1 Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa K1H 8M5, Canada; and 2 Institute of Normal Human Morphology-Anatomy, Faculty of Medicine, University of Ancona, 60020 Ancona, Italy
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Multilocular,
mitochondria-rich adipocytes appear in white adipose tissue (WAT) of
rats treated with the 3-adrenoceptor agonist, CL-316243 (CL).
Objectives were to determine whether these multilocular adipocytes
derived from cells that already existed in the WAT or from
proliferation of precursor cells and whether new mitochondria contained
in them were typical brown adipocyte mitochondria. Use of
5-bromodeoxyuridine to identify cells that had undergone mitosis during
the CL treatment showed that most multilocular cells derived from cells
already present in the WAT. Morphological techniques showed that at
least a subpopulation of unilocular adipocytes underwent conversion to
multilocular mitochondria-rich adipocytes. A small proportion of
multilocular adipocytes (~8%) was positive for UCP1 by
immunohistochemistry. Biochemical techniques showed that mitochondrial
protein recovered from WAT increased 10-fold and protein isolated from
brown adipose tissue (BAT) doubled in CL-treated rats. Stained gels
showed a different protein composition of new mitochondria isolated
from WAT from that of mitochondria isolated from BAT. Western blotting showed new mitochondria in WAT to contain both UCP1, but at a much
lower concentration than in BAT mitochondria, and UCP3, at a higher
concentration than that in BAT mitochondria. We hypothesize that
multilocular adipocytes present at 7 days of CL treatment have two
origins. First, most come from convertible unilocular adipocytes that
become multilocular and make many mitochondria that contain UCP3.
Second, some come from a cell that gives rise to more typical brown
adipocytes that express UCP1.
uncoupling proteins; white adipose tissue; brown adipose tissue; thermogenesis; ultrastructure; morphology; bromodeoxyuridine; mitochondrial biogenesis; obesity; 3-adrenoceptor; endothelial cells; brown adipocytes; convertible adipocytes
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
BROWN ADIPOCYTES in brown adipose tissue (BAT) contain a unique protein not found in any other cell type, the uncoupling protein (UCP1). Indeed, the presence of this protein in an adipocyte usually defines that adipocyte as brown (24). Recent work has disclosed the existence of a family of related proteins, also now referred to as uncoupling proteins (3, 23, 31, 34). However, these proteins are present in tissues other than BAT, as well as in brown adipocytes, and cannot be used to define any particular cell type. BAT has traditionally been regarded as occurring in specific depots in rodents, such as interscapular, perirenal, and axillary. The distribution differs from that of white adipose tissue (WAT) depots in these animals, which are in periovarian, retroperitoneal, inguinal, and other locations (8). However, unilocular white adipocytes that do not express UCP1 are usually present in BAT depots (5, 9). Moreover, under certain circumstances, cells with the morphological appearance of mature brown adipocytes can appear among the characteristic unilocular white adipocytes within WAT depots, a location where they are normally not seen.
Multilocular brown adipocytes, positive for UCP by
immunohistochemistry, have been seen in parametrial WAT depots of
cold-acclimated mice (43) and in periovarian WAT of
cold-acclimated rats (11, 12). They also
appear in WAT depots of rats (16, 17,
38) and of mice (20, 26,
32, 41) that have been treated with a
selective 3-adrenoceptor (
3-AR) agonist.
In mice, the
3-AR agonist-induced appearance of brown
adipocytes in WAT varies considerably from one strain to another and is
under complex genetic control (20, 26). We
initially defined these adipocytes as brown adipocytes at a time when
the existence of other UCPs was not known and on the basis of
immunohistochemistry with an antiserum that the present work shows to
be not entirely selective for UCP1.
It is well known that cold acclimation induces hyperplastic hypertrophy
in BAT (33). Thus norepinephrine induces proliferation of
precursor cells, via an action on 1-ARs, and
differentiation and mitochondrial biogenesis in mature brown
adipocytes, via an action on
3-ARs (4).
Chronic stimulation by a
3-AR agonist does not induce
cellular proliferation in BAT (there is no change in DNA content) but
does stimulate mitochondrial biogenesis in brown
adipocytes(21, 22). The result of chronic
stimulation of WAT by a
3-AR agonist is more complex,
and extensive remodeling of WAT occurs as its mass decreases and the
unilocular white adipocytes become smaller. DNA content decreases,
probably due to loss of vascular cells, since the adipocyte number
remains the same (16, 17). In periovarian WAT
the appearance of brown adipocytes during acclimation to cold is not
associated with cell proliferation (11), despite the
presence of sympathetic innervation to these brown adipocytes
(18).
Several hypotheses can be advanced for the origin of the multilocular adipocytes that appear in WAT under the circumstances outlined above. First, it is possible that they arise from brown adipocyte precursors already present in the WAT. It is known that 10-15% of precursors isolated from WAT become brown adipocytes rather than white adipocytes when cultured (25), and precursors to cells of the brown adipocyte lineage are present in human WAT depots (14). Second, it has been hypothesized that they arise by direct conversion of white adipocytes present in the tissue (27, 28) and that they do not necessarily express UCP1 (29, 30). Third, it has been suggested that some unilocular white adipocytes are in reality "masked" brown adipocytes that revert to the brown adipocyte phenotype in response to stimulation (6).
The present research started with two principal questions. First, are
the multilocular adipocytes that appear in WAT depots in response to
stimulation by a 3-AR agonist derived from cells that
already exist in the tissue or has cell proliferation occurred in a
subpopulation of precursor cells? We used 5-bromo-2'-deoxyuridine (BrdU) to label cells that had undergone mitosis in the tissue during the treatment with the
3-AR agonist CL-316243
(CL) and identified the cells that were labeled. Second, are the
multilocular adipocytes that appear in WAT the same as the multilocular
brown adipocytes expressing UCP1 that are present in BAT or are they another cell type masquerading as brown adipocytes? We quantitated the
changes in mitochondrial protein content of interscapular BAT (IBAT)
and retroperitoneal WAT (RWAT) and compared the proteins, including
UCP1 and UCP3, in the mitochondria isolated from these tissues using
gel electrophoresis and Western blotting. We also used electron
microscopy, to compare the ultrastructure of the multilocular
adipocytes and their mitochondria with that of brown adipocytes
in BAT, and immunohistochemistry, using a UCP1 selective antiserum, to
assess the presence of UCP1 in individual multilocular adipocytes.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Animals
Two sets of rats were studied, one in Ottawa, Canada, and the other in Ancona, Italy. In Ottawa, 10 male Sprague-Dawley rats were purchased (Charles River, St. Constant, Quebec, Canada) at 12 wk of age and housed individually at 24°C in wire mesh cages with free access to food (Agway R-M-H 4020 chow, 14.5% energy from fat) and water until they were 20 wk old. They were then separated into two groups of five rats of equivalent weights (561.4 ± 14.7 g for those to be treated with saline and 564 ± 5.7 g for those to be treated with CL). Both groups had osmotic minipumps (Alzet 2002; Alza, Palo Alto, CA) implanted subcutaneously under halothane anesthesia and received a volume of 0.49 µl/h. One group received saline, the other group received CL (dose was 1 mg · kgIn Ancona, 20 male Sprague-Dawley rats were purchased from Harlan
(Correzzana, Milan, Italy) at 20 wk of age and housed in individual
cages at 20-24°C, with free access to chow and water. After 1 wk, 12 rats, under anesthesia, had mini-osmotic pumps (Alzet 2001)
implanted subcutaneously at a median dorso-thoracic level and received
1 µl/h of a saline solution of CL 314, 243 (dose was 1 mg · kg1 · day
1) for 7 days. The control group of eight animals received only saline. The rats
receiving CL treatment were divided in two groups: seven rats were
injected daily with BrdU in saline (Sigma, St. Louis, MO), at a dose of
50 mg/kg for the first 3 days of CL treatment (CL 1-3 animals),
and another five rats were injected with BrdU during the last 4 days of
the CL treatment (CL 4-7). In the control group, four rats
received BrdU on days 1-3 (SAL 1-3) and four on
days 4-7 (SAL 4-7).
For the biochemical studies, rats were killed by decapitation, and IBAT and RWAT were removed and placed in ice-cold isolation medium (13). They were then cleaned of adherent connective tissue and muscle and, in the case of BAT, of visible WAT, and weighed. The remaining tissue was weighed and homogenized in isolation medium, and mitochondria were isolated as described previously (13). Samples of homogenates and of mitochondria were frozen for later analyses. RWAT, rather than epididymal WAT, was chosen because in previous studies we found change in epididymal WAT to be less marked than in RWAT (16, 17).
For morphological studies, animals were weighed, anesthetized with xylazine-ketamine (0.4%-0.1%/500 g body wt), and perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Tissues dissected were IBAT and RWAT. For control tests of BrdU incorporation, small intestine and esophagus were also sampled. Samples for light microscopy and immunohistochemistry were further fixed by immersion in the same fixative overnight at 4°C, and then tissues were embedded in paraffin blocks. For electron microscopy, small fragments of IBAT and RWAT were fixed in 2% glutaraldehyde-2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 4 h, postfixed in 1% osmium tetroxide, and embedded in an Epon-Araldite mixture. Semithin sections (2 µm) were stained with toluidine blue, and thin sections were obtained with an MT-X ultratome (RMC, Tucson, Arizona), stained with lead citrate, and examined with a CM10 transmission electron microscope (Philips, Eindhoven, Netherlands).
Immunohistochemistry
BrdU.
Incorporated BrdU was detected with a mouse monoclonal antibody
(Sigma, clone BU-33; St. Louis, MO) and visualized by the avidin and
biotinylated horseradish peroxidase macromolecular complex
(ABC) method. Three-micrometer sections of tissue were attached
on clean glass slides treated with poly-L-lysine solution (Sigma) and left to dry at 40°C for 2 days. After deparaffinization and rehydration with distilled water, sections were first treated with
3% hydrogen peroxide in distilled water for 5 min to inactivate endogenous peroxidase. To detect incorporated BrdU, the DNA must be
denatured to allow antibody access. We used microwave antigen retrieval
(36) with the following schedule: double irradiation in a
650-W microwave oven (Whirlpool, MWO 104) for 5 min in 0.01 M citrate
buffer, pH 6.0, with 5 min cooling between the first and second
irradiation, after which the slides were left in hot citrate buffer for
an additional 15 min and finally rinsed in phosphate-buffered saline
(PBS) for 30 min. The sections were incubated successively in:
1) primary antibody against BrdU, diluted 1:100 in PBS
containing 1% normal horse serum, to block nonspecific staining, for
1 h at 40°C, in a humid chamber; 2) PBS, twice for 15 min; 3) biotinylated secondary antibody, horse anti-mouse
IgG (Vector Labs, Burlingame, CA) 1:200, 30 min at room temperature; 4) PBS, twice for 15 min; 5) ABC complex (Vector
Labs) for 1 h; 6) PBS twice for 15 min; 7)
enzymatic development of peroxidase with 0.05% diaminobenzidine
hydrochloride (Sigma, St. Louis, MO) and 0.02% hydrogen peroxide in
0.05 Tris, pH 7.6, for 4 min. Tests of the specificity of reaction were
performed for the immunological sequence and the BrdU incorporation.
For the immunological sequence, negative control slides were prepared
by substitution of mouse IgG for the primary antibody. For the BrdU
incorporation in replicating cells, tissues of the same animals, with
known turnover time of cells, were studied (small intestine in which
labeled cells broaden and pass up the crypt, nearing the villus base by
14 h and the villus tip by 48 h and esophagus whose turnover
time is ~8 days) (40). In animals treated with BrdU in
the first 3 days (CL and SAL 1-3 groups), the interstitial villi
were negative and the superficial layer of the epithelium of the
esophagus mucosa showed BrdU-positive nuclei as expected. In animals
treated with BrdU in the last 4 days (CL and SAL 4-7 group), the
intestinal villi (base and apex) and the basal layer of epithelium of
the esophagus mucosa showed BrdU-positive nuclei as expected (Fig.
1C).
|
UCP1. Polyclonal sheep antibodies raised against rat UCP1 were used as previously described (5) for UCP1 immunohistochemistry.
Morphometric Analysis
One midline sagittal section of the entire depot on one side of each animal was used to quantitate the occurrence of different cell types. Ten different fields, viewed at ×400 magnification, were used to count ~800 adipocytes per CL-treated animal in superior, middle, and inferior regions of the depot. More fields were needed in saline-treated animals because the adipocytes were larger. Distribution of multilocular adipocytes in CL-treated rats was mostly diffuse, but a few patches composed mainly of multilocular cells, were observed.The following morphometric quantitations were performed at the light microscopic level on RWAT sections of CL-treated and control animals after 7 days of treatment. The percentages of multilocular adipocytes in all adipocytes (~800 adipocytes/animal), of UCP1-positive multilocular adipocytes in the total multilocular adipocytes, of BrdU- positive cells in all the cell types present, of BrdU-positive endothelial cells in the total BrdU-positive cells, of BrdU-positive endothelial cells in the total endothelial cells, and of BrdU-positive multilocular adipocytes in the total multilocular adipocytes were counted.
Assays
Protein was measured by a modified Lowry method, and cytochrome oxidase was measured in tissue homogenates as described before (13, 21).Gel Electrophoresis and Western Blotting
Samples of mitochondria from all five animals in each group were assessed. Figures show one representative blot or stained gel for each antiserum used. Mitochondrial proteins were separated using SDS-PAGE with 16% acrylamide gels. Standard molecular mass markers, detectable by enhanced chemiluminescence, were from Santa Cruz Biotechnology (132, 90, 55, 43, 34, and 23 kDa). Proteins were transferred to nitrocellulose membranes that were then stored at ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Origin of Multilocular Adipocytes
After 7 days of treatment with CL, an average of 17 ± 3.6% of the RWAT adipocytes had a multilocular appearance, whereas 0.3 ± 0.22% were multilocular in saline-treated animals (P < 0.001). To find out whether the multilocular cells derived from proliferation of precursors, we treated the animals with BrdU, a substance that is incorporated into the DNA of replicating cells and that subsequently persists in the nuclei of the cells deriving from that replication and can be revealed by immunohistochemistry. BrdU can be toxic if administered for long periods; therefore, we used two groups of animals: the first received BrdU during the first 3 days (group 1-3) and the second during the last 4 days (group 4-7) of treatment with CL . An average of 2.9 ± 0.65% in group 1-3 and 17.8 ± 1.5% in group 4-7 of all the RWAT cells in CL-treated animals had BrdU-positive nuclei (vs. 0.8 ± 0.25% and 3.3 ± 0.31%, respectively, in the two saline-treated groups). Most of the BrdU-positive cells were endothelial cells: 97.3 ± 1.08% (group 1-3) and 98.6 ± 0.4% (group 4-7; Fig. 1). In fact, 8.7 ± 1.51% (group 1-3) and 27.1 ± 7% (group 4-7) of all endothelial cells were labeled by BrdU in CL-treated animals. In contrast, of all the multilocular cells, 94.1 ± 1% in group 1-3 and 93.6 ± 2.8% in group 4-7 cells were negative for BrdU, strongly suggesting that the multilocular cells do not derive from a mitotic proliferation of precursors (Fig. 1).Morphology and Immunohistochemistry
Immunohistochemistry at the light microscopy level showed a markedly heterogeneous appearance of the adipocytes in CL-treated rats (Fig. 2). An average of only 8.4% of the multilocular cells expressed UCP1 by immunohistochemistry, with a maximum of 33.3% and a minimum of 0% in the different animals. Electron microscopy showed that very marked mitochondrial biogenesis must have occurred in many of the adipocytes (see Figs. 3-6). Many of the multilocular cells had the typical ultrastructure of brown adipocytes with numerous small lipid droplets and mitochondria (Fig. 3), but only a minority of them had the complete differentiation of the mitochondria characteristic of brown adipocytes. A close juxtaposition of mitochondria and lipid droplets was often observed (Fig. 3). Some of the multilocular cells showed villous extroflexions of the cytoplasm typical for white adipocytes in late stages of the delipidation process (Fig. 4) and never described for brown adipocytes during active thermogenesis (7, 8). No attempt was made to quantitate the proportion of multilocular cells showing this morphology. It was evident mainly in the cells with little remaining lipid. Electron microscopy also showed an aspect of some unilocular cells not found in controls: a thickened peripheral cytoplasmic rim rich in mitochondria (Fig. 5), suggesting that mitochondrial biogenesis had been stimulated in these cells also.
|
|
|
|
|
IBAT morphology after 7 days of CL treatment differed from that of saline-treated animals, in that the brown adipocytes of CL-treated rats showed smaller cytoplasmic lipid droplets and numerous mitochondria with abundant cristae (Fig. 6). Immunohistochemistry showed that brown adipocytes of CL-treated rats were more intensely stained than controls by the UCP1 antiserum (not shown). In a previous study, a greater density of immunogold staining was seen in BAT mitochondria of CL-treated rats than in BAT mitochondria of saline-treated rats (21).
Biochemical Indications of Mitochondrial Biogenesis
In both IBAT and RWAT, CL treatment increased protein content, total cytochrome oxidase activity, and the amount of protein recovered in the isolated mitochondria (Fig. 7, A-C). At the same time, the wet weight of IBAT did not change (0.688 ± 0.0624 g in CL-treated rats vs. 0.736 ± 0.085 g in saline-treated rats; not significant), whereas the wet weight of RWAT, a much larger tissue, decreased by ~50% (4.28 ± 0.642 g in CL-treated rats vs. 8.25 ± 0.454 g in saline-treated rats; P = 0.001). In absolute terms, the changes in protein, cytochrome oxidase, and mitochondrial protein recovered were similar for the two tissues. Protein content increased by 58 mg in RWAT and by 32 mg in IBAT. Cytochrome oxidase content increased by 110 µmol/min in RWAT and by 114 µmol/min in IBAT. Mitochondrial protein recovered increased by 2.0 mg in RWAT and by 1.9 mg in IBAT. Results are consistent with a similar stimulation of total mitochondrial biogenesis in the two tissues above a baseline content of mitochondria that was much higher in the IBAT than in the RWAT. The very low amount of mitochondrial protein recovered from RWAT of saline-treated rats and the low level of cytochrome oxidase in this tissue are consistent with the sparse and small mitochondria usually present in unilocular white adipocytes. Note that there was a 10-fold increase in mitochondrial protein recovered from RWAT in the CL-treated rats compared with the saline-treated rats, but only a 1-fold increase in the mitochondrial protein recovered from the IBAT. Thus ~90% of the mitochondrial protein in RWAT of CL-treated rats was newly made under the influence of CL stimulation, whereas ~50% of the mitochondrial protein in IBAT of CL-treated rats was newly made.
|
Because it was clear that more mitochondria had been generated in
both WAT and BAT in response to CL, the increase being 10 times more
marked in the WAT because of the low amount present without treatment,
the next question was whether these two tissues were making the same
kind of mitochondria. Gel electrophoresis of identical amounts of
mitochondrial protein followed by Coomassie blue staining revealed
several differences between the banding pattern for RWAT mitochondria
of CL-treated rats and that of IBAT mitochondria of the same animals
(Fig. 8A), indicating that the new mitochondria in WAT were not typical BAT mitochondria. The banding
pattern was similar for BAT mitochondria from saline and CL-treated
rats, indicating that the new mitochondria made in BAT in response to
CL closely resemble those already present in the BAT. Gel
electrophoresis of identical amounts of IBAT and RWAT mitochondrial
proteins followed by Western blotting using an antiserum to human
UCP3 peptide showed the presence of UCP3 in BAT mitochondria of
saline-treated rats and a selective increase in UCP3 in BAT
mitochondria of CL-treated rats (Fig. 8B). (Because the
antiserum is not totally selective for UCP3 and UCP3 standard protein
was not available, we used also BAT mitochondria from transgenic mice
with deficiency of UCP3 as well as mitochondria from wild-type mice to
identify which band was UCP3.) UCP3 was not detected in RWAT
mitochondria of saline-treated rats but was present in RWAT
mitochondria of CL-treated rats. The increase was more marked in the
RWAT mitochondria than in the IBAT mitochondria. Gel electrophoresis
followed by Western blotting using an antiserum raised in rabbits to
purified UCP1 from hamster BAT showed a selective increase in UCP1 in
BAT mitochondria of CL-treated rats (Fig. 9). No UCP1 was detected in RWAT
mitochondria from saline-treated rats, whereas UCP1 did appear in RWAT
mitochondria from CL-treated rats. However, the level of UCP1 detected
in RWAT mitochondria (10 µg protein on gel in Fig. 9) was less than
in IBAT mitochondria (1 µg on the gel), indicating again that the new
mitochondria in RWAT either were not typical BAT mitochondria or that
they were a mixture of a few typical BAT mitochondria with many
mitochondria of another kind.
|
|
Because neither antiserum used was totally selective, numerous other bands appeared on the blots. The anti-UCP3 peptide antiserum recognized more and different lower molecular mass proteins in RWAT mitochondria than in the same amount of IBAT mitochondria (Fig. 8B). Conversely, the rabbit anti-purified hamster UCP antiserum recognized more higher molecular mass proteins in the small amount of BAT mitochondria than in the larger amount of WAT mitochondria (Fig. 9). It is not possible to identify at present these many other proteins that differ in IBAT and RWAT mitochondria of CL-treated rats. However, the lack of selectivity of the antisera allows also the conclusion that there are differences in protein composition between the mitochondria from these two sources.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
There are two principal findings in this study of
remodeling of RWAT induced by chronic stimulation with the
3-AR agonist CL and associated with a marked reduction
in wet weight of the RWAT. First, the mitochondria-rich multilocular
adipocytes that appear among the unilocular adipocytes in RWAT arise
mostly from direct conversion of a subpopulation of preexisting
unilocular white adipocytes. Second, the protein composition of the
abundant new mitochondria in the multilocular adipocytes in RWAT is not the same as that of the mitochondria in multilocular adipocytes in IBAT
in the same animals. Many of the multilocular adipocytes in RWAT of the
CL-treated rats have the morphological appearance of brown adipocytes,
with numerous mitochondria and lipid droplets, but only a few of them
have a complete differentiation of the mitochondria to that
characteristic of stimulated brown adipocytes, and only a few are
positive for UCP1 by immunohistochemistry. Moreover, some of the
multilocular adipocytes in RWAT showed villous extroflexions of the
cytoplasm, typical of white adipocytes during the late stages of loss
of lipid (7, 8) but never described for brown
adipocytes during active thermogenesis. Differences in composition
between RWAT mitochondria of CL-treated rats and IBAT mitochondria of
CL-treated rats are indicated by the banding pattern of stained gels
and the pattern of proteins detected by immunoblotting, using
nonselective antisera to either UCP purified from hamster BAT or to a
peptide of human UCP3. An increase in both UCP1 and UCP3
concentration was detected in IBAT mitochondria. These two uncoupling
proteins were present in mitochondria isolated from RWAT of CL-treated
rats, but UCP1 was present at a much lower level than in BAT
mitochondria of the same animals, whereas UCP3 was more abundant in
RWAT mitochondria than in IBAT mitochondria. Neither UCP1 nor UCP3 was
detectable in RWAT mitochondria of saline-treated animals.
It seems likely, therefore, that most of the multilocular, mitochondria-rich adipocytes present in RWAT of CL-treated rats differ from the typical brown adipocytes present in IBAT of the same animals. We previously defined them as brown adipocytes (16, 17) before the existence of other uncoupling proteins was known and on the basis of reactivity with the nonselective antiserum shown now in this study to react with other mitochondrial proteins in CL-treated rats. These adipocytes should perhaps not be named "brown" if that term defines an adipocyte that expresses UCP1 but is rather convertible, meaning derived from unilocular adipocytes that do not express UCP1, following the nomenclature of Loncar (27-30). We cannot exclude the existence of a small proportion of more typical brown adipocytes, those that were immunopositive for UCP1, that may have arisen from brown preadipocytes already present in the WAT, and the expression of abundant UCP1 and also UCP3 in the mitochondria of these cells. We also cannot exclude the eventual differentiation, under continued stimulation by CL, of the convertible adipocytes into brown adipocytes, i.e., containing UCP1. Because the isolated mitochondria were derived from a mixed population of adipocytes, both convertible and more typical brown as well as more typical unilocular white adipocytes, we are not able to conclude whether convertible adipocytes express UCP1 at a very low level or whether at least some do not express this protein at all. We are able to conclude only that the level of UCP1 in mixed mitochondria isolated from RWAT at 7 days of treatment was much lower than that in isolated IBAT mitochondria. White adipose tissue of saline-treated rats had very few mitochondria, in keeping with the sparse, slender mitochondria usually seen in the white adipocytes in this tissue (7, 8, 24).
The finding of endothelial cell proliferation in response to the CL treatment is probably secondary to the release of growth factors from neighboring adipocytes stimulated by the CL. It is known that vascular endothelial growth factor is secreted by both brown (1, 2, 37) and white (10, 37, 44) adipocytes and is a potent mitogen for endothelial cells.
Our finding of an increase in UCP3 protein in mitochondria isolated
from RWAT of rats after chronic CL treatment would be in agreement with
reports of an increase in mRNA for UCP3 in WAT of 3-AR
agonist-treated rats (15, 19,
23, 42) and in leptin-treated rats
(35). UCP3 was initially reported to be expressed
exclusively in BAT and muscle in rats (39). However, at
least a subpopulation of WAT cells, those referred tohere as convertible adipocytes, has the ability to make this protein when stimulated.
A fundamental part of the long-term thermogenic response of brown
adipocytes to 3-AR agonists is stimulation of
mitochondrial biogenesis. This can apparently occur not only in brown
adipocytes in BAT but also in a subpopulation of white adipocytes in
WAT. Still unresolved are the nature and regulation of the gene(s) involved in the coordinated synthesis of the multitude of proteins needed for mitochondrial biogenesis, which necessarily accompanies the
proliferation of mitochondria as well as the selective increase in
synthesis of UCP-1 and of UCP-3 induced by norepinephrine.
If the multilocular adipocytes in RWAT of CL-treated rats are not typical brown adipocytes, can we be sure that they have an adaptive thermogenic function? Are they, as has been suggested, the location of part of the whole body thermogenic response that occurs during CL-treatment of rats (16, 17, 21)? The answer to this question cannot be ascertained at present and will require the isolation of these cells and their mitochondria and the study of their metabolic properties. Also, elucidating the many protein differences between BAT and WAT mitochondria in CL-treated rats will clearly take much further investigation.
Perspective
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Mary-Ellen Harper, Department of Biochemistry, Microbiology & Immunology, University of Ottawa, Canada, for providing BAT mitochondria from wild-type and UCP3 knockout mice to use as standards for Western blotting. We are also grateful to Dr. Daniel Ricquier for providing the sheep anti-UCP1 antiserum used in immunohistochemistry (in Ancona, Italy). We are grateful to Dr. Kurt Steiner, at Wyeth-Ayerst, for the supply of CL-316243.
![]() |
FOOTNOTES |
---|
* J. Himms-Hagen and S. Cinti share equally the senior authorship of this work.
This work was supported by grant MT-0857 of the Medical Research Council of Canada (to J. Himms-Hagen), by grants CHRX-CT94-0490 of the European Community and Ministero dell' Università e della Ricerca Scientifica e Tecnologica 1998 (to S. Cinti), and by the University of Ancona, Italy.
Technical assistance was provided by Linda Jui (in Ottawa, Canada).
Address for correspondence: J. Himms-Hagen, Dept. of Biochemistry, Microbiology and Immunology, Faculty of Medicine, Univ. of Ottawa, 451 Smyth Road, Ottawa ON K1H 8M5, Canada (E-mail:jhhagen{at}uottawa.ca).
Address for reprint requests: S. Cinti, Institute of Normal Human Morphology-Anatomy, Faculty of Medicine, Via Tronto 10/A, 60020 Ancona, Italy.
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.
Received 10 February 2000; accepted in final form 8 March 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Asano, A,
Kimura K,
and
Saito M.
Cold-induced mRNA expression of angiogenic factors in rat brown adipose tissue.
J Vet Med Sci
61:
403-409,
1999[ISI][Medline].
2.
Asano, A,
Morimatsu M,
Nikami H,
Yoshida T,
and
Saito M.
Adrenergic activation of vascular endothelial growth factor mRNA expression in rat brown adipose tissue: implication in cold-induced angiogenesis.
Biochem J
328:
179-183,
1997[ISI][Medline].
3.
Boss, O,
Muzzin P,
and
Giacobino JP.
The uncoupling proteins, a review.
Eur J Endocrinol
139:
1-9,
1998[ISI][Medline].
4.
Bronnikov, G,
Bengtsson T,
Kramarova L,
Golozoubova V,
Cannon B,
and
Nedergaard J.
1 to
3 switch in control of cyclic adenosine monophosphate during brown adipocyte development explains distinct
-adrenoceptor subtype mediation of proliferation and differentiation.
Endocrinology
140:
4185-4197,
1999
5.
Cancello, R,
Zingaretti MC,
Sarzani R,
Ricquier D,
and
Cinti S.
Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue.
Endocrinology
139:
4747-4750,
1998
6.
Casteilla, L,
Cousin B,
Viguerie-Bascands N,
Larrouy D,
and
Pénicaud L.
Hétérogénéité et plasticité cellulaires des tissus adipeux.
Médecine/Sciences
10:
1099-1106,
1994.
7.
Cinti, S.
Adipose tissue morphology: basic concepts and insights.
In: Progress in Obesity Research: 8, edited by Guy-Grand B,
and Ailhaud G.. London: Libbey, 1999, p. 3-12.
8.
Cinti, S.
The Adipose Organ. Milan, Italy: Editrice Kurtis, 1999.
9.
Cinti, S,
Frederich RC,
Zingaretti MC,
De Matteis R,
Flier JS,
and
Lowell BB.
Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue.
Endocrinology
138:
797-804,
1997
10.
Claffey, KP,
Wilkison WO,
and
Spiegelman BM.
Vascular endothelial growth factor. Regulation by cell differentiation and activated second messenger pathways.
J Biol Chem
267:
16317-16322,
1992
11.
Cousin, B,
Bascands-Viguerie N,
Kassis N,
Nibbelink M,
Ambid L,
Casteilla L,
and
Penicaud L.
Cellular changes during cold acclimatation in adipose tissues.
J Cell Physiol
167:
285-289,
1996[ISI][Medline].
12.
Cousin, B,
Cinti S,
Morroni M,
Raimbault S,
Ricquier D,
Penicaud L,
and
Casteilla L.
Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization.
J Cell Sci
103:
931-942,
1992
13.
Cui, J,
and
Himms-Hagen J.
Rapid but transient atrophy of brown adipose tissue in capsaicin-desensitized rats.
Am J Physiol Regulatory Integrative Comp Physiol
262:
R562-R567,
1992
14.
Digby, JE,
Montague CT,
Sewter CP,
Sanders L,
Wilkison WO,
O'Rahilly S,
and
Prins JB.
Thiazolidinedione exposure increases the expression of uncoupling protein 1 in cultured human preadipocytes.
Diabetes
47:
138-141,
1998[Abstract].
15.
Emilsson, V,
Summers RJ,
Hamilton S,
Liu YL,
and
Cawthorne MA.
The effects of the 3-adrenoceptor agonist BRL 35135 on UCP isoform mRNA expression.
Biochem Biophys Res Commun
252:
450-454,
1998[ISI][Medline].
16.
Ghorbani, M,
Claus TH,
and
Himms-Hagen J.
Hypertrophy of brown adipocytes in brown and white adipose tissues and reversal of diet-induced obesity in rats treated with a 3-adrenoceptor agonist.
Biochem Pharmacol
54:
121-131,
1997[ISI][Medline].
17.
Ghorbani, M,
and
Himms-Hagen J.
Appearance of brown adipocytes in white adipose tissue during CL 316,243-induced reversal of obesity and diabetes in Zucker fa/fa rats.
Int J Obes Relat Metab Disord
21:
465-475,
1997[Medline].
18.
Giordano, A,
Morroni M,
Santone G,
Marchesi GF,
and
Cinti S.
Tyrosine hydroxylase, neuropeptide Y, substance P, calcitonin gene-related peptide and vasoactive intestinal peptide in nerves of rat periovarian adipose tissue: an immunohistochemical and ultrastructural investigation.
J Neurocytol
25:
125-136,
1996[ISI][Medline].
19.
Gong, DW,
He Y,
Karas M,
and
Reitman M.
Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, 3-adrenergic agonists, and leptin.
J Biol Chem
272:
24129-24132,
1997
20.
Guerra, C,
Koza RA,
Yamashita H,
Walsh K,
and
Kozak LP.
Emergence of brown adipocytes in white fat in mice is under genetic control. Effects on body weight and adiposity.
J Clin Invest
102:
412-420,
1998
21.
Himms-Hagen, J,
Cui J,
Danforth E, Jr,
Taatjes DJ,
Lang SS,
Waters BL,
and
Claus TH.
Effect of CL-316243, a thermogenic 3-agonist, on energy balance and brown and white adipose tissues in rats.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R1371-R1382,
1994
22.
Himms-Hagen, J,
and
Danforth E, Jr.
The potential role of 3 adrenoceptor agonists in the treatment of obesity and diabetes.
Curr Opin Endocrinol Metab
3:
59-65,
1996.
23.
Himms-Hagen, J,
and
Harper ME.
Biochemical aspects of the uncoupling proteins: view from the chair.
Int J Obes Relat Metab Disord
23, Suppl6:
S30-S32,
1999[Medline].
24.
Himms-Hagen, J,
and
Ricquier D.
Brown adipose tissue.
In: Handbook of Obesity, edited by Bray GA,
Bouchard C,
and James WPT. New York: Dekker, 1998, p. 415-441.
25.
Klaus, S,
Ely M,
Encke D,
and
Heldmaier G.
Functional assessment of white and brown adipocyte development and energy metabolism in cell culture. Dissociation of terminal differentiation and thermogenesis in brown adipocytes.
J Cell Sci
108:
3171-3180,
1995
26.
Kozak, LP,
and
Koza RA.
Mitochondria uncoupling proteins and obesity: molecular and genetic aspects of UCP1.
Int J Obes Relat Metab Disord
23, Suppl6:
S33-S37,
1999[Medline].
27.
Loncar, D.
Convertible adipose tissue in mice.
Cell Tissue Res
266:
149-161,
1991[ISI][Medline].
28.
Loncar, D.
Development of thermogenic adipose tissue.
Int J Dev Biol
35:
321-333,
1991[Medline].
29.
Loncar, D,
Afzelius BA,
and
Cannon B.
Epididymal white adipose tissue after cold stress in rats. I. Nonmitochondrial changes.
J Ultrastruct Mol Struct Res
101:
109-122,
1988[ISI][Medline].
30.
Loncar, D,
Afzelius BA,
and
Cannon B.
Epididymal white adipose tissue after cold stress in rats. II. Mitochondrial changes.
J Ultrastruct Mol Struct Res
101:
199-209,
1988[ISI][Medline].
31.
Lowell, BB.
Uncoupling protein-3 (UCP3): a mitochondrial carrier in search of a function.
Int J Obes Relat Metab Disord
23, Suppl6:
S43-S45,
1999[Medline].
32.
Nagase, I,
Yoshida T,
Kumamoto K,
Umekawa T,
Sakane N,
Nikami H,
Kawada T,
and
Saito M.
Expression of uncoupling protein in skeletal muscle and white fat of obese mice treated with thermogenic 3-adrenergic agonist.
J Clin Invest
97:
2898-2904,
1996
33.
Nedergaard, J,
Herron D,
Jacobsson A,
Rehnmark S,
and
Cannon B.
Norepinephrine as a morphogen?: its unique interaction with brown adipose tissue.
Int J Dev Biol
39:
827-837,
1995[ISI][Medline].
34.
Ricquier, D,
and
Bouillaud F.
The uncoupling protein homologues: UCP1, UCP2, UCP3, StUCP and AtUCP.
Biochem J
345:
161-179,
2000[ISI][Medline].
35.
Sivitz, WI,
Fink BD,
and
Donohoue PA.
Fasting and leptin modulate adipose and muscle uncoupling protein: divergent effects between messenger ribonucleic acid and protein expression.
Endocrinology
140:
1511-1519,
1999
36.
Tischler, AS.
Triple immunohistochemical staining for bromodeoxyuridine and catecholamine biosynthetic enzymes using microwave antigen retrieval.
J Histochem Cytochem
43:
1-4,
1995
37.
Tonello, C,
Giordano A,
Cozzi V,
Cinti S,
Stock MJ,
Carruba MO,
and
Nisoli E.
Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats.
FEBS Lett
442:
167-172,
1999[ISI][Medline].
38.
Umekawa, T,
Yoshida T,
Sakane N,
Saito M,
Kumamoto K,
and
Kondo M.
Anti-obesity and anti-diabetic effects of CL316,243, a highly specific beta 3-adrenoceptor agonist, in Otsuka Long-Evans Tokushima Fatty rats: induction of uncoupling protein and activation of glucose transporter 4 in white fat.
Eur J Endocrinol
136:
429-437,
1997[ISI][Medline].
39.
Vidal-Puig, A,
Solanes G,
Grujic D,
Flier JS,
and
Lowell BB.
UCP3: an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue.
Biochem Biophys Res Commun
235:
79-82,
1997[ISI][Medline].
40.
Wynford-Thomas, D,
and
Williams ED.
Use of bromodeoxyuridine for cell kinetic studies in intact animals.
Cell Tissue Kinet
19:
179-182,
1986[ISI][Medline].
41.
Yoshida, T,
Umekawa T,
Kumamoto K,
Sakane N,
Kogure A,
Kondo M,
Wakabayashi Y,
Kawada T,
Nagase I,
and
Saito M.
3-Adrenergic agonist induces a functionally active uncoupling protein in fat and slow-twitch muscle fibers.
Am J Physiol Endocrinol Metab
274:
E469-E475,
1998
42.
Yoshitomi, H,
Yamazaki K,
Abe S,
and
Tanaka I.
Differential regulation of mouse uncoupling proteins among brown adipose tissue, white adipose tissue, and skeletal muscle in chronic 3 adrenergic receptor agonist treatment.
Biochem Biophys Res Commun
253:
85-91,
1998[ISI][Medline].
43.
Young, P,
Arch JR,
and
Ashwell M.
Brown adipose tissue in the parametrial fat pad of the mouse.
FEBS Lett
167:
10-14,
1984[ISI][Medline].
44.
Zhang, QX,
Magovern CJ,
Mack CA,
Budenbender KT,
Ko W,
and
Rosengart TK.
Vascular endothelial growth factor is the major angiogenic factor in omentum: mechanism of the omentum-mediated angiogenesis.
J Surg Res
67:
147-154,
1997[ISI][Medline].