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Unidad de Endocrinología Molecular, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain
Address all correspondence and requests for reprints to: Dr. M. J. Obregón, Instituto Investigaciones Biomédicas. Consejo Superior de Investigaciones Científicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: mjobregon{at}biomed.iib.uam.es
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Abstract |
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Thus, our results show 1) that a variety of growth factors and hormones induce DNA synthesis in a synergistic fashion in brown preadipocytes in primary culture; and 2) there is evidence for a role of norepinephrine in the regulation of brown adipocyte proliferation, potentiating the action of serum and mitogens, besides its role in uncoupling protein messenger RNA expression.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerning the proliferation process, several studies have shown that norepinephrine (NE) released from the sympathetic nerve endings is the main mediator of the proliferative stimulus in BAT activation (5, 6, 7). NE also stimulates the proliferation of brown fat cells in primary cultures, but the presence of serum is needed for its mitogenic effect (8). In general, the proliferation of normal cells is stimulated by a variety of ligands (hormones and growth factors) that, in general, seem to act in a combinatorial fashion to induce a full mitogenic response. However, little is known about the factors that control the growth of brown preadipocytes. Insulin-like growth factor I (IGF-I) seems to be an important mitogen for fetal brown adipocytes (9), and recently, transforming growth factor-ß1 has been reported to act as a potent mitogen in the same culture system (10). It has also been shown that brown adipocytes produce basic fibroblast growth factor (bFGF), which may contribute to the enlargement of the tissue (11), and that NE increases bFGF messenger RNA (mRNA) levels (12). It appears, therefore, that NE released from sympathetic nerves represents the initial stimulus in a chain of metabolic events that leads to increased proliferation of precursor cells, but there is no further evidence of other growth factors and hormones that stimulate DNA synthesis in brown fat cells in primary culture being needed for the mitogenic effect of NE.
In this study we have developed a cell culture system of quiescent brown preadipocytes that allows study of growth factors and hormones that may stimulate DNA synthesis in rat brown adipocytes and of the role of NE in the regulation of cell proliferation. We also tested whether the mitogenic stimulus affects the ability of those cells to express the UCP mRNA, which is used as a marker of its thermogenic capacity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-32P]deoxy-CTP (3000 Ci/mmol) and
[3H]thymidine were obtained from Amersham International
(Aylesbury, UK). Formamide was purchased from Merck (Darmstadt,
Germany), and the oligo labeling system was purchased from Pharmacia
(Uppsala, Sweden). Nytran membranes for RNA blotting were
purchased from Schleicher and Schuell (Dassel, Germany), and the
glass-fiber filter mats for thymidine incorporation were obtained from
Skatron (Sterling, VA) or Wallac Oy (Turku, Finland). All other
chemicals were reagent or molecular biology grade.
Cell isolation and culture
Brown fat precursor cells were isolated from the interscapular
brown adipose tissue of 20-day-old rats as described by Néchad
et al. (13), except that the hypoosmotic shock was not
performed. The process involves collagenase digestion, separation of
mature adipocytes by flotation, and subsequent filtration through
25-µm silk filters, and precursor cells were obtained by
centrifugation. The precursor cells obtained from each animal were
divided into two culture flasks (25 cm2; Nunclon, Nunc,
Roskilde, Denmark), each containing 5 ml culture medium consisting of
DMEM supplemented with 3.5 nM insulin, 10 mM
HEPES, 50 IU penicillin/ml, 50 µg streptomycin/ml, 15
µM sodium ascorbate (culture medium), and 10% NCS. The
cells were grown at 37 C in an atmosphere of 5% CO2 in air
with 95% humidity. Cells were washed on day 1 (2000
cells/cm2), and culture medium was changed every second
day.
For proliferation assays, preconfluent cells (days 2–3) were subcultured in 24-multiwell tissue culture plates (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) at a density of 8000 cells/cm2, using 1 ml culture medium supplemented with 10% NCS. After 6 h, the cells were rinsed twice with medium and maintained for 48 h in culture medium supplemented with 2% NCS (time zero). Study of the cell cycle at time zero using cytofluorometric analysis revealed that 95% of cells were in the G0/G1 phase (quiescent cells). This time was the starting point for mitogenic stimulation. Those cells, subcultured at low density, keep the ability to express UCP mRNA (our unpublished results).
For RNA analysis, preconfluent cells (day 3) were used. After being washed and maintained for 48 h in medium supplemented with 2% NCS, quiescent cells were stimulated by the addition of various growth factors and hormones. After 48 h in culture, 10 nM T3 was added, and the RNA isolation was performed 16 h later, with a 4-h pretreatment with 10 µM NE before RNA isolation.
Proliferation assays
Growth factors, hormones, or serum were added to the cells at
time zero at the concentrations indicated in each experiment. Cell
number was determined essentially as previously described (14).
Briefly, after 40 h of treatment, the culture medium was
discarded, and the cells were fixed by adding 1% glutaraldehyde in PBS
at room temperature for 10 min and thereafter stained with 0.1%
crystal violet solution in deionized water for 30 min. After washing
with a continuous slow stream of deionized water, plates were allowed
to air dry, and the remaining dye was solubilized into a suitable
volume of 10% acetic acid solution. Quantification of the
cell-absorbed dye was carried out by determining the optical density at
590 nm by spectrophotometry. For [3H]thymidine
incorporation assays, quiescent cells were stimulated with mitogenic
agents at time zero in the presence of [3H]thymidine (1
µCi/ml). After 40 h of exposure, the medium was discarded, and
the cells were removed from the plate using a trypsin-EDTA solution.
Thereafter, the contents of each well were harvested onto glass-fiber
filters using a Skatron cell harvester (Skatron Instruments, Lier,
Norway), and the radioactivity incorporated into DNA was determined
using a ß-scintillation counter. Alternatively, a cell harvester from
Inotech (Dottikon, Switzerland) or a MicroBeta from Wallac Oy was used
in these studies.
Flow cytometry analysis
One million cells were centrifuged, suspended in 300 µl
ice-cold PBS, and fixed with 900 µl ice-cold ethanol, added dropwise
with continuous vortexing. After two washes with PBS, the cells were
suspended in 850 µl PBS containing 5 µg ribonuclease/ml and
incubated for 30 min at room temperature. The cells were then stained
by adding 125 µl propidium iodide solution (50 µg propidium
iodide/ml in 50 mM sodium citrate, pH 7.2, and 0.1% Triton
X-100) and analyzed on a FACScan flow cytometer (Becton Dickinson, San
Jose, CA) using the CellQuest software from Becton Dickinson
Immunocytometry Systems (Mansfield, MA).
RNA analysis
At the end of the experiments, the cells were dissolved in 1 ml
hot guanidine-HCl extraction buffer, and RNA was isolated after ethanol
precipitation as previously described (15). The RNA concentration was
determined by measuring the optical density at 260 nm, and the ratio
260 nm/280 nm was always around 2.0. Samples of total RNA (20 µg)
were electrophoresed in 1% agarose gels containing 2.2 M
formaldehyde. Methylene blue staining of the gels revealed the presence
of equal amounts of RNA in each lane. RNA was blotted into nylon
filters (Nytran, NY13). Hybridization and washing were carried out as
previously described, using a rat UCP complementary DNA as probe (16)
labeled by random oligo priming.
Statistics
The mean values (±SE) reported were obtained from
at least three different culture flasks or wells. All experiments
presented were repeated at least three times. When not visible in the
figures, the SE was smaller than the size of the symbols.
One-way ANOVA was applied using SSPS software (SSPS, Chicago, IL).
Statistically significant differences between mean values of different
groups were then identified by the least significant difference method.
All calculations were performed as described by Snedecor and Cochran
(17).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Stimulation of DNA synthesis by serum and growth factors
Brown fat cells arrested in the G0/G1
phase of cell cycle can reenter the S phase in response to serum or a
variety of growth factors and hormones. Several mitogens were tested
for the ability to stimulate DNA synthesis in quiescent brown
preadipocytes. As expected for primary cell cultures, none of them
induced full mitogenic activation.
When quiescent cultures were stimulated with 10% serum for 40 h,
a substantial stimulation of DNA synthesis was obtained (27-fold
increase) compared with that in untreated cells (Fig. 1
). The physiological activator of BAT,
NE, when added alone induced a small increase (3.86-fold) in thymidine
incorporation at a concentration of 1 µM. The
neuropeptide vasopressin added at 20 nM produced a 5.5-fold
increase in stimulation of DNA synthesis. Within the group of
polypeptide growth factors that bind and activate tyrosine kinase
receptors, we found that aFGF, bFGF, PDGF, and EGF stimulate DNA
synthesis at concentrations between 1–10 ng/ml. Although PDGF has been
shown to be a potent mitogen for many cells types, such as fibroblast
Swiss 3T3, in our cells, when added at 10 ng/ml, it only produced a
5-fold increase in thymidine incorporation compared with that in
untreated cells. EGF added at 5 ng/ml to cells produced a similar
increase in DNA synthesis. We found that aFGF and bFGF are potent
mitogens for brown preadipocytes. At 1 ng/ml, aFGF induced an 11-fold
increase in thymidine incorporation in the presence of heparin, which
is a prerequisite for its biological action. bFGF added at 10 ng/ml had
a smaller effect, but it did not require the presence of heparin to
exert its mitogenic stimulation (data not shown). By contrast, IGF-I
had no effect on thymidine incorporation.
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As shown, increasing amounts of serum up to 10% NCS led to increased
thymidine incorporation. When added at 0.1 or 1 µM, NE
caused a shift in the dose-response curve of serum on thymidine
incorporation, measured after 40 h of incubation. A synergistic
effect was observed between NE and low concentrations of serum, and the
stimulation was saturated at 1 µM NE. The
growth-promoting activity of NE could also be demonstrated when cell
number was measured within the same incubation period (Fig. 2B). NE
added at 1 and 10 µM was able to increase the
proliferative effect of serum, and there was a synergistic effect
between NE and the lower concentrations of serum. The concentration
range required to promote an increase in cell number was higher than
that required for DNA synthesis stimulation.
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shows dose-response curves using
different mitogens in the absence or presence of 1 µM NE.
All of the mitogens stimulated DNA synthesis in a dose-dependent
manner, although at different concentration ranges. We found that bFGF,
EGF, and PDGF stimulated DNA synthesis at concentrations ranging
between 1–10 ng/ml, and aFGF stimulated DNA synthesis at
concentrations between 0.1–1 ng/ml. Vasopressin concentrations between
1–10 nM stimulated DNA synthesis, which was markedly
potentiated by the addition of NE at either low or high concentrations
of vasopressin, reaching an 8-fold increase at 20 nM
vasopressin.
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Stimulation of DNA synthesis and proliferation by different
combinations of mitogens
Given that in most culture systems the addition of more than one
growth factor is required for complete mitogenic activation, we studied
whether vasopressin was able to affect the stimulation of DNA synthesis
elicited by mitogenic concentrations of polypeptide growth factors.
As shown in Fig. 4, the addition of 20
nM vasopressin to the cell culture enhanced the mitogenic
effect elicited by the four polypeptide growth factors. Thymidine
incorporation in response to EGF increased from 5- to 17-fold when
vasopressin was added. The potentiation of PDGF and bFGF by vasopressin
was low, but the effect on DNA synthesis stimulation elicited by aFGF
was greatly enhanced. Given that NE potentiates the growth-promoting
effect of vasopressin, we tested the effect of the addition of NE to
the combination of polypeptide growth factors and vasopressin. A marked
potentiation of thymidine incorporation was observed when 1
µM NE was added for all growth factors combinations. In
all cases, thymidine uptake was comparable to that obtained in the
presence of 10% serum; the most mitogenic combinations were those
including the presence of EGF or aFGF. Cell number was measured after
the same incubation period to monitor whether these mitogenic
combinations stimulate proliferation. Addition of the mitogenic
combinations (polypeptide growth factor plus vasopressin plus NE)
resulted in a duplication of cell number, as shown in Table 1. The presence of 10% serum increased
cell number from 19,000 to 112,000, corresponding to nearly three
cycles of cell division.
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shows that, as described
extensively, brown adipocytes express UCP mRNA when stimulated
adrenergically in the presence of T3. The 16-h treatment
with T3 was sufficient to obtain a detectable signal in the
cells exposed chronically to NE (data not shown), but the addition of
NE 4 h before RNA isolation strongly increased UCP mRNA levels. As
shown, the expression level attained varied depending on the mitogen
combinations used. In the presence of aFGF, the expression of the
transcript was greatly enhanced compared with that elicited by the
other mitogens, including serum. Thus, aFGF could play a role not only
in stimulation of DNA synthesis, but also in acquisition of the
differentiated state.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Besides fetal life, cold exposure is another situation in which increased thermogenesis is required. In this situation, adrenergic stimulation increases the thermogenic capacity of BAT, and this involves an increase in the number of brown fat cells as well as an increase in the thermogenic capacity of each cell, which is accounted for by an increase in the UCP content of the tissue (1, 2, 3, 4).
In the present study we describe for the first time a culture system of quiescent brown preadipocytes obtained from newborn rats that allows study of the proliferative capacity of several hormones and growth factors that might contribute to BAT hyperplasia. The cell density we used is 5–15 times lower than that in previous reports (8,000 vs. 125,000 cells/cm2) (8, 9), allowing a wider margin for study of the proliferative activity than in previous reports.
Using this system, we show that aFGF and bFGF are potent inducers of DNA synthesis in brown fat cells; the former is the most potent growth factor tested. Both of them are potent mitogens for a variety of normal mammalian cell types from mesoderm and neuroectoderm origin and are also potent mitogens for a large number of established cell lines. They are also capable of inducing angiogenesis in vivo (21). Recently, bFGF has been shown to be involved in BAT enlargement induced by cold exposure (11), and NE considerably increased the levels of bFGF mRNA in brown adipocyte primary cultures (12). bFGF is also produced by endothelial cells, an important event for BAT growth because during BAT activation there is a large increase in endothelial cell proliferation (5) due to the need for de novo capillary formation. Our results are in accordance with these data, as we found that bFGF is a true mitogen for rat brown preadipocytes in primary cultures, and its stimulatory effect on DNA synthesis is potentiated by the addition of NE. Furthermore, aFGF is the most potent growth factor tested in our cell culture system. Besides its mitogenic and angiogenic actions, some studies suggest that both bFGF and specially aFGF participate in the central regulation of food intake (22, 23). In fact, the concentration of aFGF in rat cerebrospinal fluid markedly increases after the start of feeding, and food intake is suppressed by the infusion of aFGF (22). BAT is important for the maintenance of energy balance, as transgenic mice in which the amount of UCP is decreased develop obesity (24). Also, chronic stimulation by long term overfeeding with a palatable "cafeteria diet" causes BAT hypertrophy (2). Thus, many hormones and peptides involved in the control of food intake may be important in BAT activation.
Another hormone related to food intake control is vasopressin. It is produced in the ventromedial nucleus of the hypothalamus, which is known to be the satiety control area, and it has anorectic effects when administered centrally (25). Vasopressin, which stimulates DNA synthesis in Swiss 3T3 cells (26), is produced by small cell lung carcinoma and has been shown to stimulate the clonal growth of small cell lung carcinoma cells in soft agar (27). In our system, vasopressin stimulation of DNA synthesis is low, but increases up to 8-fold after the addition of NE.
PDGF and EGF are polypeptide growth factors of wide spectrum. None of them stimulates DNA synthesis to a great extent, as shown previously for fetal brown adipocytes (9). In that report, EGF required the presence of neuropeptides such as vasopressin and bombesin to induce a full mitogenic response. Our results show that EGF in the presence of vasopressin and NE induces a substantial increase in both DNA synthesis and cell proliferation. However, the polypeptide growth factor IGF-I does not exert a significant stimulation of DNA synthesis in our cells despite the fact that it is a complete mitogen for fetal brown adipocytes (9, 28). Also, NE addition does not alter its effect on thymidine incorporation (data not shown). It might be possible that the growth factors that lead to cell proliferation during fetal development of BAT are different from those involved in the enlargement of the tissue during the neonatal and adult periods as well as with cold exposure.
When BAT activation takes place in the rat in response to cold acclimation or after the intake of a hypercaloric diet, hyperplasia of the tissue is seen, mainly due to the increased proliferation of brown fat precursor cells and endothelial cells (5). This effect is mediated through sympathetic innervation (29). NE has been shown to be necessary for brown fat recruitment (7, 29), and various studies, including ours, show that it directly activates the proliferation of brown fat cells (7, 8, 12), but it seems to require the presence of unknown growth factors and hormones present in serum that contribute to the increased proliferation observed (8). Our results show that NE per se does not greatly stimulate DNA synthesis, but does potentiate the growth-promoting activity of serum and growth factors on both thymidine incorporation and cell proliferation. These results are consistent with previous reported data (8). Thus, some factors present in serum are required for the NE effect. Our data suggest that bFGF, EGF, and vasopressin could be involved in this stimulatory effect of NE. The growth-promoting activity of aFGF does not increase in the presence of NE, but addition of the combination of vasopressin plus NE produces a substantial increase in DNA synthesis stimulation. Thus, it seem clear that each growth factor tested has a peculiar pattern of potentiation by NE, vasopressin, or their combination.
The acquisition of a total thermogenic capacity also involves an augmented content of UCP. In this context, we tested the ability of cultured cells to express UCP mRNA as an indication of its thermogenic capacity in the presence of the different mitogen combinations. Brown preadipocytes maintained in the continuous presence of polypeptide growth factor, vasopressin, and NE express UCP mRNA when T3 is present. The addition of NE 4 h before RNA isolation increases the amount of the UCP transcripts. Thus, it seems that the presence of NE not only potentiates the stimulatory effect of other mitogens, but also increases the ability of cells to express differentiation markers. NE stimulates both proliferation and differentiation in brown fat cells, and this effect seems to be caused by an increase in intracellular cAMP levels (6, 7, 8). Canine thyrocytes in primary culture also undergo both proliferation and differentiation when stimulated by TSH (30, 31). On the other hand, the different growth factor combinations exert different effects on UCP mRNA expression. Some growth factors, such as EGF and PDGF, could interfere with the induction of UCP mRNA expression in response to differentiating conditions. Further work is needed to elucidate the role of NE in the stimulation of proliferation and differentiation as well as the effects of the different mitogens on expression of the differentiation marker, UCP.
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Acknowledgments |
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Footnotes |
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Received May 1, 1997.
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