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Division of Endocrinology, Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Québec, Canada H3T 1E2
Address all correspondence and requests for reprints to: J. Enrique Silva, M.D., Jewish General Hospital, Division of Endocrinology, Room E-162, 3755 Cote-Ste-Catherine Road, Montreal, QC, H3T 1E2, Canada. E-mail: mdsi{at}musica.mcgill.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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. A TZD, darglitazone
(darg), can rapidly induce differentiation of HIB-1B
cells, as judged by the expression of the adipocyte lipid binding
protein (aP2), lipoprotein lipase (LPL), uncoupling protein (UCP) and
ß3-adrenergic receptors. UCP messenger RNA (mRNA)
responsive to NE is evidenced as early as one day after exposure to
darg. While UCP-CAT vectors (+114/-3673 bp of rat UCP
gene) are barely responsive to NE in HIB-1B preadipocytes, both
darg and StD markedly enhance NE responsiveness of such
constructs. However, by 3 days of exposure to darg, the
responses were less vigorous than in StD cells (4- to 10-fold
vs. 20- to 50-fold), and the deletion of R90 did not
affect the response to NE in darg-differentiated cells,
whereas this deletion caused a 75% reduction in StD cells.
Prolongation of darg exposure to 5–7 days resulted in
greater response of UCP mRNA to NE and 50–80% inhibition of the
response of UCP-CAT vectors by the deletion of R90. Thus,
darg-induced differentiation of HIB-1B cells suggests
that the NE-dependent expression of the UCP gene takes place in a
step-wise manner: first, the gene is "enabled," as no UCP mRNA is
detected in HIB-1B preadipocytes; thereafter and transiently, the
response of the gene to NE is sustained by dnCRS; finally, as
differentiation progresses, a cell-specific and more powerful
cis-acting sequence, upCRS, is recruited, accounting in
the fully differentiated cell for most of the response to NE. These
results also suggest that TZDs might increase energy expenditure by
inducing terminal differentiation of BAT, and that these drugs may be
useful in the differential cloning of the factors involved in the
recruitment of the BAT specific cAMP response sequence. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of the UCP-1 gene is under complex regulation. BAT sympathetic stimulation, via norepinephrine (NE) and cAMP, is the primary signal to activate its expression but other factors, such as thyroid hormone, are critical for a full physiological response (recently reviewed in Ref.5). In spite of the progress made in the knowledge of the regulation of the expression this gene, still the sequences and mechanisms whereby cAMP stimulates the gene are poorly understood. In the mouse UCP gene, Kozak et al. (6) identified several potential cAMP response elements (CRE) of which the most important is located upstream in the gene in a 210-bp enhancer called "activator element" by the group of Ricquier (7) in the rat UCP gene. Notably, in this latter, it has not been possible to define cAMP response sequences (5, 7). The major difficulty in defining this aspect of the gene regulation has been the lack of suitable experimental models.
We have in recent years witnessed rapid progress in the understanding
of adipose tissue differentiation or adipogenesis (8). A number of
observations in this rapidly expanding field are relevant to the
studies reported here. The early stages of the adipogenic programs
appear to be common to WAT and BAT. At least in these early stages of
adipogenesis, the peroxisome proliferator-activated receptor gamma
(PPAR), more specifically PPAR2 (9, 10) seems to play a critical
role. Additional interest in these receptors has come from the
observation that the insulin-sensitizing drugs thiazolidinediones (TZD)
bind specifically to PPAR (11, 12). Most importantly, these drugs
show that PPAR2 may also participate in the terminal differentiation
of BAT. The TZD pioglitazone accelerates UCP messenger RNA (mRNA)
appearance in cultured brown adipocytes precursors and increases BAT
weight and UCP mRNA content in ob/ob and C57+/+ mice (13), whereas the
TZD BRL49653 given to adult CD1 rats increases BAT weight but not UCP
mRNA concentration (14). These, and the early observations that TZDs
can increase energy expenditure in rodents (15, 16) suggest that they
may stimulate both terminal BAT differentiation and function. TZDs have
also been reported to differentiate HIB-1B cells (17, 18), a BAT cell
line derived from a mouse hibernoma generated by transgenic expression
of the SV40 early region genes (19, 20).
HIB-1B cells exposed to TZDs could thus provide a useful model to unravel terminal differentiation of BAT, the recruitment of the UCP regulatory elements and the identification of the CRE(s). Characterizing the effect of the TZD darglitazone (darg) on the differentiation and UCP expression in HIB-1B cells, we have made interesting observations regarding the sequences involved in the stimulation of the rat UCP gene by cAMP. Results reported here suggest that the acquisition of the full expression of UCP and its responsiveness to NE occurs in a step-wise manner during differentiation and that at least two sequences participate in the mediation of cAMP stimulation of the rat UCP gene, one located in the minimal promoter and another, more powerful and tissue specific, located in a complex upstream enhancer element. Not only these results are important with regard to the regulation of the UCP gene but the capacity of TZDs to affect BAT function and differentiation adds an interesting dimension to the potential therapeutic of these drugs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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0.1%), was added for 1 to 7 days, as indicated along
with the experiments described. After differentiation, the adipogenic
media were substituted by fresh medium containing either plain FBS or
cds-FBS, as appropriate, and cells were exposed to various experimental
conditions, as described with the individual experiments. Cells were
then harvested for mRNA analysis or transfected with various UCP-CAT
expression vectors. JEG-3 and CHO cells were cultured as described
previously (21).
Transient transfection assays
Differentiated or nondifferentiated HIB-1B cells as well as
JEG-3 or CHO cells were transfected by the calcium phosphate method as
described previously (21). The preadipocytes were transfected at
80–90% confluence, and the differentiated HIB were transfected at the
end of the either of the specific differentiation protocols described
above. After darg differentiation, cells were placed in
fresh DMEM-cds-FBS and transfected 5 h later. Transfected plasmids
routinely included the 4–8 µg of UCP-CAT constructs (different
regions of the UCP gene promoter cloned upstream of the chloramphenicol
acetyl transferase [CAT] reporter gene) (21, 22, 23) and 1 µg of a
plasmid encoding the ß-galactosidase gene [pCMV-ß, ClonTech (Palo
Alto, CA) or pCH110, Pharmacia (Baie d’Urfe, Quebec, Canada)] as
internal standard. Test treatments, described with the individual
experiments, were added in fresh medium, approximately 16 h
(overnight) after transfection. Cells were harvested 24 h later
and the CAT assay performed as described elsewhere (21, 24).
RNA analysis
Total RNA was isolated from cultured HIB-1B pre-adipocytes and
adipocytes by a popular acid guanidinium thiocyanate-phenol-chloroform
extraction method previously described (25). Five or 10 µg of RNA
were denatured in formamide and formaldehyde and electrophoresed in
agarose gels for Northern blotting, following published guidelines
(26). Ethidium bromide staining, probing with an 18S rRNA or GAPDH
specific oligonucleotide was used to control for equivalence of RNA
loading. cDNAs probes for rat UCP, mouse PPAR2 (gift
from Dr. B. Spiegelman), and mouse aP2 and LPL (gift of Dr. V.
Giguère) were labeled with [
-32P]dCTP and/or
[-32P]dATP by the random primer method (27). Other
probes were commercially available and equally labeled.
Statistical analysis
Results are expressed as mean ± SEM.
Significance was assessed by the Student’s t test or ANOVA,
followed by Newman-Keul’s test for the comparison of multiple means
within an experiment. Some Northern blots were quantified using the
ImageQUant software (Molecular Dynamics, Sunnyvale, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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depicts the most salient results
regarding the acquisition of UCP expression. Cells were exposed to
darg or the darg diluent dimethyl sulfoxide
(DMSO) for the indicated times. After the induction period, cells were
challenged with various adrenergic agonists and antagonists for 4
h, as indicated, after which they were harvested and the RNA probed for
UCP, aP2 or LPL mRNAs. Note first in panels A and C of Fig. 1 that,
regardless of the exposure to darg, there was not detectable
UCP mRNA expression unless the cells were stimulated with an adrenergic
agent, suggesting that darg per se does not
significantly stimulate UCP expression. This was corroborated in RNA
obtained immediately after the incubation with darg as well
as by acutely adding the drug after the induction period (data not
shown). In Fig. 1A, cells were exposed for approximately 21/2
days to 30 µM darg and challenged with 1
µM norepinephrine (NE) or the ß3-adrenergic
receptor agonist CL316,243 (CL). As shown in lanes 3, 4, and 5, UCP
mRNA weakly if at all responded to NE or CL in the control cells,
whereas the darg-treated cells had a vigorous response
(lanes 8, 9, and 10). The effect of NE was abolished by 10
µM propranolol (lane 7). Figure 1B shows that these cells
have functional ß1- or ß2-, as well as
ß3-adrenergic receptors (ß3-AR). Cells were
stimulated for 4 h with 10 or 1 µM NE, 1
µM CL, 0.5 µM isoproterenol, or 1
µM forskolin. The receptor agonists were given ±
0.5 µM propranolol, a concentration sufficient to block
ß1-AR and ß2-AR without significantly
blocking the ß3-AR (28). Propranolol partially inhibited
the effect of 10 µM NE, markedly reduced the effect of 1
µM NE (lanes 2 and 4) and isoproterenol (lane 8) but
failed to reduce the effect of CL (lane 6). These results are
consistent with the presence of functional ß1 -AR and
ß3-AR in darg-induced HIB-1B cells, as
observed in mature brown adipocytes (29). The presence of both
receptors explain the greater response to NE than to CL, as the former
binds and activates are ß-ARs, whereas CL is a pure
ß3-AR agonist. Figure 1C shows the time dependency of the
darg effect on UCP, aP2 and LPL mRNAs. While within 1 day
there was evidence of adipose differentiation, as judged by the markers
of adipogenesis, the response of UCP mRNA was delayed, consistent with
this being a later event in the process of brown adipose
differentiation. Thus, aP2 and LPL nearly reached a plateau within a
day of exposure to darg, whereas the response of UCP mRNA to
NE was very weak at 1 day and not detectable for CL. By 2 days the
response to NE was vigorous and that to CL clear, yet weaker than that
to NE. Note, finally that neither adrenergic agent stimulated the
expression of aP2 or LPL during this short exposure. Altogether, these
results indicate that darg stimulates both the basal
adipogenesis and the brown-adipocyte specific adipogenic program in
HIB-1B cells as it makes the UCP gene responsive to NE and other
adrenergic agonists. Both ß1-AR and ß3-AR
seem to be present as in mature brown adipocytes (28).
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both constructs responded to NE,
8-Br-cAMP and forskolin indicating the presence of an intact NE
signaling pathway. The lack of UCP mRNA expression in HIB-1B
preadipocytes is thus due to other mechanism, probably repression of
the gene expression or lack of a factor necessary to enable the UCP
gene. The difference in CAT activity between 3.7 UCP-CAT and SS-70-CAT
probably reflects the greater strength of the somatostatin promoter in
HIB-1B preadipocytes because ß-galactosidase expression was the same
in cells transfected with either CAT vector (data not shown).
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shows that in JEG-3 cells the sequence
mediating cAMP response, in this case stimulated by forskolin, is
located in the minimal promoter of the UCP gene. Similar results (not
shown) were obtained in CHO cells. These observations suggest that the
90 bp, -2490/-2,399, called by us R90 as it also contains a complex
retinoic acid response element (23), contributes little or nothing to
the cAMP response in these cells. In contrast, in differentiated HIB-1B
cells, but not in JEG-3 cells, the upstream enhancer can confer
responsiveness to cAMP to a CAT vector driven by the thymidine kinase
promoter (Fig. 3B).
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shows two additional aspects of
the UCP gene response to NE in HIB-1B cells. Panel A shows the effect
of time of exposure to darg. The response to a 4-h challenge
with NE approaches a plateau by 5 days of exposure to the
darg. Note again that there was no detectable UCP mRNA in
the cells not stimulated with NE. In panel B, we show the response of
cells exposed for 3 days to darg in comparison to that of
cells differentiated with the standard protocol. The response to NE of
these latter cells was about three times more vigorous.
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and 6
suggest that this is likely the case. In Fig. 5 we have compared
UCP-CAT constructs responses to NE in cells exposed to darg
for 3 days with standard differentiated cells. The response of the
intact 3.7 UCP-CAT construct to NE in standard differentiated cells was
much more vigorous, over 20-fold, and the deletion of R90 caused
approximately a 75% drop in the response. In contrast, in
darg-differentiated cells the response of 3.7 UCP-CAT was
weaker, about 6 fold, not different from that in nondifferentiated
cells (Fig. 3
), the deletion of R90 did not affect the response, and,
moreover, the response mediated by the minimal promoter -57/+114 was
not significantly different from that of the other constructs. These
observations indicate that by 3 days of exposure to darg the
response of UCP mRNA to cAMP is mediated largely by the downstream cAMP
response sequence (dnCRS) contained in the minimal promoter of the
gene, whereas standard differentiated cells have recruited the upstream
cAMP response sequence (upCRS) contained in R90.
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. These
are two representative experiments wherein darg exposure was
prolonged for up to 10 days. After 3 days of darg, the
deletion of R90 had no effect on the CAT response to NE, in agreement
with the results just described, whereas after 5 days of
darg this deletion was associated with 50–80% drop in the
response. More prolonged exposure to darg, in
charcoal-dextran strip serum, results in cell deterioration and reduced
efficiency of transfection so that basal CAT expression in the cells
transfected with 3.7 
90 UCP-CAT was too low to calculate accurately
the fold stimulation (hence the label "error" in the figure).
Results of two other experiments in darg-differentiated
cells compared with cells differentiated with the standard protocol are
summarized in Fig. 7. Results have been
expressed as percent fall in the response to NE with the deletion of
R90, and the days on darg have been averaged. During the
first days of darg exposure, the deletion of R90 has no
effect on the response to NE, whereas in standard differentiated cells
the average drop in the response to NE with such deletion was 72%.
After 4.7 days in darg the drop in response caused by the
deletion of R90 averaged 55%, but as shown in other experiments,
e.g. Fig. 6, it could be as much as 80%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism of action of TZDs in adipogenesis is being the focus of
intensive investigation, but there is consensus that these drugs act as
ligands of PPAR [(8, 32, 33) for recent reviews]. It is possible
that ligand-activated PPARs stimulate the expression of other key
adipogenic gene products, such as C/EBP (8, 9, 10, 34, 35), but these
receptors could also act directly on some of the genes encoding adipose
tissue proteins, for example aP2, which can be directly stimulated by
liganded PPAR (36). Even though the details have to await further
research, it is reasonable to postulate that TZDs can induce adipose
differentiation by regulating both the expression of key regulatory
genes, as well as by directly affecting the expression of gene products
responsible for the mature adipose phenotype, via PPAR response
elements (PPREs) yet to be defined. How could these drugs bring about
terminal differentiation of brown adipocytes?
The UCP gene is expressed uniquely in BAT [the expression of the gene in skeletal muscle of KK yellow mice reported by Nagase et al. (37) will need reevaluation in the light of the newly discovered, more ubiquitous uncoupling protein-2 (2) or -3 (3)]. This is probably the result of gene repression in non-BAT tissues and brown adipocytes precursors. Such repression would be removed sometime during the process of differentiation of these cells. The mechanism of repression probably involves the concealing in the chromatin of the upstream enhancer described in the mouse and rat genes between approximately -2.28/-2.49 kb (6, 7), because a DNAse-I hypersensitive has been identified in this area, in chromatin from mature brown adipocytes but not in nuclei from other cells or tissues (38). In addition to the exposure of this specific part of the gene sequence, probably trans-acting factors are also necessary to bring about the expression of the gene. TZDs could enable the expression of the UCP gene and its stimulation by cAMP either by facilitating the exposure of this critical sequence, by inducing some critical transactivation factor or both, all of which is matter of active research in our laboratory.
In addition to enabling the expression of the UCP gene, does darg directly stimulate the UCP gene? Our results show that, in the absence of an adrenergic agonist, there is no detectable levels of UCP mRNA after exposing the cells for as long as 10 days to darg suggesting that drug does not directly stimulates the gene. This is in apparent contradiction with the reports by Graves and colleagues indicating that TZDs stimulate reporter gene expression driven by UCP gene sequences in HIB-1B preadipocytes (14, 18). However, it should be noted that these authors did not examine the response in differentiated HIB-1B cells nor did they examine the effect of acutely added thiazolidinediones on UCP mRNA levels. In agreement with them, we find that darg acutely added to HIB-1B preadipocytes can stimulate reporter gene expression mediated by upstream UCP gene sequences, and we also find a putative PPRE in R90, but the responsiveness is lost when the cells have entered the process of differentiation (data not shown). Besides, as shown here, darg alone does not increase UCP mRNA to a detectable level. Thus, there is no evidence this far that TZDs directly stimulate the UCP gene expression in differentiated adipocytes and our results are better interpreted as indicating that these drugs enable the expression of the gene by mechanisms to be determined, and probably also induce transactivating factors involved in the recruitment of upCRS, as discussed below.
The results reported here suggest that to make the gene fully responsive to NE, the recruitment of two cAMP response sequences is necessary. In transient transfection studies on the rat UCP gene, Cassard-Doulcier et al. obtained modest and variable responses to cAMP or adrenergic receptor agonists with virtually all their constructs spanning 4.2 kb of 5' flanking region, but no discrete sequence was deemed to be or contain a CRE (7). With transient transfection assays, Kozak et al. (5) found that, out of four possible CREs, two of them, CREs 2 and 4, were probably important for NE responses of the mouse of UCP gene. While CRE4, located in the minimal promoter was necessary for NE stimulation (its mutation reduced the responses by >90%), this sequence in isolation could not confer significant cAMP responses. This was interpreted as this sequence being "required for a promoter function" rather than being a cAMP-dependent enhancer. Only CRE2, located in between approximately -2,400 and -2,500, could confer cAMP responsiveness to both homologous and heterologous promoters (6). Our results are similar but not identical to those obtained by these authors. The upCRS is located in the same area of the gene where Kozak et al. (6) localized their CRE2. As this latter, upCRS requires the mature brown fat phenotype to be recruited and accounts for most (70–80%) of the responsiveness of the gene to cAMP in mature cells, although not as much as in the mouse gene (>90%). At variance with the results obtained by Kozak et al. (6), our data suggest that the CRE contained in dnCRS is more important than they estimated and can be considered a bona fide cAMP-dependent enhancer. Thus, we find it can sustain up to 25–30% of the reporter gene response in fully differentiated cells and seemingly is responsible for the NE-induced increase in UCP mRNA before any evidence of recruitment of the upstream element is available.
Altogether, our findings are consistent with the model we proposed in a
recent review (5). According to this model, cAMP responses of the UCP
mRNA gene would be mediated by two of several possible CRE motifs found
in the 5' flanking sequence of the gene. The downstream one (in dnCRS),
is not cell specific, weaker and probably activated by CREB, as its
response closely parallels that of the somatostatin promoter (Fig. 2),
and the overexpression of CREB enhances the response in JEG-3 cells
(data not shown). The upstream CRE contained in upCRS or R90, is cell
specific, stronger, and activated by a cell-specific protein alone or
in conjunction with CREB. The induction of such a protein could be a
direct or an indirect effect of PPAR2 activation by TZDs or
endogenous ligands such as certain prostaglandins (39). Our data
further suggest that upCRS recruitment may be facultative, in contrast
to dnCRS which is enabled in HIB-1B preadipocytes as well as
differentiated cells. Being the CRE contained in upCRS a stronger one,
the facultative recruitment may be of importance in the responses of
the tissue to stimulation. Endogenous ligands, such as those just
mentioned, could play a critical role in the recruitment of this
regulatory element. This is an area that deserves further study.
Certainly the potential of TZDs to differentiate committed precursors into brown adipocytes adds another perspective to the clinical use of these drugs. For one thing, this is consistent with early observations that TZDs stimulate energy expenditure in lean and obese rodents (15, 16). This effect of these drugs might explain the observation in human trials that the use of troglitazone has not been thus far associated with an increase in weight or adiposity (40, 41, 42, 43), even though the drug is clearly adipogenic and stimulates the expression of lipogenic enzymes. It will be critical to study the effect of these drugs on resting energy expenditure in humans. Another implication of the present results is that TZDs and HIB-1B cells could serve as a potentially valuable model to unravel the factors involved in the expression of UCP and in the cell-specific regulation by cAMP. Such a model has not been available, which explains our limited knowledge in this area in spite of the gene being cloned nearly ten years ago (44, 45).
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Acknowledgments |
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Footnotes |
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Received June 27, 1997.
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References |
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). J Biol Chem 270:12953–12956. Mol Endocrinol 10:1457–1466[Abstract]
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