Insulin and the ß3-Adrenoceptor Differentially Regulate Uncoupling Protein-1 Expression
Johannes Klein1,
Mathias Fasshauer1,
Manuel Benito and
C. Ronald Kahn
Research Division Joslin Diabetes Center (J.K., M.F., C.R.K.)
Harvard Medical School Boston, Massachusetts 02215
Department of Internal Medicine I (J.K.) Medical University
of Lübeck 23538 Lübeck, Germany
Facultad de
Farmacia (M.B.) Universidad Complutense 28040 Madrid, Spain
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ABSTRACT
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Cross-talk between insulin and the adrenergic
system is important in the regulation of energy homeostasis. In
cultured, differentiated mouse brown adipocytes, ß3-adrenergic
stimulation induced a 4.5-fold increase in uncoupling protein-1 (UCP-1)
expression, which was diminished by 25% in the presence of insulin.
ß3-Adrenergic stimulation also activated mitogen-activated protein
(MAP) kinase by 3.5-fold and caused a decrease in basal
phosphoinositide (PI) 3-kinase activity detected in p110
- and
Gß-subunit-immunoprecipitates in a time-dependent manner, whereas
insulin stimulated p110
- and phosphotyrosine-associated PI 3-kinase
activity. Inhibition of MAP kinase or PI 3-kinase potentiated the
ß3-adrenergic effect on UCP-1 expression, both alone and in the
presence of insulin. Thus, insulin inhibits ß3-adrenergic stimulation
of UCP-1, and both MAP kinase and PI 3-kinase are negative regulatory
elements in the ß3-adrenergic control of UCP-1 expression. Cross-talk
between the adrenergic and insulin signaling systems and impaired
regulation of UCP-1 might contribute to the development of a reduced
energy balance, resulting in obesity and insulin resistance.
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INTRODUCTION
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Cross-talk between hormonal stimuli promoting energy storage, such
as insulin, and those increasing energy expenditure, such as activation
of the sympathetic nervous system, may play a crucial role in the
pathophysiology of obesity and the insulin resistance syndrome
(1).
In small mammals and infants, brown adipose tissue is important in the
regulation of energy homeostasis by virtue of expression of the
uncoupling protein-1 (UCP-1), which uncouples mitochondrial oxidative
phosphorylation, thereby dissipating energy as heat (2, 3, 4). Recent
studies with selective agonists for the ß3-adrenergic receptor, a
ß-adrenergic receptor expressed mainly in brown adipocytes and
mediating induction of UCP-1, have found significant positive effects
on energy expenditure and fat metabolism in adult humans and primates
(5, 6). Moreover, mutations in the gene for UCP-1 alone (for a recent
review see Ref. 7) or in combination with mutations in the
ß3-adrenergic receptor (8, 9) have been reported to be associated
with changes in energy homeostasis favoring weight gain.
Obesity is an important component of type 2 diabetes. Obesity induces
insulin resistance and hyperinsulinemia, both of which, in turn, may
contribute to alterations in energy expenditure (10, 11, 12). In the
present study, we have investigated mechanisms of cross-talk between
insulin and the ß3-adrenergic receptor controlling UCP-1 expression
in a mouse brown adipocyte cell model. We find that insulin exerts a
mitogen-activated protein (MAP) kinase-dependent bidirectional
effect on the ß3-adrenergic control of UCP-1. Moreover, we provide
evidence that phosphoinositide (PI) 3-kinase and MAP kinase are
regulatory elements for UCP-1 expression in both insulin and
ß3-adrenergic signaling pathways. Imbalance in the regulation of
these signaling intermediates might have pathophysiological
implications for the development of obesity, insulin resistance, and
type 2 diabetes.
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RESULTS
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ß3-Adrenergic Stimulation of UCP-1 Expression Is Protein Kinase A
(PKA) Dependent and Inhibited by Insulin Treatment
Adrenergic stimulation increases UCP-1 expression via a
protein kinase A-dependent pathway (13, 14, 15, 16). In differentiated,
cultured brown adipocytes, Western blot analyses with a UCP-1-specific
antibody showed a 4.5-fold increase of UCP-1 protein levels by
ß3-adrenergic stimulation using the ß3-selective agonist CL316243
at a concentration of 10 µM. This effect was
dose-responsive with half-maximal stimulation at a concentration of 100
nM (data not shown). The ß3-adrenoceptor-induced increase
was reduced by about 40% using the membrane-permeable PKA-inhibitor
H-89 (Fig. 1
, A and B). The efficacy of
H-89 was demonstrated in PKA activity assays, which revealed a more
than 90% decrease in endogenous PKA activity after treatment of
adipocytes with this compound for 19 h (Fig. 1C
). Furthermore, the
ß3-receptor-mediated increase of UCP-1 could be mimicked by the
PKA-activating compound forskolin (Fig. 1D
). This increase was reduced
by more than 60% after pretreatment of adipocytes with H-89 (Fig. 1D
).
These experiments further confirm the importance of a PKA-dependent
signaling pathway for UCP-1 induction. When the cells were incubated
with 100 nM insulin alone for 6 h before stimulation
with CL316243, there was a significant decrease of about 25% in
ß3-adrenoceptor-induced UCP-1 protein levels (Fig. 2
, A and B). Further time course studies
showed that this decrease was first detectable after 3 h of
insulin pretreatment and was most prominent at 6 h (data not
shown). Similar results were found using epidermal growth factor
at 20 ng/ml during the prestimulation period (data not shown).
Insulin stimulation alone produced no significant effect, but there was
a trend (P = 0.08) toward a positive effect over basal
UCP-1 protein levels (Fig. 2
, A and B). Furthermore, short-term
stimulation with 100 nM insulin significantly
increased tyrosine phosphorylation of the insulin receptor but not
insulin-like growth factor I receptor (data not shown), indicating that
the observed effects of insulin are mediated primarily by the insulin
receptor.

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Figure 1. ß3-Adrenergic Induction of UCP-1 Expression Is
PKA Dependent
Differentiated brown adipocytes were either nontreated or stimulated
for 18 h with CL316243 (CL-ß3, 10 µM) alone or in
combination with the PKA inhibitor H-89 (10 µM, 1 h
pretreatment). Cell lysates were prepared as described in
Materials and Methods. A, UCP-1 was visualized by
blotting with a specific antibody and autoradiography of a
representative experiment is shown. B, UCP-1 protein levels relative to
CL316243 induction alone (Control, mean ±
SEM) are shown as determined by PhosphorImager
quantitation of two independent experiments. Statistical significance
was calculated with Students t test comparing CL-ß3
treatment alone with CL-ß3 + H-89; ** denotes differences of high
statistical significance (P 0.01). C, Representative
PKA activity assay. D, Cells were stimulated with forskolin (50
µM) for 18 h, either with or without prior
H-89-treatment for 1 h (10 µM).
Representative blot probed with UCP-1 antibody.
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Figure 2. Bidirectional Effect of Insulin and Influence of
MAP Kinase Inhibition on ß3-Adrenergic Induction of UCP-1 Expression
A, Differentiated brown adipocytes were either nontreated or treated
with insulin (24 h, 100 nM), the MEK inhibitor PD098059
(PD, 19 h, 50 µM), or CL316243 (CL-ß3, 18 h,
10 µM) alone or in the combinations indicated. UCP-1
immunoblots were prepared as described in Material and
Methods. Representative autoradiography. B, The statistical
analysis of at least three independent experiments with the
SEM is shown. C, Autoradiography of cells treated with the
indicated concentrations of PD. D, Brown adipocytes were stimulated
with 10 µM dobutamin (Dob), clenbuterol (Clen), or
CL316243 (CL-ß3) for 16 h alone or in combination with PD098059
(PD, 1 h pretreatment, 50 µM). Statistical
significance in panels B and D was calculated with Students
t test comparing CL-ß3 treatment alone (Control) with
either insulin + CL-ß3, or PD + CL-ß3, or comparing treatments with
insulin + PD + CL-ß3 and PD + CL-ß3, or insulin + CL-ß3,
respectively; * Denotes differences of statistical significance
(P 0.05); ** denotes high statistical
significance (P 0.01).
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The Role of MAP Kinase in the Regulation of UCP-1 Expression
Stimulation of different G protein-coupled receptors, including
Gs-coupled ß-adrenergic receptors (17), has been shown to regulate
both MAP kinase and PI 3-kinase pathways (for a recent review see
Ref. 18). These two pathways are also major contributors to insulin
signaling (19, 20). In the differentiated brown adipocytes, the
ß3-selective agonist CL316243 increased MAP kinase activity in a
time-dependent manner with a 3.5-fold maximum after 20 min, as assessed
with a phosphospecific antibody against the activated MAP kinase
isoforms p42 and p44 (Fig. 3
, A and B).
Inhibition of PKA with the membrane-permeable PKA-specific
inhibitor H-89 produced an almost 50% decrease in ß3-adrenergic
activation of MAP kinase (Fig. 3
, C and D). Conversely, the
adenylyl-cyclase-activating compound forskolin and the cAMP-analog
(Bu)2cAMP increased MAP kinase activation in a
time-dependent manner (Fig. 3E
). As expected, the MAP kinase kinase
(MEK) inhibitor PD098059 completely abolished MAP kinase
activation (Fig. 3
, C and D), whereas pharmacological inhibition of PI
3-kinase (LY294002), protein kinase C (GF109203X), and
Gi-coupled receptors (pertussis toxin) had no
significant effect on MAP kinase activation in these cells (data not
shown). Furthermore, LY294002 pretreatment had no significant effect on
insulin-induced MAP kinase phosphorylation (Fig. 3F
).

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Figure 3. ß3-Adrenergic Stimulation Leads to Activation of
MAP Kinase in a Time- and PKA-Dependent Manner
A, Fully differentiated brown adipocytes were either nontreated or
treated with CL316243 (CL-ß3, 10 µM) for the indicated
times. Cell lysates and MAP kinase immunoblots were prepared as
described in Material and Methods. Representative
autoradiography. After incubating immunoblots with a phosphospecific
MAP kinase antibody (P-MAP K., upper panel) blots were
stripped and reprobed with MAP kinase antibody to control for equal
loading (MAP K., lower panel). B, The statistical
analysis of four independent experiments with the SEM is
shown. Statistical significance was calculated with the Student s
t test comparing nontreated cells (Control) with CL-ß3
treatment; * denotes statistical significance (P
0.05); ** denotes high statistical significance (P
0.01). C, Brown adipocytes were either nontreated or treated with
CL316243 (CL-ß3, 20 min, 10 µM), the MEK inhibitor
PD098059 (PD, 1 h, 50 µM), the PI 3-kinase inhibitor
LY294002 (LY, 1 h, 10 µM), the PKA inhibitor H-89 (1
h, 10 µM), alone or in different combinations as
indicated. MAP kinase immunoblots were prepared as described in panel
A. D, The statistical analysis of four independent experiments with the
SEM is shown. Statistical significance was calculated using
Student s t test comparing CL-ß3 treatment (Control)
with H-89 + CL-ß3; * denotes statistical significance
(P 0.05). E, Brown adipocytes were nontreated,
treated with CL316243 (CL-ß3, 20 min, 10 µM), or
treated for the indicated times with forskolin (50 µM)
and (Bu)2cAMP (DB-cAMP, 1 mM). Immunoblots were
prepared as described in panel A with a phosphospecific MAP kinase
antibody. F, Brown adipocytes were either nontreated or stimulated with
insulin (Ins, 5 min, 100 nM) alone or in combination with
LY294002 (LY, 1 h, 10 µM). Immunoblots were prepared
as described in panel A with antiphospho MAP kinase antibody.
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As both insulin and epidermal growth factor are also activators of MAP
kinase, we investigated whether inhibition of MAP kinase would modify
the effects of either insulin or ß3-adrenergic agents on UCP-1
expression. Surprisingly, inhibition of MAP kinase by PD098059 resulted
in a significant 1.5-fold increase in the ß3-adrenergic stimulation
of UCP-1 (Fig. 2
, A and B). The positive effect of MAP kinase
inhibition on ß3-adrenoceptor-induced UCP-1 expression followed a
dose-response curve (Fig. 2C
). The inhibitor alone had no effect on the
basal level of UCP-1 expression (Fig. 2
, A and B). Interestingly,
insulin pretreatment, in the presence of a pharmacological MAP kinase
inhibitor (MEK inhibitor PD098059) further enhanced the ß3-adrenergic
stimulation of UCP-1 levels by approximately 2.5-fold (Fig. 2
, A and
B).
Furthermore, we determined whether UCP-1 protein was also up-regulated
by ß1-adrenergic stimulation with dobutamin or ß2-receptor
activation with clenbuterol. Both compounds were able to increase UCP-1
protein levels about 3-fold over basal; however, the same concentration
of Cl-ß3 was about 2 times as effective in inducing UCP-1 (Fig. 2D
).
Surprisingly, no significant change in ß1- or
ß2-adrenoceptor-induced UCP-1 protein could be observed after
inhibition of MAP-kinase, whereas ß3-adrenergic stimulation of
UCP-1 protein was again significantly enhanced by PD098059 pretreatment
(Fig. 2D
). Furthermore, forskolin-induced UCP-1 expression was not
altered by MAP kinase inhibition (data not shown).
The Role of PI 3-Kinase in the Regulation of UCP-1
Expression
To determine whether PI 3-kinase-dependent signaling pathways
might play a role in the ß3-adrenergic control of UCP-1 expression,
differentiated cells were pretreated with an inhibitor of PI 3-kinase
(LY294002) before ß3-adrenergic stimulation. Inhibitor treatment
resulted in a 2-fold increase in UCP-1 protein over the levels achieved
by ß3-adrenergic treatment alone (Fig. 4
, A and B). Insulin pretreatment did not
have a significant effect on this increase (Fig. 4
, A and B). The
inhibitor alone had no effect on the basal level of UCP-1 expression
(data not shown). The positive effect of PI 3-kinase inhibition on
ß3-adrenoceptor-induced UCP-1 expression followed a dose-response
curve (Fig. 4C
). Furthermore, in contrast to ß3-adrenergic
treatment, ß1- and ß2-adrenoceptor- as well as forskolin-induced
UCP-1 protein was not significantly changed after inhibition of PI
3-kinase by LY294002 pretreatment (Fig. 4D
and data not shown).

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Figure 4. ß3-Adrenergic Induction of UCP-1 Expression Is
Regulated by PI 3-Kinase-Dependent Pathways
A, Fully differentiated brown adipocytes were either nontreated or
treated with the ß3-selective agonist CL316243 (CL-ß3, 18 h,
10 µM), the PI 3-kinase inhibitor LY294002 (LY, 19
h, 10 µM), or insulin (24 h, 100 nM) alone or
in the combinations indicated. UCP-1 immunoblots were prepared as
described in Materials and Methods. Representative
autoradiography. B, Bar graph depicting UCP-1 protein
levels relative to CL316243 induction alone (mean ±
SEM) as determined by PhosphorImager quantitation of at
least three independent experiments. C, Autoradiography of cells
treated with the indicated concentrations of LY294002. D, Brown
adipocytes were nontreated or incubated with 10 µM
dobutamin (Dob), clenbuterol (Clen), or CL316243 (CL-ß3) for 16
h alone or in combination with LY294002 (LY, 1 h pretreatment, 10
µM). Statistical significance in panels B and C was
calculated with Students t test comparing CL-ß3
treatment alone with either CL-ß3 + LY or CL-ß3 + LY + insulin
treatments, respectively; * denotes statistical significance
(P 0.05); ** denotes high statistical
significance (P 0.01).
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As inhibition of PI 3-kinase enhanced the ß3-adrenoceptor-induced
UCP-1 protein expression, we performed in vitro PI 3-kinase
assays to identify relevant isoforms involved in the mediation of this
effect. Previous studies have shown that PI 3-kinase activity
associated with p110
is stimulated by insulin (20), whereas G
protein-coupled receptors have been shown to stimulate PI 3-kinase
activity associated with p110
acting via ß
-subunits (21). As
expected, in brown adipocytes, insulin produced a 3-fold increase in
p110
-associated PI 3-kinase activity and a more than 10-fold
increase in PI 3-kinase activity in antiphosphotyrosine
immunoprecipitates. ß3-Adrenergic stimulation produced no change in
either of these fractions (Fig. 5
, A and
B). By contrast to effects of other G-protein-coupled receptors,
ß3-adrenergic stimulation failed to increase, and actually reduced,
PI 3-kinase activity in both p110
and Gß-subunit
immunoprecipitates. This effect was time dependent and represented more
than a 60% decrease by about 40 min. Insulin had no significant
effect on PI 3-kinase associated with either p110
or Gß-subunits
(Fig. 5
, C and D).

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Figure 5. Stimulation of ß3-Adrenergic Receptors Inhibits
PI 3-Kinase p110 Activity
Fully differentiated brown adipocytes were either nontreated, treated
with CL316243 (CL-ß3, 10 µM) for the indicated periods
of time, or treated with insulin (Ins, 5 min, 100 nM).
Protein lysates were subjected to immunoprecipitation with antibodies
against p110 (A), phosphotyrosines (B), p110 (C), and
Gß-subunit (D), respectively, and PI 3-kinase activities were
measured as described in Materials and Methods. The
statistical analysis of four independent experiments with the
SEM is shown. * Denotes statistical significance
(P 0.05); ** denotes high statistical
significance (P 0.01) comparing nontreated cells
(Control) with CL-ß3 treatment. Representative experiments are
shown as insets on top of the respective bar graph
analyses in panels C and D.
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DISCUSSION
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The characteristic function of brown adipose tissue is
to regulate adaptive thermogenesis by virtue of expression of the
mitochondrial protein UCP-1 (2, 3, 4). Brown fat is also a target for both
insulin and ß-adrenergic regulation. In immortalized brown
adipocytes, we find that cross-talk between insulin and the
ß3-adrenergic receptor impacts on the regulation of UCP-1
expression. The approximately 4- to 5-fold induction of UCP-1
expression in response to ß3-adrenergic stimulation is
blocked by inhibition of PKA, consistent with a classical signaling
pathway from ß-adrenergic receptors via
G
s-mediated activation of PKA (13, 14, 15, 16).
Furthermore, specific activation of ß1- and ß2-adrenoceptors by
dobutamin and clenbuterol, as well as direct activation of PKA by
forskolin, was also able to increase UCP-1 protein levels, confirming
results obtained by other investigators (16). Insulin pretreatment
diminishes the ß3-adrenergic induction of UCP-1. This direct
inhibitory effect of insulin on ß3-adrenergic stimulation of UCP-1
may have important pathophysiological implications. Indeed, in several
animal and human studies, hyperinsulinemia has been found to precede
increased body weight gain and obesity (10, 11, 12, 22, 23). Although the
negative effect of insulin on the ß3-adrenergic UCP-1 induction is
small, in the context of chronic hyperinsulinemia, a small reduction in
energy expenditure caused by diminished UCP-1 expression could
represent a potential mechanism favoring a significant cumulative
weight gain over time.
As MAP kinase and PI 3-kinase are important signaling elements common
to both insulin and G protein-coupled receptor-signaling pathways
(18, 19, 20), we have explored their role as potential mediators of
ß3-adrenergic and insulin effects on UCP-1. In brown adipocytes, MAP
kinase is activated by ß3-adrenergic stimulation via a PKA-dependent
pathway (this study and Ref. 24). Although this might not be the only
route of activation with respect to the ß3-adrenergic receptor (25),
studies in several other systems, including 3T3-F44 preadipocytes (26),
cardiac myocytes (27), and PC12 pheochromocytoma cells (28, 29), have
also demonstrated a PKA-dependent MAP kinase activation. On the other
hand, inhibition of PI 3-kinase, protein kinase C, and
Gi-coupled receptors does not significantly
reduce the ß3-adrenergic activation of MAP kinase, indicating that,
in this cell model, these signaling intermediates are not upstream of
MAP kinase. Interestingly, inhibition of MAP kinase enhances the
ß3-adrenergic stimulation of UCP-1 and reverses the inhibitory effect
of insulin. These observations suggest a negative role for MAP kinase
in both ß3-adrenergic and insulin pathways. This is the first study
to show the relevance of a MAP kinase-mediated pathway in the
ß3-adrenergic regulation of UCP-1. A negative role for MAP kinase on
UCP-1 expression has also been described in insulin-like growth
factor-mediated pathways in fetal brown adipocytes (30).
Another interesting finding of this study is that inhibition of MAP
kinase unmasks a second stimulatory effect of insulin on the
ß3-receptor-induced UCP-1 expression. Although we cannot present
direct evidence in this study, a possible candidate mediating this
positive effect of insulin is PI 3-kinase p110
. PI 3-kinase activity
appears to also play a role in ß3-adrenergic pathways, as indicated
by the fact that ß3-adrenergic induction of UCP-1 is enhanced by PI
3-kinase inhibition. In Gi-coupled muscarinic
receptors, the catalytic PI 3-kinase isoform p110
has been shown to
be activated by direct binding to ß
-subunits (21, 31). We detect
PI 3-kinase activity in both Gß-subunit- and
p110
-immunoprecipitates. In contrast to effects of
Gi-coupled muscarinic receptors, however,
activation of Gs-coupled ß3-adrenergic
receptors decreases PI 3-kinase activity in both fractions in a
time-dependent manner. The mechanism responsible for this inhibition of
p110
activity by ß3-adrenergic stimulation is unknown at present.
A possible explanation for this novel observation might be a divergent
effect of different Gß
-subunits on their
effector.
Furthermore, MAP kinase and PI 3-kinase appear to be signaling
intermediates specific in the ß3-adrenergic control of UCP-1 inasmuch
as ß1- and ß2-adrenoceptor- as well as forskolin-induced UCP-1
protein content is not significantly altered by pharmacological
inhibition of either molecule. It has been proposed that each
ß-adrenoceptor subtype may be coupled to one or more specific
adenylyl cyclase isoforms leading to compartmentalization of the
functional adrenergic responses (16). Furthermore, it has been shown
that ß-adrenergic receptors can interact with different G proteins
(25). Further studies will be needed to clarify this issue.
Finally, both animal and cell culture studies have indicated a
pretranslational control of UCP-1 (32). Depending on conditions,
half-life of UCP-1 protein is between 1 and 7 days (32, 33). Since the
stimulation period for these experiments was only 1618 h, the
observed effects of MAP kinase and PI 3-kinase inhibition are most
likely caused by transcriptional up-regulation, as an effect mainly
mediated by changes of the protein half-life would be expected to take
much longer to be this prominent. Furthermore, ß1- and ß2-selective
stimulation of UCP-1 could not be enhanced using PD098059 or LY294002,
demonstrating a specific effect of ß3-selective stimulation and
making it unlikely that the observed augmentation after ß3-selective
stimulation is due to an effect of the pharmacological inhibitors on
the protein half-life.
Taken together, we propose a model in which the ß3-adrenoceptor
stimulates UCP-1 expression via a PKA-mediated pathway.
ß3-Adrenoceptor-mediated inhibition of the p110
isoform of PI
3-kinase adds to this effect, while the PKA-dependent activation of MAP
kinase provides a concurrent autoinhibition. Activation of MAP kinase
also functions as an important switch for mediating bidirectional
cross-talk with the insulin receptor (Fig. 6
). Dysregulation of these cross-talking
signaling elements, notably in hyperinsulinemic states, might
contribute to a decrease in energy expenditure resulting in obesity and
insulin resistance.
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MATERIALS AND METHODS
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Materials
The selective ß3-receptor agonist CL316243 was a generous gift
of Dr. Kurt Steiner (Wyeth-Ayerst Research, Princeton, NJ). The
UCP-1 specific antibody was purchased from Alpha Diagnostic
International (San Antonio, TX) and tested for specificity using a
yeast recombinant mouse UCP-1 protein (kindly provided by Dr. Bradford
Lowell, Beth Israel Deaconess Medical Center, Boston, MA).
Antiphosphotyrosine 4G10 antibody was kindly provided by Dr. Morris
White (Joslin Diabetes Center, Boston, MA). Polyclonal anti-PI 3-kinase
p110
, anti-Gß, anti-p110
, and anti-MAP kinase antibodies were
obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz,
CA); phosphospecific MAP kinase antibody and PD098059 (PD) were from
New England Biolabs, Inc. (Beverly, MA); LY294002 (LY),
H-89, GF109203X, and pertussis toxin were obtained from
Calbiochem (La Jolla, CA). BSA was from Arnel Products
Co., Inc. (New York, NY); the PKA assay system was from Life Technologies, Inc. (Gaithersburg, MD);
[
32P]-ATP was purchased from NEN Life Science Products (Boston, MA); protein A-Sepharose was from
Pharmacia Biotech (Piscataway, NJ); and
[125I]-protein A was from ICN Biochemicals, Inc. (Costa Mesa, CA). Phosphoinositol was
purchased from Avanti Polar Lipids (Alabaster, AL); TLC plates were
obtained from VWR Scientific Products (Bridgeport, NJ);
nitrocellulose was from Schleicher & Schuell, Inc. (Keene,
NH); and electrophoresis supplies were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). All other supplies were from
Sigma (St. Louis, MO).
Cell Isolation And Culture
Interscapular brown adipose tissue was isolated from newborn FVB
mice (Taconic Farms, Inc.) as previously described (34).
Preadipose cells were immortalized by infection with the retroviral
vector pBabe encoding SV 40 T antigen (kindly provided by Dr. J.
DeCaprio, Dana Farber Cancer Institute, Boston, MA) and selected with
puromycin (1 µg/ml) for at least 3 weeks. For differentiation,
selected preadipocytes were grown to confluence in culture medium
supplemented with 20 nM insulin and 1 nM T3
(differentiation medium). After induction of differentiation in
confluent cells for 24 h in differentiation medium further
supplemented with 0.5 mM isobutylmethylxanthine, 0.5
µM dexamethasone, and 0.125 mM indomethacin,
cells were maintained in differentiation medium for 45 days until
exhibiting a fully differentiated phenotype with massive accumulation
of multilocular fat droplets. The different stimulation experiments
were carried out after starving the cells in serum-free medium for
1618 h.
Western Blot Analysis
At the end of the stimulation period, cells were washed twice
with ice-cold PBS and lysed in extraction buffer (50 mM
HEPES, 137 mM NaCl, 1 mM
MgCl2, 1 mM
CaCl2, 10 mM Na pyrophosphate, 10
mM NaF, 2 mM EDTA, 10% glycerol, 1% Igepal
CA-630, 2 mM vanadate, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 2 mM PMSF, pH 7.4). Cell lysates were
centrifuged at 12,000 x g for 10 min at 4 C, and the
protein amount in the supernatant was determined by the Bradford method
(35) using BSA as standard and the dye reagent concentrate from
Bio-Rad Laboratories, Inc. (Richmond, CA). Equal amounts
of protein (100 µg) were directly solubilized in Laemmli sample
buffer and separated by SDS-PAGE and transferred to nitrocellulose
membranes. After blocking with TBS (10 mM Tris,
0.15 M NaCl, 0.05% Tween, pH 7.2) containing 3%
BSA for 30 min, membranes were incubated with the appropriate
antibodies for 2 h, washed three times for 5 min each in TBS, and
incubated with [125I]protein A for 45 min.
After an additional three washes, the immunoblots were exposed on a
PhosphorImager screen, and signals were quantified using a densitometer
(Molecular Dynamics, Inc., Sunnyvale, CA).
PI 3-Kinase Assays
Lysates were obtained as described above. Supernatants
containing 300 µg protein were immunoprecipitated for 2 h at 4 C
with the appropriate antibodies, and the immune complexes were
collected by adding 50 µl of a 50% slurry of protein A-Sepharose in
PBS for 1 h at 4 C. After washing the immune complexes twice with
PBS containing 1% Igepal-CA 630, twice with 0.5 M LiCl-0.1
M Tris (pH 7.5) and twice in reaction buffer (10
mM Tris, pH 7.5, 100 mM NaCl, 1 mM
EDTA), Sepharose beads were resuspended in a mixture containing 50 µl
of reaction buffer, 10 µl of 100 mM
MgCl2, and 10 µl of phosphatidylinositol (2
µg/µl). Reactions were initiated by adding 5 µl of reaction
mixture [880 µM ATP, 20 mM
MgCl2, and 10 µCi of
[
32P]ATP (3,000 Ci/mmol)] per tube and
stopped after 10 min by adding 20 µl of 8 N HCl and 160
µl of CHCl3-methanol (1:1). After a brief
centrifugation, 50 µl of the lower organic phase of each sample were
spotted on a silica gel TLC plate. The plate was developed in
CHCl3-methanol-H2O-NH4OH
(120:94:23:2.4), dried, exposed to a PhosphorImager screen, and
quantitated with a densitometer (Molecular Dynamics, Inc.).
PKA Assays
Lysates were obtained after 18 or 19 h (H-89-pretreated
cells) of stimulation, respectively, as described above, and PKA
activity was measured according to the manufacturers
instructions.
Statistical Analysis
Students t tests were used for analysis of
differences between treatments. P values < 0.05 are
considered statistically significant and values <0.01 are considered
highly significant. The SEM is indicated in all
bar graphs.
 |
ACKNOWLEDGMENTS
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We are grateful to Dr. Kurt Steiner (Wyeth-Ayerst Research, CN
8000, Princeton, NJ 08543) for providing us with the ß3-adrenoceptor
agonist CL316243. We gratefully acknowledge Dr. James DeCaprio (Dana
Farber Cancer Institute, Boston, MA) for the generous gift of the
retroviral pBabe vector coding for SV40T. We thank Dr. Brad Lowell for
providing us with the yeast recombinant mouse UCP-1 protein. We are
indebted to Terri-Lyn Azar and Catherine Remillard for excellent
secretarial assistance.
 |
FOOTNOTES
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Address requests for reprints to: C. Ronald Kahn, M.D., Joslin Diabetes Center, One Joslin Place, Boston, Massachusetts 02115;
This work was supported by NIH Grants DK-33201 and DK-36836 (Joslins
Diabetes and Endocrinology Research Center grant). J.K. was supported
by a grant from the Deutsche Forschungsgemeinschaft (Kl-1131/1). M.F.
was supported by a grant from the Studienstiftung des deutschen
Volkes.
1 These authors equally contributed to this work. 
Received for publication October 22, 1999.
Revision received February 28, 2000.
Accepted for publication March 15, 2000.
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