Differentiation-dependent inhibition of proteolysis by
norepinephrine in brown adipocytes
M.
Desautels and
S.
Heal
Department of Physiology, College of Medicine, University of
Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E5
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ABSTRACT |
The objective was to
evaluate whether norepinephrine (NE) and other hormonal factors have
direct effects on protein degradation in brown fat cells. NE inhibited
proteolysis by 35-45% in mouse brown adipocytes differentiated in
culture. Insulin also inhibited protein degradation but significantly
less than NE, whereas glucagon and leptin had no effect. The inhibitory
effect of NE was partially antagonized by propranolol but not by
prazosin, and dose-response curves with BRL-37344 (a
3-agonist), isoproterenol (a
1/
2-agonist) and dobutamide (a
1-agonist)
were consistent with the involvement of a
3-adrenergic receptor.
Furthermore, forskolin mimicked the effects of NE, whereas additions of
A-23187 or phorbol esters had no effect, alone or in combination with
NE or forskolin. Thus inhibition of proteolysis by NE likely involves a
3-adrenergic receptor-mediated
increase in cAMP. In contrast, NE, BRL-37344, and dobutamide had no
effect on proteolysis in preadipocytes. Inhibition of proteolysis by NE
was due at least in part to inhibition of autophagy. Thus inhibition of
proteolysis by NE and insulin in mature brown adipocytes is likely an
important process contributing to brown fat growth and atrophy under
many physiological or pathological conditions.
brown fat; insulin; autophagy;
3-adrenergic receptor; lysosomes; protein turnover
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INTRODUCTION |
BROWN ADIPOSE TISSUE (BAT) is a sympathetically
innervated heat-producing tissue involved in the control of energy
homeostasis and body temperature (24, 25). Its capacity to produce heat is dependent on specialized mitochondria with a regulated uncoupling protein (UCP) pathway that dissipates as heat the energy normally conserved as ATP on oxidation of metabolic substrates (7, 25, 34). The
capacity of BAT for thermogenesis varies with immediate or anticipated
needs for energy dissipation. Growth and atrophy of the tissue is
marked and rapid and occurs usually in synchrony with activation or
suppression of sympathetic input to BAT (25). Indeed, norepinephrine
not only stimulates metabolic activity but also promotes proliferation
and differentiation of preadipocytes to the terminally differentiated
phenotype (7).
Changes in BAT thermogenic capacity result from alterations in cell
number as well as cell content of mitochondrial proteins and UCP-1 (7,
25, 34). The thermogenic contribution of two other mitochondrial UCP
homologs, UCP-2 and UCP-3, expressed in brown fat and other
tissues, is yet uncertain (4, 18, 40). Obviously, cellular
hypertrophy/atrophy must result from changes in rates of protein
synthesis and/or degradation. In mammalian cells, several
proteolytic pathways are responsible for the degradation of
intracellular proteins under basal conditions, with some becoming more
active under certain physiological or pathological conditions. For
instance, autophagy is an important route of delivery of cell constituents to lysosomes and is regulated by nutrients and hormones, allowing cells to match their degradative status to the environmental conditions (3, 16, 36). In contrast, nonlysosomal proteolysis as found
in the cell cytosol or within certain organelles, such as mitochondria
or endoplasmic reticulum, appears responsible for the breakdown of
proteins with short half-lives, like some transcription factors, key
enzymes in metabolic pathways, or proteins with structural damage from
mutations or posttranslational modifications (20, 23, 27). In contrast
to other organs such as liver or muscles, there is very little known
about what controls proteolysis in brown fat cells.
Mice without food show marked BAT atrophy, characterized primarily by
bulk loss of mitochondrial proteins without change in tissue
cellularity (10, 14, 30). After some delay, loss of UCP-1 from the
mitochondria is also observed (10, 30). At least two pathways are
likely involved in the enhanced intracellular proteolysis occurring in
the brown fat cells of fasting mice, an autophagic process and a change
in proteolysis within mitochondria. BAT has a large complement of
lysosomal enzymes, and the tissue atrophy can be reduced in vivo with
blockers of lysosomal function (10). Proteolysis within mitochondria
likely contributes also to fasting-induced tissue atrophy, because BAT
mitochondria contain protease activity (11, 12) that may be involved in
the selective loss of UCP-1 from the mitochondria (13) and the
reduction in the apparent abundance of cristae (8). The neuroendocrine
factors responsible for fasting-induced BAT atrophy are poorly defined. Food deprivation is associated with a suppression of sympathetic activity to BAT, reductions in circulating insulin and leptin levels,
and an increase in circulating glucagon (24, 25, 29, 37). All of these
are known to alter the thermogenic activity and/or capacity of the
tissue by direct actions on brown adipocytes and/or modification of
sympathetic activity to BAT (7, 21, 25, 35, 38). The observation that
denervation of BAT mimics the tissue atrophy observed in fasting mice
suggests that norepinephrine (NE) may exert an inhibitory influence on
proteolysis (10, 14). However, there are currently no direct estimates
of the effects of NE and other factors controlling the tissue
thermogenic capacity on overall rates of protein degradation in brown
fat cells.
The objective of this work was to evaluate whether NE and other
hormonal factors directly influence proteolysis in brown fat cells
differentiated in culture.
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MATERIALS AND METHODS |
Isolation of preadipocytes and cell cultures.
The isolation, culture, and differentiation of brown preadipocytes were
carried out as described previously (31, 32). Newborn mice (CD1 strain,
2-4 days old) were killed by decapitation, and the interscapular
BAT was removed under sterile conditions and cut into small pieces with
a scalpel blade. The tissue was digested for 30 min at 37°C under a
95% O2-5%
CO2 gas phase (vortex shaking
every 2.5 min) with 1 mg/ml collagenase in DMEM (GIBCO) supplemented
with 33 µM biotin, 17 µM pantothenic acid, 100 µM ascorbic acid,
100 µg/ml streptomycin, 6 µg/ml penicillin G, and 4% (wt/vol)
bovine serum albumin. The digestion mixture was filtered through Nitex
mesh (Thompson, 243 µM) into a sterile 50-ml centrifugation tube,
rinsed with 30 ml of media, and allowed to stand at room temperature
for 20 min. After centrifugation (800 g, 5 min), the top layer and
supernatant phase containing mature brown fat cells were discarded, and
the pellet was resuspended in 40-50 ml of culture media.
Cells were inoculated in 35-mm Falcon culture dishes and incubated in a
CO2 incubator (5%
CO2-95% air) at 37°C and
100% relative humidity. After 1-2 h, the media were aspirated
off, and fresh culture media were added. The standard culture media
consisted of DMEM with antibiotics, biotin, pantothenic acid, ascorbic
acid, and 10% (vol/vol) fetal bovine serum (ICN, CELLect-Silver) and were replaced every 2nd to 3rd day. Near or at confluence
(day 5),
3,5,3'-triiodothyronine
(T3; 1 nM) and insulin (50 nM)
were added to the culture media, and cells were allowed to
differentiate until day 10.
Differentiation to brown adipocytes was followed by the appearance of
fat vacuoles by light microscopy and of UCP-1 by immunofluorescence
microscopy and Western blots (12).
Cell protein labeling and measurements of protein degradation.
Standard culture media were replaced by culture media with reduced
methionine content made of 10% (vol/vol) fetal bovine serum, 10%
(vol/vol) DMEM, and 80% (vol/vol) methionine-free DMEM.
Supplementation with vitamins and antibiotics was as before.
[35S]methionine was
added at 10 µCi/ml. After 3 days, cell monolayers were washed 3 times
with 2 ml of DMEM + 2 mM unlabeled methionine and were incubated with
2.5 ml of DMEM + 2 mM unlabeled methionine and other additions, as will
be detailed in captions to Figs. 1-6. At time
0 and various times after addition, 0.25 ml of culture media was collected into microcentrifuge tubes and mixed with 0.035 ml
trichloroacetic acid (TCA, 100% wt/vol) and 0.015 ml BSA (5% wt/vol).
After
1 h at 4°C, samples were centrifuged, and 0.15 ml of
acid-soluble supernatant was added to 4 ml of Amersham PCS scintillator
fluid and counted. Protein degradation was measured as the release of
acid-soluble radioactivity from the cells into the cell culture media
per unit time after subtraction of residual acid-soluble radioactivity
at time 0. Specific activity of
labeled cell proteins was estimated at time
0 after cell collection and disruption by sonication
into 1 ml TCA (10% wt/vol) + 5 mM methionine (TCA+Met). An aliquot (50 µl) was collected by filtration (Whatmann GF/C glass fiber filters),
washed 5 times with 5 ml TCA+Met, and counted, while another aliquot
was centrifuged, solubilized in 0.5 N NaOH, and used for protein
estimation by the method of Bradford (5).
Statistical analysis.
Differences between groups were examined by one-way and two-way ANOVA
and Duncan's multiple range test for comparisons between means. NCSS
statistical software (Kaysville, UT) was used for computation. The
results were considered statistically significant when
P < 0.05. Results are presented as
means ± SE.
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RESULTS |
Norepinephrine and insulin inhibit protein degradation in brown
adipocytes differentiated in culture.
Our cell cultures had the pattern of differentiation typically observed
by others (31, 32). Preadipocytes grew rapidly with a fibroblast-like
appearance until confluence at about day 5 and thereafter started to round up and fill with
lipid droplets. By day 10, almost all
cells had the appearance of mature brown adipocytes. These cells
expressed UCP-1, as visualized by immunofluorescence microscopy with an
antiserum specific to UCP-1 (data not shown). Western blots showed no
expression of UCP-1 on day 5, some
expression on day 7, and maximal
expression on day 10 (see Fig.
2A). Thus our brown fat cells did
acquire the terminal differentiation phenotype typical of mature brown adipocytes.
A pulse-chase approach was then used to measure rates of proteolysis.
Cells were labeled with
[35S]methionine over
72 h to label all cell proteins uniformly. Typically, the specific
activity of labeled proteins was 30,000-40,000
counts · min
1 · µg
protein
1, with 290 ± 11 µg proteins/culture dish. The protein content per culture dish was
similar within and between experiments. Labeling conditions (reduced
methionine content of the culture media) between days
7 and 10 did not
affect the apparent differentiation of the cells. After three washes to
remove the labeled amino acid, the residual radioactivity in the
supernatant was <0.02% of acid-precipitable radioactivity at
time 0. Release of acid-soluble
radioactivity from the cells into the culture media over time in the
presence of 2 mM unlabeled methionine (to minimize reincorporation of
labeled precursors) was then used to estimate rates of protein
degradation. In DMEM without serum or hormonal additions, release of
acid-soluble radioactivity was linear over a 6-h period
(r2 = 0.64-0.75) and averaged 5.9 ± 1.6 µg proteins
degraded · h
1 · culture
dish
1 (Fig.
1). The extent of proteolysis represents
~2% of the labeled cell proteins turning over per hour. Addition of
1 µM NE reduced protein degradation significantly (Fig. 1). However,
inhibition of proteolysis by NE was observed only in fully
differentiated brown fat cells (day 10 cultures, Fig. 2B), with no effect
of NE in preadipocytes (day 3 and
day 5 cultures). There was some inhibition of proteolysis by NE in day
7 cultures, in keeping with some differentiation of the
cells (low expression of UCP-1; Fig.
2A) that
was not significant. Addition of insulin also inhibited protein
degradation but was less effective than NE (Fig. 1). The combination of
insulin and NE did not reduce protein degradation any more than NE
alone. Both NE and insulin were effective inhibitors of proteolysis,
with maximal inhibition observed at concentrations of 50 nM for insulin
and 100 nM for NE (Fig. 3). Two other
hormones, glucagon and leptin, had no effect on proteolysis (Fig. 3).

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Fig. 1.
Insulin and norepinephrine (NE) inhibit protein degradation. Brown
adipocytes were labeled with
[35S]methionine for 72 h (days 7-10), washed 3×
in DMEM + 2 mM unlabeled methionine, and then incubated without
addition ( ), with 50 nM insulin ( ), with 1 µM NE ( ), or with
both insulin and NE ( ). Protein degradation was measured as
described in MATERIALS AND METHODS,
and results are expressed as µg of labeled proteins released from
cells as acid-soluble products over 0-6 h. Results are means ± SE, with n = 3 separate cell cultures.
Analysis of variance indicates significant accumulation of labeled
material in the culture medium over time
[F(2,72) = 57;
P < 0.0001] and a significant
effect of insulin [F(1,72) = 5;
P < 0.04] and NE
[F(1,72) = 29;
P < 0.0001].
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Fig. 2.
Effect of differentiation on inhibition of proteolysis by NE.
A: expression of uncoupling protein-1
(UCP-1) as visualized by Western blots after 5, 7, and 10 days in
culture. Ten micrograms of cell proteins were separated by SDS-PAGE,
followed by immunoblotting with a polyclonal antibody against rat UCP-1
and detection by enhanced chemoluminescence (13).
B: proteolysis was measured in cells
in culture for 3, 5, 7, and 10 days incubated with (solid bar) or
without (open bar) 1 µM NE. Results are expressed as µg labeled
proteins degraded to acid-soluble products in 4 h
(left) and as % of control
[(proteolysis + NE/proteolysis NE) × 100;
right] for each time point. A
significant effect of NE
[F(3,11) = 14, * P < 0.002;
n = 3] was observed on
day 10 only.
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Fig. 3.
Effects of increasing concentrations of NE, insulin, glucagon, and
leptin on protein degradation. Protein degradation in brown adipocytes
(day 10 cultures) without addition was
54.5 ± 6.4 µg labeled proteins released as acid-soluble products in
4 h (control, 100%). Significant reductions in proteolysis were
observed with increasing concentrations of NE [ ;
F(6,21) = 3, P < 0.04] and insulin
[ ; F(9,30) = 11, P < 0.001] but not with
glucagon [ ; F(4,9) = 0.1, P < 1.0] and leptin
[ ; F(4,9) = 0.1, P < 1.0]. Results are means ± SE, with n = 2-5 separate
cell cultures.
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A
3-adrenergic
receptor-mediated increase in cAMP inhibits proteolysis.
In day 10 cultures, additions of
BRL-37344 (a selective
3-agonist), isoproterenol (a
1/
2-agonist),
and dobutamide (a selective
1-agonist) inhibited
proteolysis with a relative potency of BRL-37344 > isoproterenol > dobutamide (Fig.
4A). The
apparent EC50 values were, for
BRL-37344, 3.9 nM, for isoproterenol, 6.4 nM, and for dobutamide, 100 nM. Addition of propranolol, a
-adrenergic antagonist, significantly
prevented the reduction in protein degradation caused by 0.1 µM NE
(Fig. 4B). However, even at a
concentration 100 times that of NE, propranolol reduced the effect of
NE by only ~60%. Addition of prazosin, a
1-adrenergic receptor
antagonist, had no effect on NE-dependent inhibition of proteolysis
(Fig. 4B). In keeping with the lack
of effect of NE in preadipocytes (Fig. 3), neither BRL-37344 nor
dobutamide had any effect in day 4 cultures (data not shown). Thus the inhibitory effect of NE is
predominantly a
3-adrenergic-mediated process,
known to be linked to activation of adenylate cyclase (43).
Accordingly, addition of forskolin, which activates adenylate cyclase,
inhibited protein degradation to the same extent as NE (Fig.
5). In contrast, additions of A-23187, a
calcium ionophore, or phorbol 12-myristate-13-acetate (PMA), a phorbol
ester known to activate kinase C, had no effect. When A-23187 or PMA
was added, together with either NE or forskolin, there were no additive
effects, suggesting that increased cAMP levels by NE or forskolin are
sufficient to inhibit proteolysis. Concentrations of forskolin,
A-23187, and phorbol esters in this study were similar to or slightly
higher than those used by others in brown adipocytes (39, 43).

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Fig. 4.
Effects of adrenergic agonists (A)
and antagonists (B) on NE-dependent
inhibition of protein degradation. A:
brown adipocytes (day 10 cultures)
were incubated with increasing concentrations of BRL-37344 ( ),
isoproterenol ( ), or dobutamide ( ) for up to 4 h, and rates of
proteolysis were measured as described in MATERIALS
AND METHODS. Proteolysis in the absence of drugs was
65.4 ± 3.1 µg labeled proteins released as acid-soluble products in
4 h (Control = 100%). Results are means ± SE, with
n = 3 separate cell cultures. There
were significant differences among drugs
[F(3,215) = 4;
P < 0.01] and among
concentrations [F(12,215) = 6;
P < 0.0001].
B: brown adipocytes were incubated for
4 h with 0.1 µM NE in the presence of increasing concentrations of
propranolol ( ) or prazosin ( ). Proteolysis was 56.3 ± 4.2 µg
labeled proteins degraded to acid-soluble products in 4 h in the
absence of NE (control, 100%) and 33.8 ± 2.4 with 0.1 µM NE.
Results are means ± SE, with n = 7 separate cell cultures. There was a significant effect of propranolol
at concentrations of 5, 10, and 25 µM
(P < 0.05) but not with prazosin.
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Fig. 5.
NE inhibition of protein degradation is mediated by a cAMP-dependent
process. Brown adipocytes were incubated for 4 h in the presence of
forskolin (10 µM), A-23187 (5 µM), and phorbol esters
[phorbol 12-myristate-13-acetate (PMA), 5 µM] alone or in
combination with NE (1 µM) or forskolin (10 µM). Results (µg
labeled proteins degraded to acid-soluble products in 4 h) are shown as
means ± SE, with n = 3-6
separate cell cultures. * Significant difference
(P < 0.05) relative to cell cultures
without addition.
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Inhibition of proteolysis by NE is mediated at least in part by
inhibition of autophagy.
The effect of NE was mimicked by the addition of 3-methyladenine
(3-MA), a widely used inhibitor of autophagy (3, 15, 22, 36), at
concentrations of 1-5 mM (Table 1).
Because recent work in hepatocytes suggests that 3-MA exerts its action
from inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) (2, 3),
we tested also two structurally unrelated inhibitors of PI 3-kinase,
wortmannin and LY-294002 (Table 1). They inhibited proteolysis in brown
fat cells, as did the inhibition observed with 3-MA. The doses of
wortmannin and LY-294002 were the lowest doses causing maximal
inhibition of proteolysis (dose-response curves not shown) and are
consistent with doses required to inhibit PI 3-kinase (2). Addition of
chloroquine, an acidotropic agent that raises the pH of acidic
compartments such as lysosomes (14, 16, 22), and leupeptin, a thiol
protease inhibitor that inactivates cathepsins B, H, and L also present
in BAT (14), inhibited proteolysis markedly, chloroquine more
effectively than leupeptin at the concentrations used (Table 1).
Addition of NE, together with inhibitors of autophagy and lysosomal
function, did reduce proteolysis somewhat further, but the differences
did not reach statistical significance (Table 1).
In a second set of experiments, we made use of the observations that
proteins with short half-lives are not broken down primarily by
autophagy (20, 23), and thus the inhibitory effects of NE should be
sensitive to the length of the labeling period in a pulse-chase
experiment. In mature brown adipocytes under basal conditions, reducing
the period of labeling to 1 h is expected to increase the relative
proportion of the total labeled protein pool made of proteins with
short half-lives. Such proteins are degraded mainly by proteolytic
systems present in the cytosol or within organelles (20, 23). After 72 h of labeling, all proteins are uniformly labeled, and proteins with
short half-lives should constitute a relatively smaller fraction of the
total labeled protein pool. Thus, if NE inhibits some aspect of
autophagy, one would expect a greater inhibition after 72 h of labeling
than after 1 h, which is exactly what was observed (Fig.
6).

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Fig. 6.
Effects of reducing labeling period on subsequent inhibition of protein
degradation by NE. Brown adipocytes were incubated with
[35S]methionine in
methionine-free DMEM for 1 h. After removal of labeled precursor,
measurement of protein degradation was carried out with ( ) or
without ( ) NE (1 µM) as described in MATERIALS
AND METHODS.
[35S]methionine
incorporation after 1 h was 2,448 ± 305 counts · min 1 · µg
protein 1 with
n = 3 separate cell cultures. There
was significant release of acid-soluble radioactivity over 4 h
[F(3,72) = 115, P < 0.0001] that was reduced
by NE [F(3,72) = 7.5, P < 0.001].
Inset, %inhibition of protein
degradation by NE over 4 h in cells previously labeled with radioactive
methionine for 1 or 72 h
[F(1,15) = 6.4, P < 0.03].
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DISCUSSION |
There are very few studies that examine the control of proteolysis in
adipocytes in general and brown fat cells in particular. This is a
first survey of what controls proteolysis in brown adipocytes. Brown
fat cells differentiated in culture had a high rate of protein turnover
(~2%/h; Fig. 1), consistent with the net protein loss observed by
others when brown fat cells were incubated with cycloheximide to block
protein synthesis (31). This proteolysis has both an autophagic and a
nonautophagic component, as shown by partial inhibition with blockers
of autophagy and lysosomal function (Table 1). For instance, 3-MA is an
effective autophagy inhibitor in all mammalian cell types (3, 16, 22,
36) and reduced proteolysis by 40-45% in brown fat cells (Table
1). However, because 3-MA was shown recently to inhibit PI 3-kinase,
likely underlying its mechanism of action as an inhibitor of autophagy (2, 3), it may have numerous other effects. In hepatocytes, classical
inhibitors of PI 3-kinase, wortmannin and LY-294002, inhibit the
sequestration step in the formation of autophagic vacuoles without the
cytotoxic effects associated with high concentrations of 3-MA (2). Like
3-MA, wortmannin and LY-294002 were also effective inhibitors of
proteolysis in brown adipocytes (Table 1), suggesting that, as in
hepatocytes (2), some inositol phosphate metabolites are required for proteolysis.
Proteolysis was markedly inhibited by NE (Figs. 1, 2, 3, 5; Table 1).
NE appears to inhibit autophagy, as shown by a comparable inhibition by
3-MA, wortmannin, and LY-294002, without net additive effects when
added together with NE, suggesting that they are affecting the same
pathway (Table 1). Furthermore, reducing the labeling period from 72 to
1 h, which biases the measurements of proteolysis toward proteins with
short half-lives [not degraded primarily by autophagy (20,
23)], reduced the inhibitory effect of NE (Fig. 6). This is not
to say that reduction of autophagy is the only way by which NE inhibits
proteolysis. When NE was added together with inhibitors of autophagy or
lysosomal function, proteolysis was always slightly less than from
additions of inhibitors alone (Table 1). This slightly greater effect
of NE on proteolysis did not reach statistical significance but is
suggestive that NE may be affecting other proteolytic pathways in
addition to autophagy. This nonautophagic component is likely cytosolic
and/or mitochondrial, as both compartments have proteolytic systems
capable of breaking down proteins to amino acids (20, 23, 27). There are extensive changes in mitochondrial structure and protein
composition (e.g., loss of UCP-1) under conditions of brown adipose
tissue atrophy, and it is possible that NE may affect the expression or
activity of proteases located within mitochondria.
A
3-adrenergic
receptor-mediated increase in intracellular cAMP is likely responsible
for the inhibitory action of NE. First, propranolol, a
-adrenergic
antagonist, partially antagonized the effect of NE, whereas prazosin,
an
1-adrenergic antagonist, had
no effect (Fig. 4B), indicating a
role for
-adrenergic receptors. The concentration of propranolol
required to inhibit even partially the effect of 0.1 µM NE on
proteolysis was high (10-50 µM). This result points to a
3-receptor-mediated effect,
which is known to be more resistant to propranolol than
1- or
2-adrenergic receptor-mediated
events (43). Second, the order of potency of adrenergic agonists in
reducing proteolysis (BRL-37344 > isoproterenol > dobutamide; Fig.
4A) is consistent with similar
results by others showing an important role of the
3-adrenoreceptor in mediating the lipolytic and thermogenic effects of NE (1, 43). Third, forskolin,
an activator of adenylate cyclase, inhibited proteolysis to the same
extent as NE, likely underlying its mechanism of action (Fig. 5). The
lack of effect of A-23187 and phorbol esters on proteolysis with or
without NE (Fig. 5) is also consistent with the inability of prazosin
to inhibit NE's effects on proteolysis (Fig.
4B). Altogether, these results point
to a
3-receptor-mediated increase in cAMP as responsible for the inhibitory effects of NE on proteolysis.
The physiologically important features of
3-adrenergic receptors are low
affinity for NE and resistance to desensitization during prolonged
agonist exposure (1). The concentration of NE that maximally inhibited
proteolysis (100 nM; Fig. 3) is in keeping with the concentration
suggested to occur in the synaptic cleft (1). Even plasma
concentrations of NE of 1-25 nM [depending on the
environmental conditions (1)] would inhibit proteolysis to some
extent (Fig. 3). Thus there should be chronic inhibition of
proteolysis with prolonged sympathetic stimulation, as during cold
exposure, and increased intracellular proteolysis when sympathetic activity is suppressed, as during deacclimation, food deprivation, lactation, and drug-induced diabetes (25). This interpretation is
consistent with in vivo data showing marked protein loss from brown
fat on surgical denervation or sympathectomy (10, 13, 30).
It was surprising that NE did not inhibit proteolysis in preadipocytes,
as it did in mature cells (Fig. 2). NE stimulates proliferation of
preadipocytes by a
1-adrenergic
receptor process, linked to an increase in cAMP (6). One possible
explanation is the appearance late in differentiation of a protein,
perhaps a protein or lipid kinase, to couple
3-adrenergic receptor function and proteolysis. Changes in protein and lipid phosphorylation are
involved in the control of autophagy (3). Another possibility is a link
between NE-dependent uncoupling of metabolism and proteolysis, because
expression of UCP-1 is also a late event in differentiation (Fig.
2A). Autophagy in hepatocytes is
sensitive to reductions in intracellular ATP (3, 16). We did not find a
significant change in ATP content of rat brown adipocytes exposed to NE
(41), but others have reported a fall of 15-20% in the ATP
content of hamster brown fat cells (28). Our current work suggests it
is unlikely that uncoupling of oxidative phosphorylation is the major mechanism of action of NE, because addition of fatty acids that are
effective activators of UCP-1 (25) has little effect on proteolysis
(unpublished observations).
Of the other three hormones tested, only insulin was able to inhibit
proteolysis (Fig. 1), at concentrations in the upper end of the
physiological range (Fig. 3). This is likely a function of the 4 h of
incubation used to measure proteolysis, during which some insulin may
be degraded or inactivated. In vivo, changes in insulin status are
known to alter sympathetic nervous system activity to BAT (24, 25), the
expression of the
3-adrenergic receptor, and the responsiveness of brown adipocytes to NE (17). It is
likely that these effects may be quantitatively more significant to BAT
growth and atrophy than insulin's direct action on proteolysis, which
was only about one-half that seen with NE (Fig. 1). The mechanism of
action of insulin, like that of NE, is uncertain. They are likely distinct.
Leptin and glucagon had no effect on proteolysis (Fig. 3). Leptin is a
hormone produced by white adipocytes and important in the control of
energy balance (19, 42). Leptin infusions increase the thermogenic and
lipolytic capacity of BAT in vivo (35), presumably because of increased
sympathetic activity (21, 29), but also from direct actions (8, 38).
Leptin receptor mRNA is present in brown fat cell cultures, and leptin
activates the Jak/Stat signal transduction pathway and transcription of many genes involved in lipid metabolism (38). This pathway is obviously
not involved in the acute control (
4 h) of protein degradation, but
longer-term effects (e.g., modulation of the cell proteolytic capacity)
are possible. Glucagon increases lipolysis and thermogenesis in BAT,
presumably via increases in cAMP, and in pharmacological doses induces
tissue growth (24). It may be that glucagon receptors are not expressed
in brown fat cells in culture, or that our incubation conditions are
not appropriate for detecting its effects. In intact liver, glucagon
increases protein degradation, whereas in isolated hepatocytes,
glucagon increases it, decreases it, or has no effect depending on the incubation conditions (26). Recent work also questions whether the
thermogenic actions of injected glucagon are due to a direct action on
brown adipocytes (15).
In summary, there is an important capacity for proteolysis in brown fat
cells kept under negative control by NE and insulin, which likely
contributes significantly to BAT growth and atrophy. For instance,
during food deprivation, reductions in sympathetic tone to BAT and in
circulating insulin levels would have the effect of both reducing
protein synthesis and enhancing protein degradation, thus contributing
to the rapid and marked atrophy of the tissue. The reverse would be
true during refeeding or cold exposure.
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ACKNOWLEDGEMENTS |
We are grateful to R. Hutchinson for artwork and photography.
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FOOTNOTES |
This work was supported by grants from the Medical Research Council of
Canada and Saskatchewan Health. S. Heal was a medical student supported
by the summer student research fund of the College of Medicine of the
University of Saskatchewan.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Desautels,
Dept. of Physiology, College of Medicine, University of Saskatchewan,
Health Sciences Bldg., 107 Wiggins St., Saskatoon, SK, Canada S7N 5E5
(E-mail: desautel{at}duke.usask.ca).
Received 14 September 1998; accepted in final form 22 April 1999.
 |
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