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(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) ( 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 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 T
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
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
These results led us to study the
pattern of UCP mRNA expression induced by T
Figure 2:
Effect of T
Figure 3:
Effect of T
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
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
Figure 5:
Effect
of T
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
These results show
that fetal rat brown adipocytes in culture respond to the T 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 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 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 Results from nuclear
run-on assays (Fig. 3) revealed that addition of T The presence of
cycloheximide in the culture media significantly reduced the induction
of UCP transcription rates (Fig. 3) in response to T T 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 In conclusion, our results show
a direct effect of T
Volume 271,
Number 4,
Issue of January 26, 1996 pp. 2076-2081
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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) ).
-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) .per 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 Tper 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.
Materials
Fetal and neonatal calf serum and
culture media were from Imperial Laboratories (Hampshire, United
Kingdom). AG-1-X8 anion exchange resin was from Bio-Rad. Proteinase K
and collagenase were from Boehringer Mannheim. Triiodothyronine,
noradrenaline, cycloheximide, and actinomycin D were from Sigma.
[H]GDP; [
-P]dCTP and
[
-P]UTP (650 Ci/mmol) were from Amersham
(Buckinghamshire, United Kingdom). Nick-translation DNA-labeling system
kit was from Life Technologies, Inc. (Middlesex, United Kingdom).
Nucleotides for transcription assays were from Pharmacia Biotech Inc.
Rabbit anti-UCP serum and rat cDNA probe for the UCP were kindly
provided by Dr. E. Rial and Dr. D. Ricquier, respectively. All other
chemicals were of reagent grade or of the highest purity available.
Isolation and Culture of Brown Adipocytes
Immature
fetal brown adipocytes were isolated from 20-day-old Wistar rat fetuses
by collagenase disgregation method according to Lorenzo et al.(16) and cultured until confluence under conditions
similar to those previously described by Guerra et
al.(17) . The neonatal calf serum used was depleted of
thyroid hormones by anion exchange chromatography, using resin AG-1-X8
from Bio-Rad(18) ; the extent of removal and final
concentrations of thyroid hormones were checked by solid-phase RIA as
described previously(15) . After confluence, cells were
maintained for 24 h in a serum-free culture medium before starting
hormonal treatments. Cells were subsequently maintained for additional
periods of time up to 72 h (as stated in the figure legends) in a
serum-free medium complemented with 100 µM ascorbate (to
prevent oxidation of adrenergic agents), in the absence or presence of
10 nM T, 1 µM NA, or T plus NA. Culture medium was always replaced every 24 h.) 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.RNA Extraction and Northern Blot Analysis
Total
RNA was isolated by the guanidinium thiocyanate-phenol-chloroform
method(19) , separated by electrophoresis on
agarose/formaldehyde gels, then transferred to membranes and hybridized
as described previously(17) . cDNA probes for UCP (20) and 18 S ribosomal RNA were labeled with
[-P]deoxy-CTP by nick translation.
Nuclear Run-on Transcription Assays
Nuclei were
isolated from the pooled cells of 10-12 plates (21, 22) and stored at -70 °C in 50 mM Hepes, pH 7.9, 150 mM NaCl, 0.1 mM EDTA, 5
mM dithiothreitol, 0.125 mM phenylmethylsulfonyl
fluoride, and 50% glycerol. The in vitro elongation reactions
were carried out as described (23, 24) . P-Labeled RNA transcripts were purified by the method of
Linial et al.(25) using Nick columns (Pharmacia) and
following the manufacturer's instructions. Denaturation of DNA
probes, application of DNA to GeneScreen membranes, hybridization of
the
P-labeled RNA transcripts to the membrane-bound DNA,
posthybridization washes, and autoradiography were carried out as
described by Roncero and Goodridge(26) .
Quantitation of Results from Northern Blots and Nuclear
Run-on Transcription Assays
Northern blots from the mRNA
experiments and slot blots from the transcription run-on assays were
subjected to autoradiography. Different exposure times were used for
computer-assisted densitometry (laser densitometer from Molecular
Dynamics, Sunnyvale, CA) being linear at all densities studied. The
results were expressed as mean ± S.E. of the number of
independent experiments, as indicated in the figure legends.Mitochondria Isolation and Determination of GDP-binding
Capacity
Mitochondria were isolated and the GDP-binding capacity
determined as described previously(27) . Protein content was
determined by the Lowry method as modified by Petterson(28) .Immunological Detection of the Uncoupling
Protein
Western blot analysis and immunological detection of the
UCP by specific rabbit anti-UCP serum were performed as described
previously(27) .Statistical Analysis
Differences between means
were statistically compared by analysis of variance, using Statview
1.03 for the Macintosh. Data were compared among them by the Scheffe F-test and considered significantly different when p < 0.05.
Effect of T
Fetal rat brown adipocyte primary cultures were used
throughout this work. After 7 days under our culture conditions, cells
reached confluence and showed a very low level of UCP mRNA when
analyzed by Northern blot (data not shown), providing a useful tool in
the study of the regulation of UCP gene expression. For this purpose,
confluent cells were serum-starved (Fig. 1, Cc) for 24
h and subsequently maintained in culture for up to 72 h in the absence
of serum and hormones (control cells, C) or in the presence of
10 nM T on UCP mRNA
Content and/or 1 µM noradrenaline
(used as known positive control for the UCP induction). Total RNA was
isolated from confluent cells, and from control or treated cells at 24,
48, and 72 h. The UCP mRNA levels determined by Northern blot analysis (Fig. 1) revealed the presence of two UCP mRNA species (1.6 and
1.9 kilobases). 24-h serum-starved confluent cells (Cc)
contained very low levels of UCP mRNA, which declined during the next
72 h of culture (control cells, C). The presence of
T, NA, or NA plus T in the culture medium
increased significantly the UCP mRNA levels reaching maximum values
after 48 h of treatment for each of the three conditions assayed:
8-fold in the NA-treated cells, and 18-fold in the T and NA
plus T-treated cells. Therefore, in the absence of
noradrenergic stimulation, T increased the UCP mRNA levels
in the cultured fetal rat cells.
-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.
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.
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).
Effect of T
Nuclear run-on assays were performed to examine the effect
of T on UCP Gene Transcription
Rate on UCP mRNA synthesis (Fig. 3).
Transcriptionally active nuclei were isolated from 24 h serum-starved
confluent cells maintained for 1 h in the absence (control cells, C) or presence of 10 nM T or 1 µM NA to induce gene transcription. Parallel experiments were carried
out in the presence of CHX. The UCP transcription rate in the
T- or NA-treated cells was 7- and 5.5-fold higher than that
obtained in the control cells (C), respectively. These data
demonstrate that T increases UCP gene transcription in
fetal rat brown adipocytes, as does the positive control (NA).
T and NA effects were significantly reduced when protein
synthesis was abolished by the presence of cycloheximide (CHX
+) (1.5- and 2-fold compared to control), confirming their
dependence on protein synthesis, as suggested by results in Fig. 2.
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.
Effect of T
24-h
serum-starved confluent cells were treated with 10 nM T on UCP mRNA Stability or 1 µM NA for 16 h to induce UCP gene expression
and obtain sufficient UCP mRNA levels for the study of its decay as a
function of time (up to 24 h), after removing T or NA from
the medium. This was performed in the presence or absence of
actinomycin D or CHX. At the indicated periods of time, total RNA was
isolated and UCP mRNA levels calculated by densitometric scanning of
the Northern blots (Fig. 4). UCP mRNA half-life values were
calculated from data shown in Fig. 4and confirmed by
semilogarithmic representations.
(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%.
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).Effect of T
The UCP levels were studied by Western blot analysis of
the mitochondrial proteins isolated from confluent cells and from cells
maintained in culture for additional 72 h period in the absence (C) or presence of 10 nM T on the Mitochondrial UCP
Levels, and/or 1
µM NA. Results from Western blot analysis (Fig. 5)
revealed that confluent cells contained a very low amount of UCP
(observed as a band of 32 kDa of molecular mass), which diminished in
those samples from C cells. However, the presence of T or/and NA in the culture medium produced an 2-2.5-fold
increase in the UCP level.
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.
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.
treatment, increasing both the amount of UCP present in the
mitochondria and its functional content.
plays a direct role on the UCP gene
expression, in the absence of noradrenergic stimulation. 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) .. 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. 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. 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) . 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 Tper se failed to increase UCP
mRNA levels. 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. 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.
)
We are grateful to Dr. E. Rial and Dr. D. Ricquier for
providing rabbit anti-UCP serum and rat cDNA probe for the UCP,
respectively, and to E. Lundin for help in the preparation of the
manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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