable of proliferating would
have responded to a
AR agonist if that receptor
subtype had been present. One way to address this problem is to have a
readily available population of brown fat cells which retain their
physiological functions,
AR subtypes, and cyclases and are capable
of being cloned in vitro to minimize phenotypic variability.
These immortalized clonal cell lines satisfy these criteria and provide
a model in which to investigate the control of brown adipocyte
physiology by the
AR system.
AR (0.1 µM) can induce Ucp expression in both the B7 and B3 cell lines. When Ucp induction was analyzed in more detail in the B7 cell line, we
found that Ucp mRNA levels were induced by the
AR-selective agonist CL316,243 and also by a
concentration of isoproterenol (100 µM) capable of
stimulating all three
AR subtypes simultaneously. Moreover,
directly increasing intracellular cAMP levels with dibutyryl cAMP or
forskolin potently stimulates Ucp expression in B7 cells, in
agreement with results obtained by other investigators studying primary
cultures of brown adipocytes
(15, 16) . Finally, using
several selective antagonists for
AR or
AR we also find that activation of either of these two
receptors can lead to stimulation of Ucp expression. From
these data it would appear that elevation of cAMP levels, stimulated by
any of the
AR subtypes, can enhance expression of Ucp,
but that the greatest response was achieved by stimulation of all three
AR subtypes simultaneously. While this work was in progress,
similar conclusions were also obtained by Klaus et al.(52) using the HIB 1B mouse hibernoma cell line. In contrast,
Zhao et al.(58) , using brown fat cells isolated from
normal Syrian hamster, reported that only the
AR is
capable of stimulating thermogenesis as measured by changes in oxygen
consumption. In order to evaluate whether the differences obtained from
the various laboratories reflect differences in the experimental cell
system or species, it would have been valuable if Zhao et al.(58) had provided direct evidence for the existence of
AR and
AR, in addition to
AR, in the cells they used by some functional criteria
such as adenylyl cyclase activation. The pharmacological data that were
presented do not fully address this point.
ARs coexisting in a population of brown
adipocytes is capable of contributing to Ucp induction. It is
not possible to extrapolate from the present results to know whether
other processes such as thermogenesis, cell proliferation, and
mitochondriogenesis will also be regulated by each of the
ARs
present in these brown fat cell lines; however, future experiments will
address these issues.
Table:
Kinetic parameters of adenylyl cyclase
stimulation by CL316,243
is the activation constant, expressed in molar concentrations,
for half-maximal stimulation of adenylyl cyclase by CL316,243. The
values in brackets indicate the 95% confidence intervals associated
with the estimates. In all cases the analysis indicated best fit to a
single class of sites.
AR,
-adrenergic receptor; BA, brown
adipocyte; CYP, cyanopindolol; BAT, brown adipose tissue; IBAT,
interscapular brown adipose tissue; PCR, polymerase chain reaction;
Ucp, mitochondrial uncoupling protein; WAT, white adipose
tissue; kb, kilobase(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.