(Received for publication, February 13, 1995; and in revised form, November 8, 1995)
From the
Confluent fetal rat brown adipocytes in primary culture showed an almost undetectable level of uncoupling protein (UCP) mRNA and a low mitochondrial content of functional UCP. Treatment of confluent cells with 10 nM triiodothyronine in a serum-free medium, in the absence of noradrenergic stimulation, increased the amount of UCP mRNA in a time-dependent manner. This effect was due to an increased UCP gene transcription rate and UCP mRNA stabilization, resulting in a higher content of immunoreactive mitochondrial UCP and functional UCP (detected by its ability to bind GDP). Thus, triiodothyronine might play a significant physiological role in the UCP expression throughout fetal development, when brown adipose tissue starts to differentiate and UCP is primarily expressed.
The main feature of brown adipose tissue (BAT) ()is
heat production by a mechanism generally called ``non-shivering
thermogenesis.'' This function is carried out by a protein known
as uncoupling protein (UCP), localized in the mitochondrial inner
membrane, which uncouples the electron transport chain from ATP
synthesis, dissipating the proton-electrochemical gradient as
heat(1) . BAT is activated under certain conditions: in newborn
mammals, during cold adaptation, in arousal from hibernation, and in
overfeeding (for review, see (2) ).
BAT starts to
differentiate in rat fetuses during late gestation and reaches its
maximal development and thermogenic capacity during the first postnatal
week(3, 4) . The postnatal development could be due to
the noradrenaline (NA) released by the sympathetic fibers as a
consequence of the environmental temperature drop after birth, when the
newborn faces extrauterine life(5) . Noradrenaline induces UCP
expression through a -adrenergic mechanism at the transcriptional
level(6, 7) . Triiodothyronine (T
) has
been reported to be required for the optimal UCP gene expression in
response to the noradrenergic stimulus in vivo(8) , in
brown adipocyte primary cultures (9) and in dispersed brown
adipocytes(10) . During the perinatal period, the earliest UCP
expression is detected in the last days of fetal life, under euthermic
conditions(4, 11) . Therefore, UCP gene expression
before birth must be controlled by mechanisms different from the
noradrenergic-dependent induction occurring after cold stimulation. At
this stage, other hormones or growth factors can influence BAT
development and UCP gene expression. One candidate is the thyroid
hormone, which can be maternally provided at early gestation (12, 13) or later produced by the fetus
itself(14) .
Studies in vivo to determine the
signal(s) involved in the development of BAT during fetal stages are
rather limited. However, primary cultures provide a useful tool for
such studies. We have recently described that Tper se induces UCP expression after long term treatment of fetal brown
adipocytes in culture(15) . The mechanism by which T
induces UCP remains to be established. Accordingly, the aim of
this work was to study the T
mechanism of action on the UCP
gene expression using rat fetal brown adipocyte primary cultures. Our
results not only confirm our previous data showing the role of T
per se in the UCP expression in the absence of
catecholamines(15) , but also show that T
increases
the transcription rate of UCP expression and stabilizes the UCP mRNA.
The UCP mRNA and functional UCP content are thereby increased.
Cells were harvested after confluence plus 24 h in the absence of serum (Cc), or after different periods (as stated in the figure legends) of subsequent culture in the absence (control cells, C) or presence of hormones.
UCP mRNA stability (half-life, t) was measured in confluent 24-h
serum-starved cells after exposure to T
or NA for 16 h in
the culture medium in the absence of serum, in order to induce UCP gene
expression. After T
or NA treatment, actinomycin D or
cycloheximide (CHX) were added to the cultures at 10 and 7 µg/ml,
respectively in the absence of hormones, and cells were harvested at
different periods of time, as indicated in the figure legends. Control
experiments established that these concentrations of inhibitors reduced
radiolabeled uridine and leucine incorporation, respectively, by more
than 96% during the course of the experimental time periods.
Figure 1:
Northern blot analysis of UCP mRNA
from fetal brown adipocytes in culture. A, total RNA (10
µg) was size fractionated in 0.9% agarose, 0.66 M formaldehyde, transferred to nylon membranes, and hybridized with
-
P-labeled cDNA probes for UCP mRNA (upper
panel) and 18 S ribosomal RNA (lower panel, used for
normalization). RNA samples were obtained from 24-h serum-starved
confluent cells (Cc) and from 24-h serum-starved confluent
cells maintained in culture in the absence (C) or presence of
10 nM T
, 1 µM NA, or NA plus T
for an additional 24, 48, and 72 h. A representative experiment
of four is shown. B, the relative quantity of the UCP mRNA
levels expressed in arbitrary units (a.u.) and obtained by
densitometric scanning of four independent experiments after
normalization (mean ± S.E.). Treatment groups were compared by
analysis of variance. Significantly different from control group:
☆, p < 0.05; ☆☆, p < 0.01;
☆☆☆, p < 0.001. Significantly different from
T
-treated cells: *, p <
0.001.
These results led us to study the
pattern of UCP mRNA expression induced by T during shorter
periods of time and to compare it to the NA treatment. In parallel, the
dependence of the hormonal action on the protein synthesis was studied
by the simultaneous presence of CHX (Fig. 2). UCP mRNA levels
increased very rapidly in the NA-treated cells with the maximum
stimulation occurring between 5 and 12 h of treatment (25- and 30-fold
increase, respectively, compared to time 0), which declined when the
hormone was present up to 24 h (8-fold). Treatment with T
progressively increased the UCP mRNA content up to 12-fold after
24 h. The increase of UCP mRNA levels induced by T
or NA
was significantly diminished by the presence of cycloheximide, which
suggests that the effect of T
or NA depends on the ongoing
protein synthesis.
Figure 2:
Effect of T or NA on the UCP
mRNA levels in fetal brown adipocytes in culture and its dependence on
protein synthesis. Total RNA (10 µg) was size fractionated in 0.9%
agarose-0.66 M formaldehyde, transferred to nylon membranes,
and hybridized with
P-labeled cDNA probes for UCP
mRNA and 18 S ribosomal RNA (used for normalization). RNA samples were
from 24-h serum-starved confluent cells maintained in culture for the
indicated periods of time (h) in the absence(-) or presence
(+) of 10 nM T
, 1 µM NA, and 7
µg/ml CHX as indicated in the upper panel. When present,
cycloheximide was added to the culture medium 15 min prior to the
addition of hormones and maintained for the indicated periods of time
(h). A representative Northern blot out of three is shown (middle
panel). Lower panel, the relative quantity of the UCP
mRNA levels expressed in arbitrary units (a.u.) and obtained
by densitometric scanning of three independent experiments after
normalization (mean ± S.E.). The graphs plot the mRNA levels
(ordinate) as a function of the hours of treatment in the absence of
hormones (
) or in the presence of T
(
) and NA
(
), and also in the absence or presence of cycloheximide (-CHX and +CHX,
respectively).
Figure 3:
Effect of T on the UCP gene
transcription rate and its dependence on protein synthesis. Nuclear
run-on transcription assays were performed using nuclei isolated from
24-h serum-starved confluent cells maintained during 1 h in the absence
(control cells, C) or presence of 10 nM T
or 1 µM NA, and in the absence(-) or presence
(+) of 7 µg/ml CHX. The labeled RNA transcripts were
hybridized to cDNAs for UCP, pGEM (vector), and
-actin that were
immobilized on nylon filters as described under ``Experimental
Procedures.'' A representative experiment of three is shown. The
graph shows the relative UCP gene transcription rate (expressed in
arbitrary units) obtained by densitometric scanning of the
autoradiograms from three independent experiments after normalization
with pGEM and
-actin (mean ± S.E.). Treatment groups were
compared by analysis of variance. Significantly different from control
group: ☆, p < 0.001. Significantly different from
NA-treated cells:
, p < 0.001. Significantly different
from T
-treated cells: *= p <
0.001.
Figure 4:
UCP
mRNA stability in fetal brown adipocytes in culture. UCP mRNA levels
(mean ± S.E.) were obtained by densitometric scanning of
Northern blots from four independent experiments, containing 10 µg
of total RNA from fetal brown adipocytes and performed as described
under ``Experimental Procedures.'' Total RNA was isolated
from 24 h serum-starved confluent cells treated for 16 h with 1
µM NA (upper panel), or 10 nM T (lower panel), to induce UCP gene expression in culture.
RNA was isolated from cells at different periods of time (0, 2, 4, 8,
12, and 24 h) after removal of hormones and addition of 10 µg/ml
actinomycin D (
) or 7 µg/ml cycloheximide (
), or in
the absence of both inhibitors (
). The graphs plot the decrease in
the relative quantity of the UCP mRNA levels versus time (h)
of treatment in the absence or presence of inhibitors. The mean UCP
mRNA level observed at time 0 was set at
100%.
The NA-induced UCP mRNA showed a
very short half-life (4 ± 1 h), which was increased in the
presence of actinomycin D (10 ± 2 h) or cycloheximide (22
± 2 h; p < 0.05). However, the UCP mRNA induced by
T was very stable, showing a calculated half-life of more
than 24 h (p < 0.001 compared to the NA value). This mRNA
stability decreased in the presence of actinomycin D (10 ± 1 h; p < 0.001) or cycloheximide (14 ± 1 h; p < 0.001).
Figure 5:
Effect
of T on mitochondrial UCP content in fetal brown adipocytes
in culture. Upper panel, representative Western blot analysis
of mitochondrial proteins (10 µg) after immunological detection of
the UCP (M
= 32,000) with an anti-UCP
rabbit serum. Lower panel, relative quantitation of the UCP
levels obtained by densitometric scanning of four independent
experiments (mean ± S.E.). Mitochondrial proteins were obtained
from 24 h serum-starved confluent cells (Cc) and from these
cells maintained in culture for additional 72 h in the absence (C) or presence of 10 nM T
, 1 µM NA, or NA plus T
, as described under
``Experimental Procedures.'' The mean UCP level observed in
Cc was set at 100%, and the UCP levels under other conditions were
expressed relative to this value. Treatment groups were compared by
analysis of variance. Significantly different from control cells (C): ☆, p < 0.01.
In order to assess the functional
activity of the UCP and its correlation with UCP content, the
mitochondrial GDP-binding capacity of the cells in culture was measured (Table 1). B values under the different
hormonal treatments were calculated and showed a good correlation with
the UCP content derived from the Western blot analysis. No
statistically significant changes in the dissociation constants (K
values) were observed.
These results show
that fetal rat brown adipocytes in culture respond to the T treatment, increasing both the amount of UCP present in the
mitochondria and its functional content.
The immature brown adipocytes from rat fetuses proliferated
and reached confluence under our culture conditions. Confluent cells
show low levels of UCP mRNA, providing useful starting material to
study the induction of UCP gene expression. These cells were used to
study whether T plays a direct role on the UCP gene
expression, in the absence of noradrenergic stimulation.
Maintenance
of confluent cells (Cc) in culture in the absence of serum for
various periods of time, up to 72 h (C) produced a decrease in
the levels of UCP mRNA (Fig. 1). However, treatment of cells
with T during these periods of time induced an increase in
the UCP mRNA content, reaching maximum levels after 48 h of treatment.
These UCP mRNA levels were significantly higher than those obtained
after a 48-h treatment with 1 µM NA used as a positive
control. When shorter periods of treatment were analyzed (Fig. 2), results showed that T
induced a slow but
progressive increase in the levels of the UCP mRNA. Klaus et al.(29) reported that treatment of brown adipocytes
differentiated in culture with T
plus insulin caused a slow
increase of UCP mRNA content. However, in their cell model T
alone did not change UCP mRNA levels but it did prevent the loss
of UCP mRNA content after serum removal. Fig. 2also shows that
NA induced a quick and potent increase in the UCP mRNA content,
reaching a maximum between 5 and 12 h of treatment. A similar pattern
of rapid response to NA or
-adrenergic agonists was reported to
occur in primary cultures of brown adipocytes (29) and in brown
adipocyte cell lines derived from brown fat tumors of transgenic
mice(30, 31) .
The UCP mRNA levels observed in
cells treated for 48 or 72 h with NA were significantly lower than
those in cells treated with T. This fact could be explained
by a shorter half-life of the UCP mRNA in the NA-treated cells, as
deduced from results shown in Fig. 4and Table 1.
Moreover, a
-adrenergic receptor desensitization during long
treatment with NA, which has been described for many
systems(32) , cannot be excluded.
Results from nuclear
run-on assays (Fig. 3) revealed that addition of T to the culture medium increased the transcription rate of UCP
gene up to values similar to those induced by NA treatment. Yeh et
al.(33) obtained indirect evidences of the T
effect on UCP expression in vivo, where T
treatment of denervated BAT produced an increment in UCP
expression. Cassard-Doulcier et al.(34) identified a
domain in the UCP gene promoter containing a directly repeated sequence
that was able to bind retinoic X and triiodothyronine receptors.
Furthermore, after submission of this manuscript, Rabelo et al.(35) reported that T
was able to directly
stimulate the rat UCP gene, via its receptors acting on two far
up-stream thyroid hormone response elements (positioned between
-2391 and -2334). In that report, transient transfection
experiments were performed in JEG-3 cells (a human placental cell line)
as well as in HIB-1B cells (a brown fat cell line established from
hibernoma induced in transgenic mice). These results obtained by means
of a transfected reporter gene support the T
effect on the
endogenous UCP gene activation here described.
The presence of
cycloheximide in the culture media significantly reduced the induction
of UCP transcription rates (Fig. 3) in response to T and NA. This suggests that the T
and NA action
depends on ongoing protein synthesis. We have shown previously (15) that T
induces the expression of its own
nuclear receptors in fetal brown adipocytes in culture after a long
term treatment, leading to an increased nuclear T
binding
capacity and to an increased ability to respond to T
.
Therefore, the requirements for the ongoing protein synthesis in the
T
-increased UCP gene transcription rate are probably
related to the T
induction of its own nuclear receptors
expression, although other explanations cannot be excluded. From in
vivo experiments carried out in adult rats, Bianco et al.(36) reported that the T
amplification of NA
stimulation of UCP gene transcription was a mechanism not requiring
protein synthesis. The discrepancy between our results obtained in
fetal cells in culture and those from in vivo can be explained
by the following; (i) in the in vivo model the T
effect was observed using rats treated also by NA, and (ii) adult
brown adipose tissue has a higher nuclear T
binding
capacity (37) than fetal brown adipocytes in culture where a
regulation of the thyroid hormone receptors by T
has also
been described(15) .
T treatment of cells
increased the UCP gene transcription rate and stabilized the UCP mRNA.
The stabilization is deduced from the maintenance of UCP mRNA levels
after removal of the hormone (Fig. 4). These experimental
conditions are different from those reported by Bianco et al.(10) using isolated rat brown adipocytes, in which UCP
mRNA levels decrease in cells in the absence of adrenergic stimulation
but the presence of T
prevented this decline. However, they
also reported that T
per se failed to increase UCP
mRNA levels.
The short half-life of the UCP mRNA in the NA-treated cells (Fig. 4) agrees with similar data obtained from in vivo experiments, both in mice (38) and rats (39) after cold stimulation, where the main action is due to the NA released from the sympathetic innervation of the tissue. Picóet al.(40) also reported that the UCP mRNA levels induced in vitro by NA treatment of mouse brown adipocytes maintained in primary cultures decayed very rapidly after NA removal from the medium (half-life was 3.7 h). From our results and those already cited(38, 39, 40) , it is deduced that NA not only increases the UCP gene transcription rate but also destabilizes the messenger, assuring a rapid decrease of UCP mRNA levels once the signal is abolished.
The NA and/or T treatment increased
the UCP mRNA content in the cells (by 8-18-fold depending on
hormonal treatment after 48-72 h) and also the UCP present in the
mitochondrial fraction (by 2-2.5 fold, Fig. 5, Table 1). In terms of quantity, the increase in the mitochondrial
UCP does not parallel the increase in UCP mRNA levels. It should be
pointed out that the amount of functional UCP reached in the
mitochondria after the in vitro treatments reported here is
similar to that described as the maximum value reached in vivo during the perinatal development of the
rat(4, 41) . The difference between the increase in
the UCP mRNA and the mitochondrial UCP levels in the NA- and/or
T
-treated cells suggests the existence of
posttranscriptional regulatory mechanisms. Puigserver et al.(42) have previously reported that the pool of newly
synthesized UCP, after NA treatment of brown adipocytes in culture, is
more susceptible to degradation than that which has become fully
incorporated into the mitochondria.
In conclusion, our results show
a direct effect of T on UCP gene transcription and UCP mRNA
stabilization, which produces increased levels of UCP mRNA in fetal
brown adipocytes as well as a higher content of functional UCP in the
mitochondrial fraction (Fig. 5, Table 1). This effect was
obtained in the absence of adrenergic stimulation, stressing the direct
role of T
in UCP gene expression in fetal cells. Thus,
T
might play a significant role in the in utero development of the fetal brown adipose tissue, where the
noradrenergic mechanisms involved in cold stimulation are not
operating. Furthermore, due to the
-adrenergic desensitization
process, thyroid hormones might have a physiological relevance in the
maintenance of high levels of UCP acutely induced by noradrenergic
stimulation of brown adipose tissue.